Targeting KRASG13C: Exploring Warhead Orientation with Cyclic Linker-Based Inhibitors

Targeting KRASG13C: Exploring Warhead Orientation with Cyclic Linker-Based Inhibitors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting KRASG13C: Exploring Warhead Orientation with Cyclic Linker-Based Inhibitors Tonia Kirschner, João Rodriguez, Emerson Gonçalves Moreira, Janina Niggenaber, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6028840/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The small GTPase KRAS is a key driver of carcinogenesis when mutated, and significant progress has been made in targeting KRAS G 12 C and other oncogenic variants. Building on our previous work demonstrating the potential of nucleotide-based inhibitors with an acrylamide warhead to target KRAS G 13 C , we designed and synthesized a library of nucleotide-based compounds with cyclic linkers to explore the effect of warhead orientation on reactivity toward Cys13. Using mass spectrometry, kinetic studies, and protein X-ray crystallography, we validated the binding and reactivity of these modulators. In addition, computational predictions of the conformational space of the linkers and warheads provided insights into their reactivity, which agreed well with the experimental data. These findings advance our understanding of the structure-reactivity relationship in these nucleotide-based KRAS inhibitors and will be the basis for further optimization. Biological sciences/Cancer Biological sciences/Chemical biology Biological sciences/Computational biology and bioinformatics Biological sciences/Structural biology KRAS G13C Nucleotide-based inhibitors Computer-aided drug design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Kirsten rat sarcoma (KRAS) is one of the most prominent oncogenes in human tumors, found in approximately 30% of cases. 1 Oncogenic mutations in KRAS cause a shift of equilibrium from the inactive guanosine diphosphate (GDP)-bound state to the active guanosine triphosphate (GTP)-bound state, thereby amplifying proliferative signaling pathways and promoting uncontrolled cell growth. 2 – 4 Historically considered "undruggable," targeting KRAS experienced a breakthrough led by Kevan Shokat's research group. 5 Their discovery of an allosteric binding site, known as the switch II pocket (SWIIP; Fig. 1 ), enabled the development of small-molecule inhibitors that modulate this small GTPase. 6 This breakthrough led to the development of covalently binding molecules such as sotorasib (AMG510, LUMYKRAS®, Amgen) 7 and adagrasib (MRTX849, KRAZATI®, Bristol Myers Squibb) 8 , 9 , which received FDA approval in 2021 and 2022, respectively. This revolutionized the treatment of KRAS G 12 C -mutated lung cancer, a condition previously lacking effective targeted therapy. Initially, acrylamide-based inhibitors targeting the acquired cysteine residue specifically addressed G12C mutations. However, recent years have seen the emergence of novel strategies utilizing SWIIP binders incorporating mutant-specific moieties for G12D, 10 – 12 G12S, 13 and G12R mutations. 14 These advancements promise innovative approaches to address cancer types harboring mutations beyond G12C. 15 Another significant mutation hotspot within the KRAS gene is codon 13, where mutations occur in approximately 14% of tumors. Within this subset, 6% feature a glycine-to-cysteine substitution (KRAS G 13 C ), presenting a potential anchor point for the design of covalent inhibitors. 16 , 17 Currently, the first KRAS G 13 C inhibitor, RMC-8839, is in preclinical development utilizing Tri-complex ("molecular glue-like" compounds) inhibitors, initially reported by Zhang et al . and subsequently advanced by Revolution Medicines. 18 , 19 In addition to the possibility of allosteric inhibition, there is also the option of occupying the guanine nucleotide-binding pocket with an inhibitor that locks the GTPase in an inactive state. Small GTPases, such as KRAS, have an extremely high affinity for GDP and GTP. 20 , 21 Unlike kinases, which have a more moderate affinity for adenosine triphosphate (ATP) and where ATP-competitive inhibitors have been successfully developed, 22 orthosteric inhibition of KRAS initially appeared challenging. 23 However, Lim et al . and Hunter et al . first demonstrated a nucleotide-based KRAS G 12 C inhibitor (SML 8-73-1) that competes for the nucleotide-binding pocket (Fig. 1 ) and irreversibly reacts with the acquired Cys12, thereby competing with the natural substrates. These modulators, however, exhibited significantly reduced protein affinity due to modifications of the β -phosphate group. 24 – 26 Our previous work introduced nucleotide-based KRAS G 13 C inhibitors featuring a linker and acrylamide moiety, specifically targeting this mutation. 27 Compared to the compound set by Lim et al ., 24 our modifications at the 2'- or 3'-position of the ribose with the linker and the warhead preserved the high reversible affinity of the nucleotide. In this study, we utilized the acrylamide warhead, renowned for its biocompatibility due to its low non-specific reactivity towards thiol groups, which necessitates an optimal orientation of the acrylamide warhead towards the target cysteines after the initial association step and in the reversibly bound state. 3 , 28 However, our previously developed molecules displayed comparatively low reactivity towards the targeted Cys13 in KRAS, potentially due to an unfavorable preorientation of the aliphatic linear linker. In this study, we report the synthesis, reactivity, and structural profiling of novel compounds featuring cyclic aliphatic linkers. Additionally, we employed in silico methods to predict the conformational space of the linker and warhead in the reversibly bound state for nucleotide-based compounds to explore the molecular basis for the compounds’ reactivities and to guide future linker design. Results and Discussion Chemistry and Biological Experiments Design and Synthesis of Acryl-bearing GDP-based Inhibitors We started with a detailed structural analysis based on the previously published co-crystal structures of KRAS G 13 C in complex with eda-GDP and bda-GDP (PDB IDs 7OK3 and 7OK4, respectively). Closer examination revealed that the linker regions of both molecules adopt a distinct bent conformation to facilitate covalent interaction with Cys13 (Fig. 2 ). This flexibility is attributed to the aliphatic linear nature and, thus, increased adaptability of the linkers. Based on these findings, we aimed to rigidify the linker by replacing the linear design with cyclic linkers. This modification is expected to promote improved pre-orientation of the warhead, thereby increasing covalent binding efficiency. We synthesized a focused library of eight compounds featuring cyclic linkers (Fig. 3 ). To achieve this, we introduced a new synthetic strategy compared to the previously established protocol by Goebel et al ., 27 where the GDP nucleotide was protected with carbonyldiimidazole (CDI). Concurrently, the linker, a cyclic diamine, was modified with acryloyl chloride, and these two units were subsequently assembled, and the phosphate-moiety further deprotected to form the desired nucleotide-based inhibitors as 2’ and 3’-isomers (Fig. 3 A, the synthesis and analysis of intermediates are described in detail in the methods section and Tables 1 and 2 ). Selective Covalent Modification of KRAS G13C In mass spectrometry (MS) studies with KRAS G13C 1−169 (Cys-light), we observed covalent modification of the desired cysteine 13 for all compounds and that the reactivity increased with rising pH levels, which can be attributed to the enhanced nucleophilicity of the cysteine. The starting molecule, acrylic-eda-GDP 7a , exhibited a reactivity profile similar to molecule 7b . Furthermore, linkers modified at the 1,3-positions on 6-membered rings ( 7b and 7c ) showed the highest reactivity, followed by those on 5- and 7-membered rings. This observation suggests that the positioning of the warhead in the 1,3 configuration promotes a preferred orientation, thereby facilitating the covalent reaction. Conversely, 1,4-modified 6-membered rings were the least effective, likely due to their high rigidity and linear nature. Additionally, the R configuration at the warhead-carrying position was slightly favored (Fig. 4 A). In addition to KRAS G 13 C , we also investigated the reactivities towards KRAS G12C 1−169 (Cys-light) and KRAS wt 1−169 , where no to very low modification was observed, indicating good overall selectivity towards Cys13 (Figure S1 ). Time-resolved studies of compounds 7b , 7c , and 7d demonstrated modification rates comparable to the parental molecule 7a (Fig. 4 B), with compound 7b exhibiting slightly superior performance compared to 7c and 7d . Inhibitory Effects of Novel Covalent Nucleotide-based Inhibitors It was also interesting to assess the synthesized compounds' inhibitory activity. For this purpose, we utilized a son of sevenless (SOS)-catalyzed nucleotide exchange assay. Consistent with previous reports, we observed an increased intrinsic exchange rate in the KRAS G 13 C mutant compared to wild-type KRAS, presumably contributing to its oncogenic nature. 27 Upon modification of the protein with the covalent nucleotide analogs, no nucleotide exchange was observed for compounds 7a and 7b , including the linear and cyclic linkers, respectively, that showed the highest reactivities in the previous experiments. This indicates that the protein is effectively blocked in its inactive state and can no longer be activated (Fig. 5 A). In a second assay, we investigated whether SOS-catalyzed nucleotide exchange to a non-hydrolyzable GTP analog (guanosine-5'-[( β,γ )-imido]triphosphate, GppNHp) and effector binding were still possible after covalent modification with the ligand. A pull-down experiment was conducted using a glutathione S-transferase (GST)-tagged Raf RAS-binding domain (Raf RBD) to assess this. Adding GppNHp (with and without SOS) enabled the pull-down of unmodified KRAS G 13 C , demonstrating the activation of the GTPase and subsequent effector binding. In contrast, KRAS G 13 C covalently modified with molecules 7a and 7b cannot become activated in the presence of GppNHp and SOS and consequently did not interact with the Raf RBD (Fig. 5 B). Co-Crystal Structure of 7b in Complex with KRAS G 13 C Mutant To further characterize the novel nucleotides with the cyclic linkers, KRAS G 13 C was co-crystallized with compound 7b to obtain detailed structural insights into ligand binding within the binding pocket (resolution:1.85 Å, R work : 18.98, R free : 22.78; Fig. 6 A; data statistics are shown in Table 3 ). Overall, the obtained structure is highly similar to previously published GDP-bound (inactive) wild-type structures (for example PDB ID 4OBE) 25 , highlighting that our design approach preserved the binding geometry of the nucleotide scaffold, which remains similarly oriented and engages in the same reversible interactions. Additionally, the covalent bond to Cys13 is resolved in the electron density. The molecule is generally similarly oriented as the compound edaGDP 7a (PDB 7OK3). Interestingly, three molecules of RAS were found in the asymmetric unit, with the orientation of the linker near the covalently modified Cys13 modeled differently in these 3 instances (Fig. 6 B), and the electron density less well-defined than in the remaining molecule (Figure S3). This suggests flexibility of the linker, which is consistent with the results of MS studies, indicating that the pre-organization of the linker still needs to be optimized by further rigidification to contribute to an efficient covalent reaction. Computational Experiments To gain a better understanding of the chemical nature of the linkers and their associated reactivities, computational predictions of the conformational space of the linkers and the warhead within the nucleotide-based inhibitors and KRAS G 13 C were performed. In general, the success associated with the use of CADD in the design of new KRAS inhibitors can be limited by several factors, such as the accuracy of the employed scoring function used for virtual screening, the difficulty in modeling the formation of covalent bonds during docking, and the identification of bioactive conformations from conformational ensembles of flexible ligand-receptor complexes. [19] To circumvent these limitations, we developed a strategy consisting of: i) using DockTScore within the DockThor docking engine to generate reliable reversible complexes between acrylamide-containing GDP-based inhibitors 7a - i and KRAS G 13 C , and ii) refining of these complexes using multi-microsecond MD simulations to explore the proximity of the warhead to Cys13 under native conditions. The behavior of GDP and Mg 2+ during simulations with KRAS G 13 C was observed to be highly stable (Figure S4), consistent with the high reversible affinities of nucleotides and Ras described previously in the literature. [15, 20] Presumably due to the water solvent dynamics and flexibility of switch-I (residues His27-Asp38), we observed a slight shift of Mg 2+ during the simulations, slightly towards the α-phosphate O3 (Figure S6, Table S2). In contrast, the linker region between GDP and the warhead shows greater structural dynamics (Fig. 7 ). The reaction of α,β-unsaturated carbonyl covalent inhibitors with KRAS G 13 C under physiological conditions likely occurs by a concerted mechanism involving proton transfer, nucleophilic addition, and solvent-assisted tautomerization. 31 To understand the differences in reactivity among the studied molecules, we evaluated the interatomic distance between the sulfur atom of Cys13 in KRAS G 13 C and the oxygen atom of the α,β-unsaturated carbonyl group of each molecule, which characterizes the initial proton transfer step. The synthesized compounds, once bound to KRAS G 13 C , adopt conformations with their warheads mostly far away from Cys13 (distances > 0.5 nm, Fig. 7 ), while the nucleotide (GDP) remains firmly inserted in the nucleotide-binding pocket (Figure S5). Notably, compounds 7a , 7b , and 7d (Fig. 7 , A) adopted warhead conformations near the sulfur atom of Cys13 more frequently than compound 7f and 7i (Fig. 7 , B), suggesting that the higher observed reactivity results from this improved pre-orientation. However, the total population of these states with conformations close to Cys13 (distances below 0.5 nm) is in the range of 5.4% ( 7a ) 6.2% ( 7b ) and 14.1% ( 7d ) to 0.5% ( 7i ) and 0.2% ( 7f ) (Table S1 ), indicating that further compound optimization is still necessary and possible to further increase the reactivity. These results are thus well in line with the high reversible affinity of the modified nucleotides while at the same time being slow to react with the thiol group to form the covalent bond. Conclusion and Outlook In this study, we synthesized a focused library of covalently binding nucleotide analogs featuring cyclic diamine linkers to systematically examine how linker rigidity and geometry impact reactivity with the Cys13 residue in mutant KRAS. The compounds were shown to disrupt KRAS activity by inhibiting GEF-catalyzed nucleotide exchange and blocking effector binding, effectively stabilizing the protein in its inactive state. While these new analogs did not exhibit enhanced reactivity compared to the parent molecules, structural characterization using X-ray crystallography and in silico modeling provided novel insights and revealed considerable conformational flexibility that explains the low reactivity at physiological pH. Our in silico modeling, developed to predict the conformational landscape of these molecules, indicates that only a minor population of the analogs adopt conformations of the warhead close to Cys13 to achieve covalent interaction. Thus, this predictive approach allows us to explain the observed low reactivity at physiological pH and offers a valuable tool for guiding the future design of nucleotide-based inhibitors with optimized preorientation and reactivity profiles, thus providing a systematic framework for enhancing these compounds’ reactivities in a targeted manner. Methods Chemistry All reagents and solvents were purchased from Acros, Activate Scientific, Alfa Aesar, Apollo Scientific, Merck, Sigma-Aldrich, TCI Chemicals or VWR and used without further purification. Dry solvents were purchased as anhydrous reagents from commercial suppliers. 1 H and 13 C NMR spectra were recorded on a Bruker Avance DRX AV400 (400 and 101 MHz), AV500 (500 and 125 MHz), AV600 (600 and 151 MHz). 1H chemical shifts are reported in δ (ppm) as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and br (broad singlet) and are referenced to the residual solvent signal: CDCl 3 (7.26), DMSO- d 6 (2.50), or MeOD- d 4 (3.34). 13 C spectra are referenced to the residual solvent signal: CDCl 3 (77.1), DMSO- d 6 (39.52), or MeOD- d 4 (49.86). High-resolution electrospray ionization mass spectra (ESI-FTMS) were recorded on a Thermo LTQ Orbitrap (high-resolution mass spectrometer from Thermo Electron) coupled to an Accela HPLC system supplied with a Hypersil GOLD column (Thermo Electron). LCMS (ESI-MS) analysis was performed using an Agilent HPLC system (1100 series) with a CC 125/4 Nucleodur C18 gravity column (3 µm) from Macherey Nagel coupled to a Thermo Scientific Finnigan LCQ Advantage Max Ion Trap and ESA Corona detector. Compounds were purified by flash chromatography on a Biotage Isolera One using Büchi Reveleris Silica Cartridges (4–120 g) monitored by UV at λ = 210 and 280 nm. Unless otherwise noted, all final products were synthesized and used as racemic mixtures of the corresponding trans -isomers. General Procedure A for Synthesis of Modified Amines 3a to 3i – The Boc-protected amine (1 equiv) is dissolved in THF and treated with the base DIPEA (2 equiv). The mixture is cooled to 0°C in an ice bath. Subsequently, the THF-diluted acryloyl chloride (1 equiv) is added dropwise with caution, and the solution is stirred for 4 hours. Upon completion of the reaction, the reaction mixture is extracted with DCM/basic H 2 O (3 x 50 ml), and the solvent is removed under vacuum. A solution of the Boc-protected amine is prepared in DCM (3 parts) and cooled to 0°C. Then, TFA (1 part) is added dropwise, stirring the mixture on ice for 30 minutes. Subsequently, the mixture is neutralized with 10 M NaCl, and the remaining solvent is removed. The desired product is obtained through column chromatography on silica gel (DCM/MeOH + 1% NH 3 ). General Procedure B for Protection of GDP 5 – Using a strongly acidic cation exchanger (Ion Exchanger I from Merck), the nucleotide was initially converted into a DMF-soluble form. Therefore, the pre-swollen column material was loaded into a column with a diameter of 2 cm and a packing height of 15 to 20 cm and then incubated for one hour in a pyridine/water mixture (1:1). After washing the column material with a total of 250 mL of distilled water, a solution of 0.5 mmol of the nucleotide in a total volume of 3 mL was applied to the column using a Pasteur pipette. The nucleotide was eluted with a mixture of methanol/water (1:1) into a round-bottom flask preloaded with 1 mL of tetrabutylammonium hydroxide (TBA). After removing the solvent mixture using a rotary evaporator, the residue was treated three times with 20 mL of DMF each and then concentrated again. The residue was dissolved in a total of 20 mL of DMF, and after the addition of 2.5 mmol of CDI under an argon atmosphere, the reaction mixture was stirred overnight at 0°C. Upon completion of the reaction, it was quenched by adding 150 µL of methanol. The compounds formed in this reaction were used directly in the subsequent reaction without further purification. General Procedure C for Coupling the modified Amine to GDP 6a to 6i – In 5 mL of dry DMF, 2.5 mmol of the amine was dissolved and slowly added to the reaction solution. The resulting precipitate was centrifuged at 10,000 rpm for 10 minutes and then washed twice with 30 mL of dry DMF each, followed by centrifugation again. Finally, the pellet was dissolved in 30 mL of distilled water. The products formed during the reaction were used directly in the subsequent reaction without further purification. General Procedure D for Deprotection of GDP 7a to 7i – The pH of the reaction solution was adjusted to 1.5 using a 0.25 molar HCl solution and stirred overnight at 4°C. Following the completion of the reaction, the pH was adjusted to 7.5 by adding 0.25 molar NaOH solution, and the nucleotide solution was diluted to a total volume of 100 mL with ddH2O. The sample was filtered using a syringe filter and subjected to purification via a Q-Sepharose column on the FPLC system. After loading the nucleotide solution onto the column equilibrated with Buffer A (50 mM TEAB), elution was performed at a flow rate of 1 mL/min using a linear gradient of 0-100% Buffer B (1 M TEAB) over 600 minutes. The collected fractions were analyzed by HPLC using TBAB as the counterion in the running buffer (50 mM KPi pH 6.6, 10 mM TBAB, 16% ACN; column: ProntoSIL® 120-5-C18-AQ, Bischoff), and fractions containing the product were combined. Following the removal of the solvent mixture using a rotary evaporator, the residue was dissolved in 10 mL ddH 2 O and lyophilized. N -(2-aminoethyl)acrylamide 3a . According to the general procedure A, 100 mg of tert -butyl (2-aminoethyl)carbamate (0.62 mmol, 1 equiv) was reacted. 3a was obtained after purification by column chromatography in a yield of 68.51 mg (0.59 mmol, 96%). 1 H NMR (600 MHz, MeOD) δ 6.27–6.26 (m, J = 1.6 Hz, 1H), 6.26–6.25 (m, 1H), 5.73–5.70 (m, 1H), 3.53 (t, J = 9.8, 3.7 Hz, 2H), 3.09 (t, J = 6.0 Hz, 2H). 13 C NMR (151 MHz, MeOD) δ 131.57, 127.49, 40.81, 38.26, 37.90. LC-MS(ESI-MS): ( m/z ) calculated for C 5 H 11 N 2 O ([M + H] + ): 115.08; found 114.8. ( R )- N -(pyrrolidin-3-yl)acrylamide 3f . According to the general procedure A, 100 mg of tert- butyl ( R )-3-aminopyrrolidine-1-carboxylate (0.53 mmol, 1 equiv) was reacted. 3f was obtained after purification by column chromatography in a yield of 36 mg (0.26 mmol, 48.5%). 1 H NMR (600 MHz, DMSO) δ 6.51–6.30 (m, J = 60.2, 41.5 Hz, 1H), 5.31–5.26 (m, J = 17.1, 10.1 Hz, 1H), 5.21–5.14 (m, J = 17.1, 2.1 Hz, 1H), 4.71–4.66 (m, J = 10.1, 5.6, 1.9 Hz, 1H), 3.46–3.30 (m, 2H), 1.74–1.68 (m, 2H), 1.22–1.10 (m, J = 21.7, 14.5, 7.4 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 164.79, 131.29, 125.90, 49.07, 48.35, 43.44, 18.67. LC-MS(ESI-MS): (m/z) calculated for C 7 H 13 N 2 O ([M + H] + ): 141.09; found 141.1. ( S )- N -(pyrrolidin-3-yl)acrylamide 3g . According to the general procedure A, 100 mg of tert- butyl ( S )-3-aminopyrrolidine-1-carboxylate (0.53 mmol, 1 equiv) was reacted. 3g was obtained after purification by column chromatography in a yield of 48 mg (0.34 mmol, 64.6%). 1 H NMR (600 MHz, MeOD) δ 6.27–6.25 (m, J = 3.1 Hz, 1H), 6.25–6.24 (m, J = 2.5 Hz, 1H), 5.75–5.67 (m, 1H), 4.50–4.46 (m, 1H), 3.57–3.52 (m, J = 12.3, 6.9 Hz, 1H), 3.51–3.45 (m, J = 11.8, 7.6 Hz, 1H), 3.41–3.36 (m, J = 10.9, 7.7, 6.1 Hz, 1H), 3.34–3.28 (m, 1H), 2.39–2.28 (m, 1H), 2.11–2.01 (m, 1H). 13 C NMR (151 MHz, MeOD) δ 168.33, 131.40, 127.59, 51.12, 50.32, 45.60, 30.86. LC-MS(ESI-MS): ( m/z ) calculated for C 7 H 13 N 2 O ([M + H] + ): 141.1; found 141.4. N -(2-aminocyclohexyl)acrylamide 3e . According to the general procedure A, 100 mg of tert -butyl (2-aminocyclohexyl)carbamate (0.46 mmol, 1 equiv) was reacted. 3e was obtained after purification by column chromatography in a yield of 32.8 mg (0.19 mmol, 42.4%). 1 H NMR (400 MHz, MeOD) δ 6.53–6.42 (m, J = 17.0, 10.2 Hz, 1H), 6.35–6.23 (m, 1H), 5.77–5.67 (m, 1H), 3.33–3.31 (m, J = 3.2, 1.6 Hz, 2H), 1.81–1.72 (m, 2H), 1.63–1.52 (m, J = 20.2, 8.6 Hz, 2H), 1.47–1.40 (m, J = 9.7 Hz, 2H), 1.37–1.32 (m, J = 6.4 Hz, 2H). 13 C NMR (101 MHz, MeOD) δ 165.45, 137.84, 131.79, 63.21, 60.88, 35.62, 33.37, 18.92. LC-MS(ESI-MS): (m/z) calculated for C 9 H 17 N 2 O ([M + H] + ): 169.1; found 169.2. ( R )- N -(piperidin-3-yl)acrylamide 3b . According to the general procedure A, 100 mg of tert -butyl (R )-3-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. 3b was obtained after purification by column chromatography in a yield of mg 61.3 mg (0.4 mmol, 81.1%). 1 H NMR (400 MHz, MeOD) δ 6.85–6.71 (m, 1H), 6.23 (dd, J = 16.8, 1.5 Hz, 1H), 5.79 (dd, J = 10.7, 1.8 Hz, 1H), 4.36–4.26 (m, J = 7.9 Hz, 1H), 3.88–3.78 (m, 1H), 3.51–3.35 (m, 2H), 2.18–2.09 (m, J = 12.9 Hz, 1H), 1.93–1.80 (m, 1H), 1.78–1.57 (m, J = 27.3, 13.1, 9.4, 4.0 Hz, 2H), 1.41–1.28 (m, 1H). 13 C NMR (101 MHz, MeOD) δ 167.18, 129.27, 128.73, 57.17, 56.09, 45.27, 35.53, 21.41. LC-MS(ESI-MS): (m/z) calculated for C 8 H 15 N 2 O ([M + H] + ): 155.1; found 156.1. ( S )- N -(piperidin-3-yl)acrylamide 3c . According to the general procedure A, 100 mg of tert -butyl (S )-3-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. 3c was obtained after purification by column chromatography in a yield 68.8 mg (0.4 mmol, 91.1%). 1 H NMR (700 MHz, MeOD) δ 6.81–6.70 (m, 1H), 6.26–6.19 (m, J = 16.7 Hz, 1H), 5.81–5.75 (m, J = 10.7 Hz, 1H), 4.35–4.27 (m, 1H), 4.13–4.01 (m, 1H), 3.90–3.80 (m, J = 13.2 Hz, 1H), 3.44–3.35 (m, J = 25.1 Hz, 1H), 3.26–3.18 (m, J = 33.6, 16.8 Hz, 1H), 2.17–2.09 (m, 1H), 1.90–1.79 (m, 1H), 1.75–1.67 (m, 1H), 1.68–1.55 (m, J = 52.1 Hz, 1H). 13 C NMR (176 MHz, MeOD) δ 178.25, 129.13, 128.75, 55.83, 46.81, 45.44, 29.32, 24.17. LC-MS(ESI-MS): ( m/z ) calculated for C 8 H 15 N 2 O ([M + H] + ): 155.1; found 156.1. N -(piperidin-4-yl)acrylamide 3i . According to the general procedure A, 100 mg of tert- butyl 4-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. 3i was obtained after purification by column chromatography in a yield of 61.3 mg (0.4 mmol, 80%) . 1 H NMR (500 MHz, DMSO) δ 8.60 (s, 1H), 8.42 (s, 1H), 8.23 (d, J = 7.4 Hz, 1H), 6.21 (dd, J = 17.1, 10.0 Hz, 1H), 6.13–6.07 (m, 1H), 5.60 (dd, J = 10.0, 2.3 Hz, 1H), 3.89 (tdd, J = 11.0, 7.4, 3.9 Hz, 1H), 3.26 (d, J = 12.9 Hz, 2H), 2.99 (dd, J = 22.2, 12.1 Hz, 2H), 1.92 (dd, J = 13.8, 3.1 Hz, 2H), 1.61–1.52 (m, 2H). 13 C NMR (126 MHz, DMSO) δ 163.98, 131.56, 125.57, 43.45, 42.14, 28.24. LC-MS(ESI-MS): (m/z) calculated for C 8 H 15 N 2 O ([M + H] + ): 155.1; found 156.1. 1-(4-aminopiperidin-1-yl)prop-2-en-1-one 3h . According to the general procedure A, 100 mg of tert- butyl 4-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. 3h was obtained after purification by column chromatography in a yield of 61.3 mg (0.39 mmol, 80%). 1 H NMR (700 MHz, MeOD) δ 6.70–6.64 (m, J = 16.8, 10.7 Hz, 1H), 6.12–6.07 (m, J = 16.8, 3.4 Hz, 1H), 5.68–5.64 (m, J = 10.7, 1.8 Hz, 1H), 4.53 (d, J = 12.7 Hz, 1H), 4.11 (d, J = 13.7 Hz, 1H), 3.34–3.27 (m, J = 13.5, 9.5 Hz, 1H), 3.17–3.09 (m, 1H), 2.03–1.94 (m, J = 13.4 Hz, 2H), 1.48–1.36 (m, 2H). 13 C NMR (176 MHz, MeOD) δ 167.63, 128.89, 128.83, 45.05, 41.41, 30.74. LC-MS(ESI-MS): ( m/z ) calculated for C 8 H 15 N 2 O ([M + H] + ): 155.1; found 155.1. ( R )- N -(azepan-3-yl)acrylamide 3d . According to the general procedure A, 100 mg of tert- butyl 4-aminopiperidine-1-carboxylate (0.47 mmol, 1 equiv) was reacted. 3d was obtained after purification by column chromatography in a yield of 62 mg (0.4 mmol, 78.5%). 1 H NMR (600 MHz, MeOD) δ 6.28–6.20 (m, 2H), 5.70 (dd, J = 6.9, 5.1 Hz, 1H), 3.42–3.38 (m, J = 13.6, 3.9, 0.7 Hz, 1H), 3.32–3.27 (m, J = 10.8, 6.3, 3.1 Hz, 2H), 3.25–3.18 (m, 2H), 2.10–1.95 (m, 2H), 1.95–1.84 (m, 2H), 1.81–1.73 (m, J = 13.0, 10.2, 3.0 Hz, 1H), 1.71–1.62 (m, 1H). 13 C NMR (151 MHz, MeOD) δ 167.70, 131.51, 127.58, 55.82, 50.39, 43.77, 33.54, 26.19, 23.59. LC-MS(ESI-MS): (m/z) calculated for C 9 H 17 N 2 O ([M + H] + ): 169.1; found 169.1. CDI-protected GDP 5 . According to General Procedure B, 5 was obtained after purification.LC-MS ( m/z ): Calculated for C 14 H 14 N 7 O 11 P 2 − : 518.0 [M-H] − , found: 518.0. According to the General Procedure C, the modified amines (1.2 mmol, 2 equivalents) were reacted with 5 in the presence of DIPEA (0.5 ml, 3.0 mmol, 5 equivalents). The following products were obtained (Table 1 ): According to the General Procedure D, molecules 6a – i were reacted. The following products were obtained (Table 2 ): Biological Experiments Protein expression and purification KRAS G 13 C 1−169 Cys-light (C51S C80L C118S) were expressed in E. coli BL21 (DE3) at 37°C. Protein expression was induced at A600 nm of 0.5 by the addition of 0.2–0.3 mM isopropyl-b-D-thiogalactoside (IPTG), and growth was continued at 19°C overnight. The bacteria were collected by centrifugation, and the obtained pellet resuspended in Ni-NTA buffer (50 mM Tris pH 8.0, 250 mM NaCl, 40 mM imidazole, 4 mM MgCl 2 , 10 µM GDP, 1 mM Tris(2-chlorehyl)phosphate (TCEP), and 5% glycerol). The cells were lysed with a microfluidizer, and after addition of protease inhibitor cocktail (Roche complete EDTA free) and 1% CHAPS (w/v) stirring was continued for 1 hr at 4°C. The lysate was cleared by centrifugation (35,000 x g, 1 h, 4°C), and the supernatant was loaded onto a Ni-affinity chromatography column (Qiagen Ni-NTA Superflow, 5 mL) pre-equilibrated with Ni-NTA buffer. The protein was eluted with a linear gradient of imidazole buffer (40 mM – 500 mM). For cleavage of the N-terminal hexahistidine-tag, TEV protease was added to the pooled elution fractions and dialyzed overnight into dialysis buffer at 4°C (25 mM Tris pH 8.0, 100 mM NaCl, 4 mM MgCl 2 , 10 µM GDP, 1 mM TCEP, and 5% glycerol). The cleaved protein was then applied to a reverse Ni-affinity chromatography column. The eluted protein fractions were concentrated to around 10 mL followed by dilution with salt-free AEX buffer (25 mM Tris pH 8.0, 2 mM MgCl 2 , 10 µM GDP, 1 mM TCEP, and 5% glycerol) for better binding conditions onto AEX column (Cytiva, QFF HiTrap, 2x1 mL). The protein was eluted with a linear gradient (0 mM – 1000 mM NaCl). Finally, the protein was purified by size-exclusion chromatography (GE HiLoad 16/60 Superdex 75 pg) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 10 µM GDP, 1 mM TCEP, and 5% glycerol. Covalent Modification of Proteins To assess the extent of covalent modification of KRAS G 13 C 1–169 , 50 µM Ras protein was incubated with a 10-fold molar excess of acryl-bearing nucleotides in a buffer containing 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM TCEP, and 1 mM EDTA. After incubation at room temperature for the appropriate duration, the extent of protein modification was analyzed by ESI-MS. MS spectra were acquired on a VelosPro IonTrap mass spectrometer (Thermo Scientific) using an AdvanceBio Desalting-RP, 2.1x2.5mm column (Agilent Technologies). A gradient elution was employed, transitioning from mobile phase A (0.1% formic acid in water) to mobile phase B (0.1% formic acid in acetonitrile). Effector Binding (Pull-Down Assays) Pull-down experiments were performed using a buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, and 2 mM MgCl₂. For each assay, 10 µg KRAS G 13 C :GDP or covalently modified KRAS G 13 C - 7a and 7b and 20 µg GST-tagged cRaf-RBD (amino acids 51–131) were incubated either in the presence or absence of 100 µM GppNHp and 1 µg SOS overnight at room temperature. Following incubation, 50 µL of glutathione magnetic beads were added to each sample and incubated for 30 minutes. Beads were then washed with 500 µL buffer, separated using a magnetic rack, and the supernatant was carefully removed. The beads were resuspended in 50 µL of 4x SDS-loading buffer, and samples were analyzed by SDS-PAGE. Guanine Nucleotide-Exchange Factor Assay SOS-catalyzed nucleotide exchange was measured at 25°C using a FluoroMax-3 spectrofluorometer. Fluorescence was monitored with an excitation wavelength of 360 nm and an emission wavelength of 440 nm. The assay buffer consisted of 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl₂, and 1 mM TCEP. In the assay, 5 µM KRAS (residues 1–169) was incubated with 10 µM mant-dGDP, followed by the addition of SOS at varying concentrations (0.25 µM and 0.5 µM). Fluorescence changes were recorded to monitor nucleotide exchange. Crystallization The KRAS G 13 C protein was diluted to 2 mg/mL and incubated with a 3-fold excess of the acryl-bearing nucleotide 7b in 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 5% glycerol at room temperature. After incubation for 24 hr, the protein modification was controlled by ESI-MS. The MS spectra were recorded on an VelosPro IonTrap (Thermo Scientific) with an AdvanceBio Desalting-RP, 2.1x2.5mm column (Agilent Technologies) and a gradient of the mobile phase A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile). The protein:inhibitor complex was purified by size-exclusion chromatography (Superdex Increase 75 pg 10/300 GL) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 5 mM TCEP, and 5% glycerol, and subsequently concentrated to 44 mg/mL and 28 mg/mL and used for crystallization. A former identified initial crystallization condition (JCSG Core III, B5) was further optimized and used for crystallization at 4, 12 and 18°C. The protein:inhibitor complex was mixed in a 1:1 ratio (1 µL protein:inhibitor complex and 1 µL reservoir solution containing 100 mM sodium acetate pH 8.0–9.0, 200 mM Tris-HCl pH 8.0–9.0, 25–35% (w/v) PEG4000). The crystals grew within 24 h with the higher protein concentration and within weeks with the lower concentration. The fast-grown crystals were much more intergrown; therefore, the slow-grown crystals were used for the following shrinking step. The crystals were transferred in a new drop containing SEC buffer condition and crystallization conditions mixed in a 1:1 ratio plus an additional 30% PEG3350/PEG4000, 20% glycerol, 20% ethylene glycol, or 20% PEG 400 for 24 h at 18°C. After incubation, the crystals were fished and flash cooled in liquid nitrogen. The data sets were collected at the ID30B beamline of the ESRF (European Synchrotron Radiation Facility, Grenoble, France, DOI: 10.15151/ESRF-ES-1581727707 ) The data were processed using XDS and scaled using XSCALE. Structure Determination and Refinement The complex crystal structure was solved by molecular replacement with PHASER using structure PDB ID: 7ok3 as template. 32 The molecules in the asymmetric units were manually adjusted using the program COOT. 33 The refinement was performed with Phenix.refine 1.21.1. 34 Inhibitor topology files were generated using eLBOW of the Phenix 1.21.1 program package. Refined structures were validated with the PDB validation server. Data collection, structure refinement statistics, PDB-ID codes, and further details for data collection are provided in Table 3 . PyMOL (W.L. DeLano, The PyMOL Molecular Graphics System) was used for generating the figures. Table 3 Data statistics for KRAS G 13 C covalently bound to compound 7b (PDB ID 9I7Y). Data collection Space group C 1 2 1(5) Cell dimensions a, b, c [Å] 70.01, 84.76, 88.77 α, β, γ [°] 90.0, 113.26, 90.0 Resolution [Å] 50.00-1.85 (1.90–1.85) R meas [%] 4.6 (126.9) I / σ I 18.37 (1.37) Completeness [%] 99.8 (99.7) CC 1/2 99.9 (61.8) Redundancy 6.86 (6.43) Refinement Resolution [Å] 42.38–1.85 (1.90–1.85) No. reflections 40651 R work / R free 18.98 / 22.78 (55.79 / 54.51) No. atoms Protein chain A = 1249 chain B = 1247 chain C = 1140 total: 3636 Ligand 3 x 41 Ions (Mg 2+ ) 3 Water 128 B -factors Protein chain A = 56.28 chain B = 62.13 chain C = 61.55 Ligand chain A = 52.72 chain B = 52.18 chain C = 62.00 Ions (Mg 2+ ) 52.85 Water 55.83 R.m.s. deviations Bond lengths [Å] Bond angles [°] 0.011 1.249 Ramachandran [%] Outliers Allowed Favored 0 1.75 98.25 Rotamer [%] Outliers Allowed Favored 0.78 2.85 96.37 Molecular Modeling The computational prediction of drug affinity is a major challenge in medicinal chemistry 35 that becomes even more complex in the case of covalent inhibitors. To mitigate such limitations, we employed an approach focused on the reversible complexes in the present work, hypothesizing that the availability of ligand conformations in close proximity to Cys13 would relate to the inhibitory efficiency of the compounds. In this sense, the crystallographic structure of KRAS G 13 C covalently bound to inhibitor 7a was retrieved from PDB under ID 7ok3, and used as a basis for the following steps of the study. Compound 7a was removed from the crystallographic complex and used to model compounds 7b-I using Maestro (Maestro, Schrödinger, LLC, New York, NY, 2024). The molecules were submitted to an energy minimization step and used as inputs for molecular docking to the target enzyme. After an accurate redocking of 7a, each compound was docked to KRAS G 13 C using DockThor web-server, which uses one of the top-scoring functions currently available, 36 maintaining the ligand’s flexibility. To access the solution ensemble of the reversible states of compounds 7a - i and explore the proximity of the warhead to Cys13, each docked complex was submitted to molecular dynamics (MD) simulations using GROMACS 37 and CHARMM36 force field. 38 Each complex was prepared using the Solution Builder tool available on the CHARMM-GUI web server, 39 inserted in cubic boxes with a minimum distance of 10 Å between the solute and the box edges, solvated with TIP3P water molecules, and neutralized with a 0.15 M NaCl concentration. Simulations were carried out at physiological pH and temperature (298 K) using an integration step of 2 fs. The LINCS algorithm was used to constrain the lengths of hydrogen bonds, while the PME method was used to calculate the long-range electrostatic interactions. The V-rescale thermostat with two coupling groups was used to maintain the temperature of the system, and the Parrinello-Rahman barostat was used to maintain the pressure of the system. The systems were initially equilibrated for 1ns for a canonic ensemble (NVT) with the atomic positions restrained using a 5,000 kJ mol − 1 force on both protein and ligand atoms. After this step, five 1 ns simulations under the isothermal–isobaric ensemble (NPT) were performed, progressively lowering the above-mentioned force in steps of 1,000 kJ.mol − 1 . After this careful equilibration process, production runs were performed for 1 µs, each complex being simulated with three replicas. Abbreviations ATP, adenosinetriphosphate; CADD, computer aided drug design; CDI, carbonyldiimidazole; DIPEA, N,N -diisopropylethylamine; DMF, dimethylformamide; GDP, guanosinediphosphate; GN, guanosine nucleotide; GppNHp, guanosine-5'-[(β,γ)-imido]triphosphate; GST, glutathione-S-transferase; GTP, guanosinetriphosphate; KRAS, Kirsten rat sarcoma; MD, molecular dynamics; MS, mass spectrometry; RBD, ras binding domain; SOS, son of sevenless; SWIIP, switch-II-pocket. Declarations Acknowledgments This work was co-funded by the German Research Foundation (DFG), the State of North Rhine-Westphalia (NRW), the European Union (European Regional Development Fund: Investing In Your Future) (EFRE-800400), DDHD (Drug Discovery Hub Dortmund), the German Federal Ministry of Education and Research (InCa, 01ZX2201B), the Mercator Research Center Ruhr (MERCUR, IGNITE (Ex-2021-0033)), the German Cancer Aid ((Deutsche Krebshilfe), Targeting Transcriptional Addiction in Cancer (TACTIC)), the "Netzwerke 2021" program, an initiative of the Ministry of Culture and Science of the State of North Rhine-Westphalia (CANcer TARgeting, NW21-062C) and, supported by the European Synchrotron Radiation Facility (ESRF, Grenoble, France, proposal MX-2580, beamline ID30B; DOI 10.15151/ESRF-ES-1581727707). We thank Andreas Arndt for his support with protein expression and purification. Supporting Information The Supporting Information is available online free of charge. The authors will release the atomic coordinates upon article publication. Data availability The data supporting the findings of this study are available in the paper and its Supplementary Information. The crystal structure data generated in this study have been deposited in the PDB database under accession code 9I7Y (DOI: https://doi.org/10.2210/pdb9I7Y/pdb). Author Contributions D.R., M.P.M., H.V. are responsible for initiating and supervising the project. T.K. designed and synthesized the compounds and performed biological and biochemical experiments including protein MS studies. J.N. performed co-crystallization experiments, data processing, structure building and structural analysis. J.R. and E.G.M. performed computational experiments including MD simulations. The manuscript was written with contributions from all authors. All authors approved the final version of the manuscript. References Scheffzek, K. et al. The Ras-RasGAP Complex: Structural Basis for GTPase Activation and Its Loss in Oncogenic Ras Mutants. Science 277 , 333-339, doi:10.1126/science.277.5324.333 (1997). Simanshu, D. K., Nissley, D. V. & McCormick, F. RAS Proteins and Their Regulators in Human Disease. Cell 170 , 17-33, doi:https://doi.org/10.1016/j.cell.2017.06.009 (2017). Goody, R. S., Müller, M. P. & Rauh, D. Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules. Cell Chemical Biology 26 , 1338-1348, doi:https://doi.org/10.1016/j.chembiol.2019.07.005 (2019). Karnoub, A. E. & Weinberg, R. A. Ras oncogenes: split personalities. Nature Reviews Molecular Cell Biology 9 , 517-531, doi:10.1038/nrm2438 (2008). Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A. & Shokat, K. M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503 , 548-551, doi:10.1038/nature12796 (2013). Goebel, L., Müller, M. P., Goody, R. S. & Rauh, D. KRasG12C inhibitors in clinical trials: a short historical perspective. RSC Medicinal Chemistry 11 , 760-770, doi:10.1039/D0MD00096E (2020). Canon, J. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575 , 217-223, doi:10.1038/s41586-019-1694-1 (2019). Fell, J. B. et al. Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer. Journal of Medicinal Chemistry 63 , 6679-6693, doi:10.1021/acs.jmedchem.9b02052 (2020). Hallin, J. et al. The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discovery 10 , 54-71, doi:10.1158/2159-8290.CD-19-1167 (2020). Wang, X. et al. Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor. Journal of medicinal chemistry 65 , 3123-3133, doi:10.1021/acs.jmedchem.1c01688 (2022). Yu, Z. et al. Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles. Journal of the American Chemical Society 145 , 20403-20411, doi:10.1021/jacs.3c05899 (2023). Zheng, Q., Zhang, Z., Guiley, K. Z. & Shokat, K. M. Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D. Nature Chemical Biology , doi:10.1038/s41589-024-01565-w (2024). Zhang, Z., Guiley, K. Z. & Shokat, K. M. Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S). Nature Chemical Biology 18 , 1177-1183, doi:10.1038/s41589-022-01065-9 (2022). Zhang, Z., Morstein, J., Ecker, A. K., Guiley, K. Z. & Shokat, K. M. Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile. Journal of the American Chemical Society 144 , 15916-15921, doi:10.1021/jacs.2c05377 (2022). Kirschner, T., Müller, M. P. & Rauh, D. Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy. Journal of Medicinal Chemistry 67 , 6044-6051, doi:10.1021/acs.jmedchem.3c02403 (2024). Forbes, S. A. et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Research 43 , D805-D811, doi:10.1093/nar/gku1075 (2015). Visscher, M., Arkin, M. R. & Dansen, T. B. Covalent targeting of acquired cysteines in cancer. Current Opinion in Chemical Biology 30 , 61-67, doi:https://doi.org/10.1016/j.cbpa.2015.11.004 (2016). Zhang, Z. & Shokat, K. M. Bifunctional Small-Molecule Ligands of K-Ras Induce Its Association with Immunophilin Proteins. Angewandte Chemie International Edition 58 , 16314-16319, doi:https://doi.org/10.1002/anie.201910124 (2019). Schulze, C. J. et al. Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS. Science 381 , 794-799, doi:10.1126/science.adg9652 (2023). Traut, T. W. Physiological concentrations of purines and pyrimidines. Molecular and Cellular Biochemistry 140 , 1-22, doi:10.1007/BF00928361 (1994). Goody, R. S., Frech, M. & Wittinghofer, A. Affinity of guanine nucleotide binding proteins for their ligands: facts and artefacts. Trends in Biochemical Sciences 16 , 327-328, doi:https://doi.org/10.1016/0968-0004(91)90134-H (1991). Thaimattam, R., Banerjee, R., Miglani, R. & Iqbal, J. Protein Kinase Inhibitors: Structural Insights Into Selectivity. Current Pharmaceutical Design 13 , 2751-2765, doi:http://dx.doi.org/10.2174/138161207781757042 (2007). Müller, M. P., Jeganathan, S., Heidrich, A., Campos, J. & Goody, R. S. Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. Scientific Reports 7 , 3687, doi:10.1038/s41598-017-03973-6 (2017). Lim, S. M. et al. Therapeutic Targeting of Oncogenic K-Ras by a Covalent Catalytic Site Inhibitor. Angewandte Chemie International Edition 53 , 199-204, doi:https://doi.org/10.1002/anie.201307387 (2014). Hunter, J. C. et al. In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C. Proceedings of the National Academy of Sciences 111 , 8895-8900, doi:10.1073/pnas.1404639111 (2014). Müller, M. P., Jeganathan, S., Heidrich, A., Campos, J. & Goody, R. S. Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. Sci Rep 7 , 3687, doi:10.1038/s41598-017-03973-6 (2017). Goebel, L. et al. Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors. eLife 12 , e82184, doi:10.7554/eLife.82184 (2023). Lategahn, J., Keul, M. & Rauh, D. Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer. Angewandte Chemie International Edition 57 , 2307-2313, doi:https://doi.org/10.1002/anie.201710398 (2018). Forbes, S. A. et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res 43 , D805-811, doi:10.1093/nar/gku1075 (2015). Wolber, G. & Langer, T. LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. J Chem Inf Model 45 , 160-169, doi:10.1021/ci049885e (2005). Yan, X. et al. Multiscale calculations reveal new insights into the reaction mechanism between KRASG12C and α, β-unsaturated carbonyl of covalent inhibitors. Computational and Structural Biotechnology Journal 23 , 1408-1417, doi:10.1016/j.csbj.2024.03.027 (2024). Read, R. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallographica Section D 57 , 1373-1382, doi:doi:10.1107/S0907444901012471 (2001). Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallographica Section D 60 , 2126-2132, doi:doi:10.1107/S0907444904019158 (2004). Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D 66 , 213-221, doi:doi:10.1107/S0907444909052925 (2010). Verli, H. & Oostenbrink, C. Status and Prospects of Molecular Simulations for Drug Discovery. Journal of the Brazilian Chemical Society 35 , doi:10.21577/0103-5053.20240119 (2024). Guedes, I. A. et al. New machine learning and physics-based scoring functions for drug discovery. Sci Rep-Uk 11 , 3198, doi:10.1038/s41598-021-82410-1 (2021). Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2 , 19-25, doi:https://doi.org/10.1016/j.softx.2015.06.001 (2015). Huang, J. & MacKerell Jr, A. D. CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data. J Comput Chem 34 , 2135-2145, doi:https://doi.org/10.1002/jcc.23354 (2013). Lee, J. et al. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J Chem Theory Comput 12 , 405-413, doi:10.1021/acs.jctc.5b00935 (2016). Table 1 and 2 Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Kirschneretal.SI.pdf Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Mar, 2025 Reviews received at journal 10 Mar, 2025 Reviews received at journal 28 Feb, 2025 Reviews received at journal 27 Feb, 2025 Reviewers agreed at journal 19 Feb, 2025 Reviewers agreed at journal 17 Feb, 2025 Reviewers agreed at journal 17 Feb, 2025 Reviewers invited by journal 17 Feb, 2025 Editor assigned by journal 17 Feb, 2025 Editor invited by journal 17 Feb, 2025 Submission checks completed at journal 14 Feb, 2025 First submitted to journal 14 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6028840","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":415776974,"identity":"dec18f60-cfcf-4b39-becf-39868100da7c","order_by":0,"name":"Tonia Kirschner","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Tonia","middleName":"","lastName":"Kirschner","suffix":""},{"id":415776976,"identity":"bfef74a0-7a4f-40fc-a80a-d7f0b8ec467b","order_by":1,"name":"João Rodriguez","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul (UFRGS)","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"","lastName":"Rodriguez","suffix":""},{"id":415776978,"identity":"13587829-5773-48ba-b197-c9809f481ac8","order_by":2,"name":"Emerson Gonçalves Moreira","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul (UFRGS)","correspondingAuthor":false,"prefix":"","firstName":"Emerson","middleName":"Gonçalves","lastName":"Moreira","suffix":""},{"id":415776979,"identity":"cb2829a6-5c33-40a2-93dc-af6e1a2310ec","order_by":3,"name":"Janina Niggenaber","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Janina","middleName":"","lastName":"Niggenaber","suffix":""},{"id":415776980,"identity":"cc9d25f4-2cba-4f66-8f35-7c5a4dad5ad3","order_by":4,"name":"Hugo Verli","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul (UFRGS)","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"","lastName":"Verli","suffix":""},{"id":415776981,"identity":"8c025a2b-e7dc-4fda-84eb-589296c973a1","order_by":5,"name":"Matthias Philipp Müller","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"Philipp","lastName":"Müller","suffix":""},{"id":415776982,"identity":"ea4c7ddb-3622-4c56-8f48-8215cf2b4eed","order_by":6,"name":"Daniel Rauh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYDCCAyBUAGLxMD4AkowNEHFmsBRuLQZgLcwGB8BamAlrYYBqYZMgSgvf8bMHD3wwYMjnn5F7rPpjG4Nsv/T5o5t5d1gDpRqwapE8k5dwcIYBg+WMG3lpNw62MRjP7Etmu817Jh0ohd0agwM5Bod5DIBOu5FjBtTyP3HDGWaglrbDDAY3ErBrOf/G4PAfoBZ5oJYCoC1IWu4/wK7lBtAWoB0GQIYZA6qWG9i9L3njjcHBHgMJA8Mzb4wlzpwD+qWH2ezm3LZ0Hskz2B3Gdz7H+MOPChsDueM5hh8qyoAhxsP47MbbNms5vuPYvQ8FEphCPPjUj4JRMApGwSjADwADiGY3LIj7AQAAAABJRU5ErkJggg==","orcid":"","institution":"TU Dortmund University","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Rauh","suffix":""}],"badges":[],"createdAt":"2025-02-14 08:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6028840/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6028840/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-22145-5","type":"published","date":"2025-10-31T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76430172,"identity":"cdab4b6f-cdfb-45fa-b8ef-d8c2111d48d6","added_by":"auto","created_at":"2025-02-17 06:30:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of important binding pockets and oncogenic mutations in KRAS.\u003c/strong\u003e Mutational frequencies of mutations occurring in KRAS (retrieved from the COSMIC database; 5\u003csup\u003eth\u003c/sup\u003e February 2025 \u003csup\u003e29\u003c/sup\u003e). The nucleotide-binding pocket (beige) is shown on the left and the SWIIP is shown on the right (grey), including the position of the oncogenic mutations at positions G12, G13 and Q61 (grey, beige and green spheres, respectively), including exemplified molecules binding to the pockets as mentioned above.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/197b2759be37559b0e8a6a01.png"},{"id":76430173,"identity":"f2ec1368-75e0-40c0-9945-f8b62513a6cc","added_by":"auto","created_at":"2025-02-17 06:30:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of novel nucleotide-based inhibitors with cyclic linkers\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e: Co-crystal structure of KRAS\u003csup\u003eG13C\u003c/sup\u003e covalently modified by eda-GDP (PDB 7OK3) indicating a bend conformation of the linker regions. \u003cstrong\u003eB\u003c/strong\u003e: Modelled pose of a GDP-based inhibitor featuring a cyclic linker in KRAS\u003csup\u003eG13C\u003c/sup\u003e (modelling carried out in LigandScout\u003csup\u003e30\u003c/sup\u003e, based on PDB ID 7OK3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/d87815bc5443558572d95b0f.png"},{"id":76430123,"identity":"56800229-e481-437b-aeac-2929e6420f22","added_by":"auto","created_at":"2025-02-17 06:30:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConvergent synthesis scheme of novel nucleotide-based inhibitors\u003c/strong\u003e.\u003csup\u003ea\u003c/sup\u003e \u003cstrong\u003eA\u003c/strong\u003e:\u003csup\u003ea\u003c/sup\u003e Reagents and conditions: (\u003cem\u003ei\u003c/em\u003e) Acryloyl chloride, DIPEA, THF, rt, 12 h; (\u003cem\u003eii\u003c/em\u003e) DCM/TFA (3:1), rt. (\u003cem\u003eiii\u003c/em\u003e) CDI, DMF, 4 °C, 12 h; (\u003cem\u003eiv\u003c/em\u003e) DIPEA, DMF, rt, 12\u0026nbsp;h; (\u003cem\u003ev\u003c/em\u003e) aq. HCl (pH 1.5) \u003cstrong\u003e1-7\u003c/strong\u003e. \u003cstrong\u003eB\u003c/strong\u003e: Acrylamide-bearing GDP-based inhibitors \u003cstrong\u003e7a-7i\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/ce6b94e2cbefbcf51a3198f0.png"},{"id":76430149,"identity":"25cfd8a2-00b2-4c6d-b4d8-6548eff3a68e","added_by":"auto","created_at":"2025-02-17 06:30:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel nucleotide-based inhibitors modify KRAS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG13C\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e covalently\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e: pH-dependent covalent modification of KRAS\u003csup\u003eG13C\u003c/sup\u003e\u003csub\u003e1-169\u003c/sub\u003e (Cys-light) proteins at pH 7.5 - 9.5 after 24 h at rt. \u003cstrong\u003eB\u003c/strong\u003e: Time-dependent analysis of the covalent modification of KRAS\u003csup\u003eG13C\u003c/sup\u003e\u003csub\u003e1-169\u003c/sub\u003e (Cys-light) at pH 9.5 and rt.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/84f7833ee3b5701d2922b12b.png"},{"id":76430120,"identity":"8e3d7b1d-a32f-4394-af38-e304da487cad","added_by":"auto","created_at":"2025-02-17 06:30:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":209177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe nucleotide-analogues lock RAS in an inactive state\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e: GEF-catalyzed nucleotide exchange of KRAS\u003csup\u003eWT\u003c/sup\u003e:GDP, KRAS\u003csup\u003eG13C\u003c/sup\u003e:GDP and KRAS\u003csup\u003eG13C\u003c/sup\u003e-\u003cstrong\u003e7a\u003c/strong\u003e and \u003cstrong\u003e7b\u003c/strong\u003e. \u003cstrong\u003eB\u003c/strong\u003e: Pull-down-Assay. Whereas KRAS\u003csup\u003eG13C\u003c/sup\u003e:GDP can be pulled down by GST-tagged Raf RBD in the presence of GppNHp and GppNHp/SOS, KRAS\u003csup\u003eG13C\u003c/sup\u003e-\u003cstrong\u003e7a/7b\u003c/strong\u003e:GDP cannot become activated and consequently cannot bind to the Raf RBD, neither in the presence of GppNHp only nor GppNHp and SOS. The unprocessed SDS gel is shown in Figure S2.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/ceb1420f770a8c528336359e.png"},{"id":76430130,"identity":"92225905-8695-4471-a7a7-d2320350a9fa","added_by":"auto","created_at":"2025-02-17 06:30:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-Ray crystallographic model of KRAS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG13C\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e covalently modified with 7b\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e: Compound \u003cstrong\u003e7b\u003c/strong\u003e covalently bound to Cys13 of KRAS\u003csup\u003eG13C\u003c/sup\u003e. \u003cstrong\u003eB\u003c/strong\u003e: 3 copies of covalently modified RAS were observed in the asymmetric unit. The figure indicates that the GDP-core adopts a similar orientation in all of these, whereas the linker is flexible and adopts different conformations \u003cstrong\u003eC\u003c/strong\u003e: The 2Fo-Fc electron density map is contoured at an r.m.s.d. of 1.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/33c585a0d2a85357ca5ed9fc.png"},{"id":76430119,"identity":"30650b93-cb17-4c25-a0a5-de46874df5d8","added_by":"auto","created_at":"2025-02-17 06:30:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":227236,"visible":true,"origin":"","legend":"\u003cp\u003eCompound’s solution ensemble when in a reversible complex with KRAS\u003csup\u003eG13C\u003c/sup\u003e, as observed in MD simulations. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB:\u003c/strong\u003e fluctuation of the distance between Cys13 sulfur atom and the oxygen atom from the α-β-unsaturated carbonyl group is presented for each compound, while the respective distributions of these interatomic distances are indicated in the upper right graph for compounds \u003cstrong\u003e7a\u003c/strong\u003e, \u003cstrong\u003e7b\u003c/strong\u003e and \u003cstrong\u003e7d\u003c/strong\u003e, representing compound with higher activity, and in the lower right graph for compounds \u003cstrong\u003e7f\u003c/strong\u003e and \u003cstrong\u003e7i\u003c/strong\u003e, representing compounds with lower activity (see population of conformers below 0.5 nm). The figures show that the less reactive compounds \u003cstrong\u003e7f\u003c/strong\u003e and \u003cstrong\u003e7i\u003c/strong\u003e adopt conformations of the warhead close to Cys13 far less often than compounds \u003cstrong\u003e7a\u003c/strong\u003e, \u003cstrong\u003e7b,\u003c/strong\u003e and \u003cstrong\u003e7c\u003c/strong\u003e. \u003cstrong\u003eC\u003c/strong\u003e, KRAS\u003csup\u003eG13C\u003c/sup\u003e in complex with ligands \u003cstrong\u003e7d\u003c/strong\u003e presenting the warhead in a conformation within 4Å to the sulfur atom from Cys13.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/aabd0671be1edb4bd36cd8c9.png"},{"id":95039959,"identity":"a4881b60-e6f2-4700-af2f-16f11c9d9a40","added_by":"auto","created_at":"2025-11-03 16:06:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2471236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/54f34827-4fe7-486a-816a-22fce60e67c1.pdf"},{"id":76430117,"identity":"ee4434ec-8772-4edb-a846-91792ba105f6","added_by":"auto","created_at":"2025-02-17 06:30:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":106245,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/6da5aa288d2df9b79f1ea054.docx"},{"id":76430140,"identity":"29185df1-2ddc-4a79-9b93-c094836c2f0b","added_by":"auto","created_at":"2025-02-17 06:30:55","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2643123,"visible":true,"origin":"","legend":"","description":"","filename":"Kirschneretal.SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6028840/v1/538fc4d45b09fca582761e55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting KRASG13C: Exploring Warhead Orientation with Cyclic Linker-Based Inhibitors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKirsten rat sarcoma (KRAS) is one of the most prominent oncogenes in human tumors, found in approximately 30% of cases.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Oncogenic mutations in KRAS cause a shift of equilibrium from the inactive guanosine diphosphate (GDP)-bound state to the active guanosine triphosphate (GTP)-bound state, thereby amplifying proliferative signaling pathways and promoting uncontrolled cell growth.\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Historically considered \"undruggable,\" targeting KRAS experienced a breakthrough led by Kevan Shokat's research group.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Their discovery of an allosteric binding site, known as the switch II pocket (SWIIP; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), enabled the development of small-molecule inhibitors that modulate this small GTPase.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e This breakthrough led to the development of covalently binding molecules such as sotorasib (AMG510, LUMYKRAS\u0026reg;, Amgen)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and adagrasib (MRTX849, KRAZATI\u0026reg;, Bristol Myers Squibb)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, which received FDA approval in 2021 and 2022, respectively. This revolutionized the treatment of KRAS\u003csup\u003eG\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003eC\u003c/sup\u003e-mutated lung cancer, a condition previously lacking effective targeted therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInitially, acrylamide-based inhibitors targeting the acquired cysteine residue specifically addressed G12C mutations. However, recent years have seen the emergence of novel strategies utilizing SWIIP binders incorporating mutant-specific moieties for G12D,\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e G12S,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and G12R mutations.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e These advancements promise innovative approaches to address cancer types harboring mutations beyond G12C.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Another significant mutation hotspot within the KRAS gene is codon 13, where mutations occur in approximately 14% of tumors. Within this subset, 6% feature a glycine-to-cysteine substitution (KRAS\u003csup\u003eG\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003eC\u003c/sup\u003e), presenting a potential anchor point for the design of covalent inhibitors.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Currently, the first KRAS\u003csup\u003eG\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003eC\u003c/sup\u003e inhibitor, RMC-8839, is in preclinical development utilizing Tri-complex (\"molecular glue-like\" compounds) inhibitors, initially reported by Zhang \u003cem\u003eet al\u003c/em\u003e. and subsequently advanced by Revolution Medicines.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e In addition to the possibility of allosteric inhibition, there is also the option of occupying the guanine nucleotide-binding pocket with an inhibitor that locks the GTPase in an inactive state. Small GTPases, such as KRAS, have an extremely high affinity for GDP and GTP.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Unlike kinases, which have a more moderate affinity for adenosine triphosphate (ATP) and where ATP-competitive inhibitors have been successfully developed,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e orthosteric inhibition of KRAS initially appeared challenging.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e However, Lim \u003cem\u003eet al\u003c/em\u003e. and Hunter \u003cem\u003eet al\u003c/em\u003e. first demonstrated a nucleotide-based KRAS\u003csup\u003eG\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003eC\u003c/sup\u003e inhibitor (SML 8-73-1) that competes for the nucleotide-binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and irreversibly reacts with the acquired Cys12, thereby competing with the natural substrates. These modulators, however, exhibited significantly reduced protein affinity due to modifications of the \u003cem\u003eβ\u003c/em\u003e-phosphate group.\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur previous work introduced nucleotide-based KRAS\u003csup\u003eG\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003eC\u003c/sup\u003e inhibitors featuring a linker and acrylamide moiety, specifically targeting this mutation.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Compared to the compound set by Lim \u003cem\u003eet al\u003c/em\u003e.,\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e our modifications at the 2'- or 3'-position of the ribose with the linker and the warhead preserved the high reversible affinity of the nucleotide. In this study, we utilized the acrylamide warhead, renowned for its biocompatibility due to its low non-specific reactivity towards thiol groups, which necessitates an optimal orientation of the acrylamide warhead towards the target cysteines after the initial association step and in the reversibly bound state.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, our previously developed molecules displayed comparatively low reactivity towards the targeted Cys13 in KRAS, potentially due to an unfavorable preorientation of the aliphatic linear linker. In this study, we report the synthesis, reactivity, and structural profiling of novel compounds featuring cyclic aliphatic linkers. Additionally, we employed \u003cem\u003ein silico\u003c/em\u003e methods to predict the conformational space of the linker and warhead in the reversibly bound state for nucleotide-based compounds to explore the molecular basis for the compounds\u0026rsquo; reactivities and to guide future linker design.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eChemistry and Biological Experiments\u003c/h2\u003e\n \u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003eDesign and Synthesis of Acryl-bearing GDP-based Inhibitors\u003c/h2\u003e\n \u003cp\u003eWe started with a detailed structural analysis based on the previously published co-crystal structures of KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e in complex with eda-GDP and bda-GDP (PDB IDs 7OK3 and 7OK4, respectively). Closer examination revealed that the linker regions of both molecules adopt a distinct bent conformation to facilitate covalent interaction with Cys13 (Fig. \u003cspan\u003e2\u003c/span\u003e). This flexibility is attributed to the aliphatic linear nature and, thus, increased adaptability of the linkers. Based on these findings, we aimed to rigidify the linker by replacing the linear design with cyclic linkers. This modification is expected to promote improved pre-orientation of the warhead, thereby increasing covalent binding efficiency.\u003c/p\u003e\n \u003cp\u003eWe synthesized a focused library of eight compounds featuring cyclic linkers (Fig. \u003cspan\u003e3\u003c/span\u003e). To achieve this, we introduced a new synthetic strategy compared to the previously established protocol by Goebel \u003cem\u003eet al\u003c/em\u003e.,\u003csup\u003e\u003cspan\u003e27\u003c/span\u003e\u003c/sup\u003e where the GDP nucleotide was protected with carbonyldiimidazole (CDI). Concurrently, the linker, a cyclic diamine, was modified with acryloyl chloride, and these two units were subsequently assembled, and the phosphate-moiety further deprotected to form the desired nucleotide-based inhibitors as 2\u0026rsquo; and 3\u0026rsquo;-isomers (Fig. \u003cspan\u003e3\u003c/span\u003eA, the synthesis and analysis of intermediates are described in detail in the methods section and Tables \u003cspan\u003e1\u003c/span\u003e and \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003e\u003cu\u003eSelective Covalent Modification of KRAS\u003csup\u003eG13C\u003c/sup\u003e\u003c/u\u003e\u003c/h3\u003e\n\u003cp\u003eIn mass spectrometry (MS) studies with KRAS\u003csup\u003eG13C\u003c/sup\u003e\u003csub\u003e1\u0026minus;169\u003c/sub\u003e (Cys-light), we observed covalent modification of the desired cysteine 13 for all compounds and that the reactivity increased with rising pH levels, which can be attributed to the enhanced nucleophilicity of the cysteine. The starting molecule, acrylic-eda-GDP \u003cstrong\u003e7a\u003c/strong\u003e, exhibited a reactivity profile similar to molecule \u003cstrong\u003e7b\u003c/strong\u003e. Furthermore, linkers modified at the 1,3-positions on 6-membered rings (\u003cstrong\u003e7b\u003c/strong\u003e and \u003cstrong\u003e7c\u003c/strong\u003e) showed the highest reactivity, followed by those on 5- and 7-membered rings. This observation suggests that the positioning of the warhead in the 1,3 configuration promotes a preferred orientation, thereby facilitating the covalent reaction. Conversely, 1,4-modified 6-membered rings were the least effective, likely due to their high rigidity and linear nature. Additionally, the \u003cem\u003eR\u003c/em\u003e configuration at the warhead-carrying position was slightly favored (Fig. \u003cspan\u003e4\u003c/span\u003eA). In addition to KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e, we also investigated the reactivities towards KRAS\u003csup\u003eG12C\u003c/sup\u003e\u003csub\u003e1\u0026minus;169\u003c/sub\u003e (Cys-light) and KRAS\u003csup\u003ewt\u003c/sup\u003e\u003csub\u003e1\u0026minus;169\u003c/sub\u003e, where no to very low modification was observed, indicating good overall selectivity towards Cys13 (Figure \u003cspan\u003eS1\u003c/span\u003e). Time-resolved studies of compounds \u003cstrong\u003e7b\u003c/strong\u003e, \u003cstrong\u003e7c\u003c/strong\u003e, and \u003cstrong\u003e7d\u003c/strong\u003e demonstrated modification rates comparable to the parental molecule \u003cstrong\u003e7a\u003c/strong\u003e (Fig. \u003cspan\u003e4\u003c/span\u003eB), with compound \u003cstrong\u003e7b\u003c/strong\u003e exhibiting slightly superior performance compared to \u003cstrong\u003e7c\u003c/strong\u003e and \u003cstrong\u003e7d\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003eInhibitory Effects of Novel Covalent Nucleotide-based Inhibitors\u003c/h3\u003e\n\u003cp\u003eIt was also interesting to assess the synthesized compounds\u0026apos; inhibitory activity. For this purpose, we utilized a son of sevenless (SOS)-catalyzed nucleotide exchange assay. Consistent with previous reports, we observed an increased intrinsic exchange rate in the KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e mutant compared to wild-type KRAS, presumably contributing to its oncogenic nature.\u003csup\u003e\u003cspan\u003e27\u003c/span\u003e\u003c/sup\u003e Upon modification of the protein with the covalent nucleotide analogs, no nucleotide exchange was observed for compounds \u003cstrong\u003e7a\u003c/strong\u003e and \u003cstrong\u003e7b\u003c/strong\u003e, including the linear and cyclic linkers, respectively, that showed the highest reactivities in the previous experiments. This indicates that the protein is effectively blocked in its inactive state and can no longer be activated (Fig. \u003cspan\u003e5\u003c/span\u003eA). In a second assay, we investigated whether SOS-catalyzed nucleotide exchange to a non-hydrolyzable GTP analog (guanosine-5\u0026apos;-[(\u003cem\u003e\u0026beta;,\u0026gamma;\u003c/em\u003e)-imido]triphosphate, GppNHp) and effector binding were still possible after covalent modification with the ligand. A pull-down experiment was conducted using a glutathione S-transferase (GST)-tagged Raf RAS-binding domain (Raf RBD) to assess this. Adding GppNHp (with and without SOS) enabled the pull-down of unmodified KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e, demonstrating the activation of the GTPase and subsequent effector binding. In contrast, KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e covalently modified with molecules \u003cstrong\u003e7a\u003c/strong\u003e and \u003cstrong\u003e7b\u003c/strong\u003e cannot become activated in the presence of GppNHp and SOS and consequently did not interact with the Raf RBD (Fig. \u003cspan\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"Underline\" name=\"Emphasis\"\u003eCo-Crystal Structure of\u003c/span\u003e \u003cspan type=\"BoldUnderline\" name=\"Emphasis\"\u003e7b\u003c/span\u003e \u003cspan type=\"Underline\" name=\"Emphasis\"\u003ein Complex with KRAS\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Underline\" name=\"Emphasis\"\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/span\u003e\u003c/sup\u003e \u003cspan type=\"Underline\" name=\"Emphasis\"\u003eMutant\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTo further characterize the novel nucleotides with the cyclic linkers, KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e was co-crystallized with compound \u003cstrong\u003e7b\u003c/strong\u003e to obtain detailed structural insights into ligand binding within the binding pocket (resolution:1.85 \u0026Aring;, R\u003csub\u003ework\u003c/sub\u003e: 18.98, R\u003csub\u003efree\u003c/sub\u003e: 22.78; Fig. \u003cspan\u003e6\u003c/span\u003eA; data statistics are shown in Table \u003cspan\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOverall, the obtained structure is highly similar to previously published GDP-bound (inactive) wild-type structures (for example PDB ID 4OBE)\u003csup\u003e\u003cspan\u003e25\u003c/span\u003e\u003c/sup\u003e, highlighting that our design approach preserved the binding geometry of the nucleotide scaffold, which remains similarly oriented and engages in the same reversible interactions. Additionally, the covalent bond to Cys13 is resolved in the electron density. The molecule is generally similarly oriented as the compound edaGDP \u003cstrong\u003e7a\u003c/strong\u003e (PDB 7OK3). Interestingly, three molecules of RAS were found in the asymmetric unit, with the orientation of the linker near the covalently modified Cys13 modeled differently in these 3 instances (Fig. \u003cspan\u003e6\u003c/span\u003eB), and the electron density less well-defined than in the remaining molecule (Figure S3). This suggests flexibility of the linker, which is consistent with the results of MS studies, indicating that the pre-organization of the linker still needs to be optimized by further rigidification to contribute to an efficient covalent reaction.\u003c/p\u003e\n\u003ch3\u003eComputational Experiments\u003c/h3\u003e\n\u003cp\u003eTo gain a better understanding of the chemical nature of the linkers and their associated reactivities, computational predictions of the conformational space of the linkers and the warhead within the nucleotide-based inhibitors and KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e were performed. In general, the success associated with the use of CADD in the design of new KRAS inhibitors can be limited by several factors, such as the accuracy of the employed scoring function used for virtual screening, the difficulty in modeling the formation of covalent bonds during docking, and the identification of bioactive conformations from conformational ensembles of flexible ligand-receptor complexes.\u003csup\u003e[19]\u003c/sup\u003e To circumvent these limitations, we developed a strategy consisting of: i) using DockTScore within the DockThor docking engine to generate reliable reversible complexes between acrylamide-containing GDP-based inhibitors \u003cstrong\u003e7a\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e and KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e, and ii) refining of these complexes using multi-microsecond MD simulations to explore the proximity of the warhead to Cys13 under native conditions.\u003c/p\u003e\n\u003cp\u003eThe behavior of GDP and Mg\u003csup\u003e2+\u003c/sup\u003e during simulations with KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e was observed to be highly stable (Figure S4), consistent with the high reversible affinities of nucleotides and Ras described previously in the literature. \u003csup\u003e[15, 20]\u003c/sup\u003e Presumably due to the water solvent dynamics and flexibility of switch-I (residues His27-Asp38), we observed a slight shift of Mg\u003csup\u003e2+\u003c/sup\u003e during the simulations, slightly towards the \u0026alpha;-phosphate O3 (Figure S6, Table S2). In contrast, the linker region between GDP and the warhead shows greater structural dynamics (Fig. \u003cspan\u003e7\u003c/span\u003e). The reaction of \u0026alpha;,\u0026beta;-unsaturated carbonyl covalent inhibitors with KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e under physiological conditions likely occurs by a concerted mechanism involving proton transfer, nucleophilic addition, and solvent-assisted tautomerization.\u003csup\u003e\u003cspan\u003e31\u003c/span\u003e\u003c/sup\u003e To understand the differences in reactivity among the studied molecules, we evaluated the interatomic distance between the sulfur atom of Cys13 in KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e and the oxygen atom of the \u0026alpha;,\u0026beta;-unsaturated carbonyl group of each molecule, which characterizes the initial proton transfer step. The synthesized compounds, once bound to KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e, adopt conformations with their warheads mostly far away from Cys13 (distances\u0026thinsp;\u0026gt;\u0026thinsp;0.5 nm, Fig. \u003cspan\u003e7\u003c/span\u003e), while the nucleotide (GDP) remains firmly inserted in the nucleotide-binding pocket (Figure S5). Notably, compounds \u003cstrong\u003e7a\u003c/strong\u003e, \u003cstrong\u003e7b\u003c/strong\u003e, and \u003cstrong\u003e7d\u003c/strong\u003e (Fig. \u003cspan\u003e7\u003c/span\u003e, A) adopted warhead conformations near the sulfur atom of Cys13 more frequently than compound \u003cstrong\u003e7f\u003c/strong\u003e and \u003cstrong\u003e7i\u003c/strong\u003e (Fig. \u003cspan\u003e7\u003c/span\u003e, B), suggesting that the higher observed reactivity results from this improved pre-orientation. However, the total population of these states with conformations close to Cys13 (distances below 0.5 nm) is in the range of 5.4% (\u003cstrong\u003e7a\u003c/strong\u003e) 6.2% (\u003cstrong\u003e7b\u003c/strong\u003e) and 14.1% (\u003cstrong\u003e7d\u003c/strong\u003e) to 0.5% (\u003cstrong\u003e7i\u003c/strong\u003e) and 0.2% (\u003cstrong\u003e7f\u003c/strong\u003e) (Table \u003cspan\u003eS1\u003c/span\u003e), indicating that further compound optimization is still necessary and possible to further increase the reactivity. These results are thus well in line with the high reversible affinity of the modified nucleotides while at the same time being slow to react with the thiol group to form the covalent bond.\u003c/p\u003e"},{"header":"Conclusion and Outlook","content":"\u003cp\u003eIn this study, we synthesized a focused library of covalently binding nucleotide analogs featuring cyclic diamine linkers to systematically examine how linker rigidity and geometry impact reactivity with the Cys13 residue in mutant KRAS. The compounds were shown to disrupt KRAS activity by inhibiting GEF-catalyzed nucleotide exchange and blocking effector binding, effectively stabilizing the protein in its inactive state. While these new analogs did not exhibit enhanced reactivity compared to the parent molecules, structural characterization using X-ray crystallography and \u003cem\u003ein silico\u003c/em\u003e modeling provided novel insights and revealed considerable conformational flexibility that explains the low reactivity at physiological pH. Our \u003cem\u003ein silico\u003c/em\u003e modeling, developed to predict the conformational landscape of these molecules, indicates that only a minor population of the analogs adopt conformations of the warhead close to Cys13 to achieve covalent interaction. Thus, this predictive approach allows us to explain the observed low reactivity at physiological pH and offers a valuable tool for guiding the future design of nucleotide-based inhibitors with optimized preorientation and reactivity profiles, thus providing a systematic framework for enhancing these compounds’ reactivities in a targeted manner.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eChemistry\u003c/h2\u003e\n \u003cp\u003eAll reagents and solvents were purchased from Acros, Activate Scientific, Alfa Aesar, Apollo Scientific, Merck, Sigma-Aldrich, TCI Chemicals or VWR and used without further purification. Dry solvents were purchased as anhydrous reagents from commercial suppliers. \u003csup\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra were recorded on a Bruker Avance DRX AV400 (400 and 101 MHz), AV500 (500 and 125 MHz), AV600 (600 and 151 MHz). 1H chemical shifts are reported in \u003cem\u003e\u0026delta;\u003c/em\u003e (ppm) as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and br (broad singlet) and are referenced to the residual solvent signal: CDCl\u003csub\u003e3\u003c/sub\u003e (7.26), DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (2.50), or MeOD-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e (3.34). \u003csup\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/sup\u003eC spectra are referenced to the residual solvent signal: CDCl\u003csub\u003e3\u003c/sub\u003e (77.1), DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e (39.52), or MeOD-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e (49.86). High-resolution electrospray ionization mass spectra (ESI-FTMS) were recorded on a Thermo LTQ Orbitrap (high-resolution mass spectrometer from Thermo Electron) coupled to an Accela HPLC system supplied with a Hypersil GOLD column (Thermo Electron). LCMS (ESI-MS) analysis was performed using an Agilent HPLC system (1100 series) with a CC 125/4 Nucleodur C18 gravity column (3 \u0026micro;m) from Macherey Nagel coupled to a Thermo Scientific Finnigan LCQ Advantage Max Ion Trap and ESA Corona detector. Compounds were purified by flash chromatography on a Biotage Isolera One using B\u0026uuml;chi Reveleris Silica Cartridges (4\u0026ndash;120 g) monitored by UV at \u0026lambda;\u0026thinsp;=\u0026thinsp;210 and 280 nm. Unless otherwise noted, all final products were synthesized and used as racemic mixtures of the corresponding \u003cem\u003etrans\u003c/em\u003e-isomers.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eGeneral Procedure A for Synthesis of Modified Amines\u003c/em\u003e \u003cstrong\u003e3a\u003c/strong\u003e \u003cem\u003eto\u003c/em\u003e \u003cstrong\u003e3i\u003c/strong\u003e \u0026ndash; The Boc-protected amine (1 equiv) is dissolved in THF and treated with the base DIPEA (2 equiv). The mixture is cooled to 0\u0026deg;C in an ice bath. Subsequently, the THF-diluted acryloyl chloride (1 equiv) is added dropwise with caution, and the solution is stirred for 4 hours. Upon completion of the reaction, the reaction mixture is extracted with DCM/basic H\u003csub\u003e2\u003c/sub\u003eO (3 x 50 ml), and the solvent is removed under vacuum. A solution of the Boc-protected amine is prepared in DCM (3 parts) and cooled to 0\u0026deg;C. Then, TFA (1 part) is added dropwise, stirring the mixture on ice for 30 minutes. Subsequently, the mixture is neutralized with 10 M NaCl, and the remaining solvent is removed. The desired product is obtained through column chromatography on silica gel (DCM/MeOH\u0026thinsp;+\u0026thinsp;1% NH\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eGeneral Procedure B for Protection of GDP\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e \u0026ndash; Using a strongly acidic cation exchanger (Ion Exchanger I from Merck), the nucleotide was initially converted into a DMF-soluble form. Therefore, the pre-swollen column material was loaded into a column with a diameter of 2 cm and a packing height of 15 to 20 cm and then incubated for one hour in a pyridine/water mixture (1:1). After washing the column material with a total of 250 mL of distilled water, a solution of 0.5 mmol of the nucleotide in a total volume of 3 mL was applied to the column using a Pasteur pipette. The nucleotide was eluted with a mixture of methanol/water (1:1) into a round-bottom flask preloaded with 1 mL of tetrabutylammonium hydroxide (TBA). After removing the solvent mixture using a rotary evaporator, the residue was treated three times with 20 mL of DMF each and then concentrated again. The residue was dissolved in a total of 20 mL of DMF, and after the addition of 2.5 mmol of CDI under an argon atmosphere, the reaction mixture was stirred overnight at 0\u0026deg;C. Upon completion of the reaction, it was quenched by adding 150 \u0026micro;L of methanol. The compounds formed in this reaction were used directly in the subsequent reaction without further purification.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eGeneral Procedure C for Coupling the modified Amine to GDP\u003c/em\u003e \u003cstrong\u003e6a\u003c/strong\u003e \u003cem\u003eto\u003c/em\u003e \u003cstrong\u003e6i\u003c/strong\u003e \u0026ndash; In 5 mL of dry DMF, 2.5 mmol of the amine was dissolved and slowly added to the reaction solution. The resulting precipitate was centrifuged at 10,000 rpm for 10 minutes and then washed twice with 30 mL of dry DMF each, followed by centrifugation again. Finally, the pellet was dissolved in 30 mL of distilled water. The products formed during the reaction were used directly in the subsequent reaction without further purification.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eGeneral Procedure D for Deprotection of GDP\u003c/em\u003e \u003cstrong\u003e7a\u003c/strong\u003e \u003cem\u003eto\u003c/em\u003e \u003cstrong\u003e7i\u003c/strong\u003e \u0026ndash; The pH of the reaction solution was adjusted to 1.5 using a 0.25 molar HCl solution and stirred overnight at 4\u0026deg;C. Following the completion of the reaction, the pH was adjusted to 7.5 by adding 0.25 molar NaOH solution, and the nucleotide solution was diluted to a total volume of 100 mL with ddH2O. The sample was filtered using a syringe filter and subjected to purification via a Q-Sepharose column on the FPLC system. After loading the nucleotide solution onto the column equilibrated with Buffer A (50 mM TEAB), elution was performed at a flow rate of 1 mL/min using a linear gradient of 0-100% Buffer B (1 M TEAB) over 600 minutes. The collected fractions were analyzed by HPLC using TBAB as the counterion in the running buffer (50 mM KPi pH 6.6, 10 mM TBAB, 16% ACN; column: ProntoSIL\u0026reg; 120-5-C18-AQ, Bischoff), and fractions containing the product were combined. Following the removal of the solvent mixture using a rotary evaporator, the residue was dissolved in 10 mL ddH\u003csub\u003e2\u003c/sub\u003eO and lyophilized.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-(2-aminoethyl)acrylamide \u003cstrong\u003e3a\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert\u003c/em\u003e-butyl (2-aminoethyl)carbamate (0.62 mmol, 1 equiv) was reacted. \u003cstrong\u003e3a\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 68.51 mg (0.59 mmol, 96%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (600 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.27\u0026ndash;6.26 (m, J\u0026thinsp;=\u0026thinsp;1.6 Hz, 1H), 6.26\u0026ndash;6.25 (m, 1H), 5.73\u0026ndash;5.70 (m, 1H), 3.53 (t, J\u0026thinsp;=\u0026thinsp;9.8, 3.7 Hz, 2H), 3.09 (t, J\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (151 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 131.57, 127.49, 40.81, 38.26, 37.90. LC-MS(ESI-MS): (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 115.08; found 114.8.\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-\u003cem\u003eN\u003c/em\u003e-(pyrrolidin-3-yl)acrylamide \u003cstrong\u003e3f\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert-\u003c/em\u003ebutyl (\u003cem\u003eR\u003c/em\u003e)-3-aminopyrrolidine-1-carboxylate (0.53 mmol, 1 equiv) was reacted. \u003cstrong\u003e3f\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 36 mg (0.26 mmol, 48.5%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (600 MHz, DMSO) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.51\u0026ndash;6.30 (m, J\u0026thinsp;=\u0026thinsp;60.2, 41.5 Hz, 1H), 5.31\u0026ndash;5.26 (m, J\u0026thinsp;=\u0026thinsp;17.1, 10.1 Hz, 1H), 5.21\u0026ndash;5.14 (m, J\u0026thinsp;=\u0026thinsp;17.1, 2.1 Hz, 1H), 4.71\u0026ndash;4.66 (m, J\u0026thinsp;=\u0026thinsp;10.1, 5.6, 1.9 Hz, 1H), 3.46\u0026ndash;3.30 (m, 2H), 1.74\u0026ndash;1.68 (m, 2H), 1.22\u0026ndash;1.10 (m, J\u0026thinsp;=\u0026thinsp;21.7, 14.5, 7.4 Hz, 2H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (151 MHz, DMSO) \u003cem\u003e\u0026delta;\u003c/em\u003e 164.79, 131.29, 125.90, 49.07, 48.35, 43.44, 18.67. LC-MS(ESI-MS): (m/z) calculated for C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 141.09; found 141.1.\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-\u003cem\u003eN\u003c/em\u003e-(pyrrolidin-3-yl)acrylamide \u003cstrong\u003e3g\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert-\u003c/em\u003ebutyl (\u003cem\u003eS\u003c/em\u003e)-3-aminopyrrolidine-1-carboxylate (0.53 mmol, 1 equiv) was reacted. \u003cstrong\u003e3g\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 48 mg (0.34 mmol, 64.6%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (600 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.27\u0026ndash;6.25 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1 Hz, 1H), 6.25\u0026ndash;6.24 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.5 Hz, 1H), 5.75\u0026ndash;5.67 (m, 1H), 4.50\u0026ndash;4.46 (m, 1H), 3.57\u0026ndash;3.52 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.3, 6.9 Hz, 1H), 3.51\u0026ndash;3.45 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.8, 7.6 Hz, 1H), 3.41\u0026ndash;3.36 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.9, 7.7, 6.1 Hz, 1H), 3.34\u0026ndash;3.28 (m, 1H), 2.39\u0026ndash;2.28 (m, 1H), 2.11\u0026ndash;2.01 (m, 1H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (151 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 168.33, 131.40, 127.59, 51.12, 50.32, 45.60, 30.86. LC-MS(ESI-MS): (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 141.1; found 141.4.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-(2-aminocyclohexyl)acrylamide \u003cstrong\u003e3e\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert\u003c/em\u003e-butyl (2-aminocyclohexyl)carbamate (0.46 mmol, 1 equiv) was reacted. \u003cstrong\u003e3e\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 32.8 mg (0.19 mmol, 42.4%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (400 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.53\u0026ndash;6.42 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 10.2 Hz, 1H), 6.35\u0026ndash;6.23 (m, 1H), 5.77\u0026ndash;5.67 (m, 1H), 3.33\u0026ndash;3.31 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.2, 1.6 Hz, 2H), 1.81\u0026ndash;1.72 (m, 2H), 1.63\u0026ndash;1.52 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20.2, 8.6 Hz, 2H), 1.47\u0026ndash;1.40 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.7 Hz, 2H), 1.37\u0026ndash;1.32 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (101 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 165.45, 137.84, 131.79, 63.21, 60.88, 35.62, 33.37, 18.92. LC-MS(ESI-MS): (m/z) calculated for C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 169.1; found 169.2.\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-\u003cem\u003eN\u003c/em\u003e-(piperidin-3-yl)acrylamide \u003cstrong\u003e3b\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert\u003c/em\u003e-butyl \u003cem\u003e(R\u003c/em\u003e)-3-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. \u003cstrong\u003e3b\u003c/strong\u003e was obtained after purification by column chromatography in a yield of mg 61.3 mg (0.4 mmol, 81.1%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (400 MHz, MeOD) \u0026delta; 6.85\u0026ndash;6.71 (m, 1H), 6.23 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.8, 1.5 Hz, 1H), 5.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.7, 1.8 Hz, 1H), 4.36\u0026ndash;4.26 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 3.88\u0026ndash;3.78 (m, 1H), 3.51\u0026ndash;3.35 (m, 2H), 2.18\u0026ndash;2.09 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.9 Hz, 1H), 1.93\u0026ndash;1.80 (m, 1H), 1.78\u0026ndash;1.57 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27.3, 13.1, 9.4, 4.0 Hz, 2H), 1.41\u0026ndash;1.28 (m, 1H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (101 MHz, MeOD) \u0026delta; 167.18, 129.27, 128.73, 57.17, 56.09, 45.27, 35.53, 21.41. LC-MS(ESI-MS): (m/z) calculated for C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 155.1; found 156.1.\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eS\u003c/em\u003e)-\u003cem\u003eN\u003c/em\u003e-(piperidin-3-yl)acrylamide \u003cstrong\u003e3c\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert\u003c/em\u003e-butyl \u003cem\u003e(S\u003c/em\u003e)-3-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. \u003cstrong\u003e3c\u003c/strong\u003e was obtained after purification by column chromatography in a yield 68.8 mg (0.4 mmol, 91.1%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (700 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.81\u0026ndash;6.70 (m, 1H), 6.26\u0026ndash;6.19 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.7 Hz, 1H), 5.81\u0026ndash;5.75 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.7 Hz, 1H), 4.35\u0026ndash;4.27 (m, 1H), 4.13\u0026ndash;4.01 (m, 1H), 3.90\u0026ndash;3.80 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.2 Hz, 1H), 3.44\u0026ndash;3.35 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25.1 Hz, 1H), 3.26\u0026ndash;3.18 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;33.6, 16.8 Hz, 1H), 2.17\u0026ndash;2.09 (m, 1H), 1.90\u0026ndash;1.79 (m, 1H), 1.75\u0026ndash;1.67 (m, 1H), 1.68\u0026ndash;1.55 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;52.1 Hz, 1H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (176 MHz, MeOD) \u0026delta; 178.25, 129.13, 128.75, 55.83, 46.81, 45.44, 29.32, 24.17. LC-MS(ESI-MS): (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 155.1; found 156.1.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-(piperidin-4-yl)acrylamide \u003cstrong\u003e3i\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert-\u003c/em\u003ebutyl 4-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. \u003cstrong\u003e3i\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 61.3 mg (0.4 mmol, 80%) .\u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (500 MHz, DMSO) \u003cem\u003e\u0026delta;\u003c/em\u003e 8.60 (s, 1H), 8.42 (s, 1H), 8.23 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 1H), 6.21 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.1, 10.0 Hz, 1H), 6.13\u0026ndash;6.07 (m, 1H), 5.60 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.0, 2.3 Hz, 1H), 3.89 (tdd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.0, 7.4, 3.9 Hz, 1H), 3.26 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.9 Hz, 2H), 2.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.2, 12.1 Hz, 2H), 1.92 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.8, 3.1 Hz, 2H), 1.61\u0026ndash;1.52 (m, 2H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (126 MHz, DMSO) \u003cem\u003e\u0026delta;\u003c/em\u003e 163.98, 131.56, 125.57, 43.45, 42.14, 28.24. LC-MS(ESI-MS): (m/z) calculated for C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 155.1; found 156.1.\u003c/p\u003e\n \u003cp\u003e1-(4-aminopiperidin-1-yl)prop-2-en-1-one \u003cstrong\u003e3h\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert-\u003c/em\u003ebutyl 4-aminopiperidine-1-carboxylate (0.49 mmol, 1 equiv) was reacted. \u003cstrong\u003e3h\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 61.3 mg (0.39 mmol, 80%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (700 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.70\u0026ndash;6.64 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.8, 10.7 Hz, 1H), 6.12\u0026ndash;6.07 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.8, 3.4 Hz, 1H), 5.68\u0026ndash;5.64 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.7, 1.8 Hz, 1H), 4.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.7 Hz, 1H), 4.11 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.7 Hz, 1H), 3.34\u0026ndash;3.27 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.5, 9.5 Hz, 1H), 3.17\u0026ndash;3.09 (m, 1H), 2.03\u0026ndash;1.94 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.4 Hz, 2H), 1.48\u0026ndash;1.36 (m, 2H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (176 MHz, MeOD) \u0026delta; 167.63, 128.89, 128.83, 45.05, 41.41, 30.74. LC-MS(ESI-MS): (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 155.1; found 155.1.\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eR\u003c/em\u003e)-\u003cem\u003eN\u003c/em\u003e-(azepan-3-yl)acrylamide \u003cstrong\u003e3d\u003c/strong\u003e. According to the general procedure A, 100 mg of \u003cem\u003etert-\u003c/em\u003ebutyl 4-aminopiperidine-1-carboxylate (0.47 mmol, 1 equiv) was reacted. \u003cstrong\u003e3d\u003c/strong\u003e was obtained after purification by column chromatography in a yield of 62 mg (0.4 mmol, 78.5%). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR\u003c/strong\u003e (600 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 6.28\u0026ndash;6.20 (m, 2H), 5.70 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.9, 5.1 Hz, 1H), 3.42\u0026ndash;3.38 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.6, 3.9, 0.7 Hz, 1H), 3.32\u0026ndash;3.27 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.8, 6.3, 3.1 Hz, 2H), 3.25\u0026ndash;3.18 (m, 2H), 2.10\u0026ndash;1.95 (m, 2H), 1.95\u0026ndash;1.84 (m, 2H), 1.81\u0026ndash;1.73 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.0, 10.2, 3.0 Hz, 1H), 1.71\u0026ndash;1.62 (m, 1H). \u003csup\u003e\u003cstrong\u003e\u003cspan\u003e13\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC NMR\u003c/strong\u003e (151 MHz, MeOD) \u003cem\u003e\u0026delta;\u003c/em\u003e 167.70, 131.51, 127.58, 55.82, 50.39, 43.77, 33.54, 26.19, 23.59. LC-MS(ESI-MS): (m/z) calculated for C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 169.1; found 169.1.\u003c/p\u003e\n \u003cp\u003eCDI-protected GDP \u003cstrong\u003e5\u003c/strong\u003e. According to General Procedure B, \u003cstrong\u003e5\u003c/strong\u003e was obtained after purification.LC-MS (\u003cem\u003em/z\u003c/em\u003e): Calculated for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e: 518.0 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e, found: 518.0.\u003c/p\u003e\n \u003cp\u003eAccording to the General Procedure C, the modified amines (1.2 mmol, 2 equivalents) were reacted with \u003cstrong\u003e5\u003c/strong\u003e in the presence of DIPEA (0.5 ml, 3.0 mmol, 5 equivalents). The following products were obtained (Table \u003cspan\u003e1\u003c/span\u003e):\u003c/p\u003e\n \u003cdiv\u003eAccording to the General Procedure D, molecules \u003cstrong\u003e6a\u003c/strong\u003e\u0026ndash;\u003cstrong\u003ei\u003c/strong\u003e were reacted. The following products were obtained (Table \u003cspan\u003e2\u003c/span\u003e):\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBiological Experiments\u003c/h2\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eProtein expression and purification\u003c/h2\u003e\n \u003cp\u003eKRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e \u003csub\u003e1\u0026minus;169\u003c/sub\u003e Cys-light (C51S C80L C118S) were expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) at 37\u0026deg;C. Protein expression was induced at A600 nm of 0.5 by the addition of 0.2\u0026ndash;0.3 mM isopropyl-b-D-thiogalactoside (IPTG), and growth was continued at 19\u0026deg;C overnight. The bacteria were collected by centrifugation, and the obtained pellet resuspended in Ni-NTA buffer (50 mM Tris pH 8.0, 250 mM NaCl, 40 mM imidazole, 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M GDP, 1 mM Tris(2-chlorehyl)phosphate (TCEP), and 5% glycerol). The cells were lysed with a microfluidizer, and after addition of protease inhibitor cocktail (Roche complete EDTA free) and 1% CHAPS (w/v) stirring was continued for 1 hr at 4\u0026deg;C. The lysate was cleared by centrifugation (35,000 x g, 1 h, 4\u0026deg;C), and the supernatant was loaded onto a Ni-affinity chromatography column (Qiagen Ni-NTA Superflow, 5 mL) pre-equilibrated with Ni-NTA buffer. The protein was eluted with a linear gradient of imidazole buffer (40 mM \u0026ndash; 500 mM). For cleavage of the N-terminal hexahistidine-tag, TEV protease was added to the pooled elution fractions and dialyzed overnight into dialysis buffer at 4\u0026deg;C (25 mM Tris pH 8.0, 100 mM NaCl, 4 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M GDP, 1 mM TCEP, and 5% glycerol). The cleaved protein was then applied to a reverse Ni-affinity chromatography column. The eluted protein fractions were concentrated to around 10 mL followed by dilution with salt-free AEX buffer (25 mM Tris pH 8.0, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M GDP, 1 mM TCEP, and 5% glycerol) for better binding conditions onto AEX column (Cytiva, QFF HiTrap, 2x1 mL). The protein was eluted with a linear gradient (0 mM \u0026ndash; 1000 mM NaCl). Finally, the protein was purified by size-exclusion chromatography (GE HiLoad 16/60 Superdex 75 pg) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 \u0026micro;M GDP, 1 mM TCEP, and 5% glycerol.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eCovalent Modification of Proteins\u003c/h2\u003e\n \u003cp\u003eTo assess the extent of covalent modification of KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e\u003csub\u003e1\u0026ndash;169\u003c/sub\u003e, 50 \u0026micro;M Ras protein was incubated with a 10-fold molar excess of acryl-bearing nucleotides in a buffer containing 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM TCEP, and 1 mM EDTA. After incubation at room temperature for the appropriate duration, the extent of protein modification was analyzed by ESI-MS. MS spectra were acquired on a VelosPro IonTrap mass spectrometer (Thermo Scientific) using an AdvanceBio Desalting-RP, 2.1x2.5mm column (Agilent Technologies). A gradient elution was employed, transitioning from mobile phase A (0.1% formic acid in water) to mobile phase B (0.1% formic acid in acetonitrile).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eEffector Binding (Pull-Down Assays)\u003c/h2\u003e\n \u003cp\u003ePull-down experiments were performed using a buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, and 2 mM MgCl₂. For each assay, 10 \u0026micro;g KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e:GDP or covalently modified KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e-\u003cstrong\u003e7a\u003c/strong\u003e and \u003cstrong\u003e7b\u003c/strong\u003e and 20 \u0026micro;g GST-tagged cRaf-RBD (amino acids 51\u0026ndash;131) were incubated either in the presence or absence of 100 \u0026micro;M GppNHp and 1 \u0026micro;g SOS overnight at room temperature. Following incubation, 50 \u0026micro;L of glutathione magnetic beads were added to each sample and incubated for 30 minutes. Beads were then washed with 500 \u0026micro;L buffer, separated using a magnetic rack, and the supernatant was carefully removed. The beads were resuspended in 50 \u0026micro;L of 4x SDS-loading buffer, and samples were analyzed by SDS-PAGE.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eGuanine Nucleotide-Exchange Factor Assay\u003c/h2\u003e\n \u003cp\u003eSOS-catalyzed nucleotide exchange was measured at 25\u0026deg;C using a FluoroMax-3 spectrofluorometer. Fluorescence was monitored with an excitation wavelength of 360 nm and an emission wavelength of 440 nm. The assay buffer consisted of 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl₂, and 1 mM TCEP. In the assay, 5 \u0026micro;M KRAS (residues 1\u0026ndash;169) was incubated with 10 \u0026micro;M mant-dGDP, followed by the addition of SOS at varying concentrations (0.25 \u0026micro;M and 0.5 \u0026micro;M). Fluorescence changes were recorded to monitor nucleotide exchange.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eCrystallization\u003c/h2\u003e\n \u003cp\u003eThe KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e protein was diluted to 2 mg/mL and incubated with a 3-fold excess of the acryl-bearing nucleotide \u003cstrong\u003e7b\u003c/strong\u003e in 100 mM CHES (pH 9.5), 50 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 5% glycerol at room temperature. After incubation for 24 hr, the protein modification was controlled by ESI-MS. The MS spectra were recorded on an VelosPro IonTrap (Thermo Scientific) with an AdvanceBio Desalting-RP, 2.1x2.5mm column (Agilent Technologies) and a gradient of the mobile phase A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile). The protein:inhibitor complex was purified by size-exclusion chromatography (Superdex Increase 75 pg 10/300 GL) in a final buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl2, 5 mM TCEP, and 5% glycerol, and subsequently concentrated to 44 mg/mL and 28 mg/mL and used for crystallization.\u003c/p\u003e\n \u003cp\u003eA former identified initial crystallization condition (JCSG Core III, B5) was further optimized and used for crystallization at 4, 12 and 18\u0026deg;C. The protein:inhibitor complex was mixed in a 1:1 ratio (1 \u0026micro;L protein:inhibitor complex and 1 \u0026micro;L reservoir solution containing 100 mM sodium acetate pH 8.0\u0026ndash;9.0, 200 mM Tris-HCl pH 8.0\u0026ndash;9.0, 25\u0026ndash;35% (w/v) PEG4000). The crystals grew within 24 h with the higher protein concentration and within weeks with the lower concentration. The fast-grown crystals were much more intergrown; therefore, the slow-grown crystals were used for the following shrinking step. The crystals were transferred in a new drop containing SEC buffer condition and crystallization conditions mixed in a 1:1 ratio plus an additional 30% PEG3350/PEG4000, 20% glycerol, 20% ethylene glycol, or 20% PEG 400 for 24 h at 18\u0026deg;C. After incubation, the crystals were fished and flash cooled in liquid nitrogen. The data sets were collected at the ID30B beamline of the ESRF (European Synchrotron Radiation Facility, Grenoble, France, DOI: \u003cspan\u003e\u003cspan\u003e10.15151/ESRF-ES-1581727707\u003c/span\u003e\u003c/span\u003e) The data were processed using XDS and scaled using XSCALE.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eStructure Determination and Refinement\u003c/h2\u003e\n \u003cp\u003eThe complex crystal structure was solved by molecular replacement with PHASER using structure PDB ID: 7ok3 as template.\u003csup\u003e\u003cspan\u003e32\u003c/span\u003e\u003c/sup\u003e The molecules in the asymmetric units were manually adjusted using the program COOT.\u003csup\u003e\u003cspan\u003e33\u003c/span\u003e\u003c/sup\u003e The refinement was performed with Phenix.refine 1.21.1.\u003csup\u003e\u003cspan\u003e34\u003c/span\u003e\u003c/sup\u003e Inhibitor topology files were generated using eLBOW of the Phenix 1.21.1 program package. Refined structures were validated with the PDB validation server. Data collection, structure refinement statistics, PDB-ID codes, and further details for data collection are provided in Table\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e. PyMOL (W.L. DeLano, The PyMOL Molecular Graphics System) was used for generating the figures.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eData statistics for KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e covalently bound to compound \u003cstrong\u003e7b\u003c/strong\u003e (PDB ID 9I7Y).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cspan type=\"SmallCaps\" name=\"Emphasis\"\u003eData collection\u003c/span\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpace group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC\u003c/em\u003e 1 2 1(5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell dimensions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ea, b, c [\u0026Aring;]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.01, 84.76, 88.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026alpha;, \u0026beta;, \u0026gamma; [\u0026deg;]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.0, 113.26, 90.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResolution [\u0026Aring;]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.00-1.85 (1.90\u0026ndash;1.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003emeas\u003c/sub\u003e [%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6 (126.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eI\u003c/em\u003e / \u0026sigma;\u003cem\u003eI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.37 (1.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCompleteness [%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.8 (99.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCC\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.9 (61.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRedundancy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.86 (6.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cspan type=\"BoldSmallCaps\" name=\"Emphasis\"\u003eRefinement\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResolution [\u0026Aring;]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.38\u0026ndash;1.85 (1.90\u0026ndash;1.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo. reflections\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40651\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003ework\u003c/sub\u003e / R\u003csub\u003efree\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.98 / 22.78 (55.79 / 54.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. atoms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echain A\u0026thinsp;=\u0026thinsp;1249\u003c/p\u003e\n \u003cp\u003echain B\u0026thinsp;=\u0026thinsp;1247\u003c/p\u003e\n \u003cp\u003echain C\u0026thinsp;=\u0026thinsp;1140\u003c/p\u003e\n \u003cp\u003etotal: 3636\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLigand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 x 41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIons (Mg\u003csup\u003e2+\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB\u003c/em\u003e-factors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echain A\u0026thinsp;=\u0026thinsp;56.28\u003c/p\u003e\n \u003cp\u003echain B\u0026thinsp;=\u0026thinsp;62.13\u003c/p\u003e\n \u003cp\u003echain C\u0026thinsp;=\u0026thinsp;61.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLigand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echain A\u0026thinsp;=\u0026thinsp;52.72\u003c/p\u003e\n \u003cp\u003echain B\u0026thinsp;=\u0026thinsp;52.18\u003c/p\u003e\n \u003cp\u003echain C\u0026thinsp;=\u0026thinsp;62.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIons (Mg\u003csup\u003e2+\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eR.m.s. deviations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBond lengths [\u0026Aring;]\u003c/p\u003e\n \u003cp\u003eBond angles [\u0026deg;]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003cp\u003e1.249\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRamachandran [%]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOutliers\u003c/p\u003e\n \u003cp\u003eAllowed\u003c/p\u003e\n \u003cp\u003eFavored\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e1.75\u003c/p\u003e\n \u003cp\u003e98.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRotamer [%]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOutliers\u003c/p\u003e\n \u003cp\u003eAllowed\u003c/p\u003e\n \u003cp\u003eFavored\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003cp\u003e2.85\u003c/p\u003e\n \u003cp\u003e96.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eMolecular Modeling\u003c/h2\u003e\n \u003cp\u003eThe computational prediction of drug affinity is a major challenge in medicinal chemistry\u003csup\u003e\u003cspan\u003e35\u003c/span\u003e\u003c/sup\u003e that becomes even more complex in the case of covalent inhibitors. To mitigate such limitations, we employed an approach focused on the reversible complexes in the present work, hypothesizing that the availability of ligand conformations in close proximity to Cys13 would relate to the inhibitory efficiency of the compounds. In this sense, the crystallographic structure of KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e covalently bound to inhibitor 7a was retrieved from PDB under ID 7ok3, and used as a basis for the following steps of the study. Compound 7a was removed from the crystallographic complex and used to model compounds 7b-I using Maestro (Maestro, Schr\u0026ouml;dinger, LLC, New York, NY, 2024). The molecules were submitted to an energy minimization step and used as inputs for molecular docking to the target enzyme. After an accurate redocking of 7a, each compound was docked to KRAS\u003csup\u003eG\u003cspan\u003e13\u003c/span\u003eC\u003c/sup\u003e using DockThor web-server, which uses one of the top-scoring functions currently available,\u003csup\u003e\u003cspan\u003e36\u003c/span\u003e\u003c/sup\u003e maintaining the ligand\u0026rsquo;s flexibility. To access the solution ensemble of the reversible states of compounds \u003cstrong\u003e7a\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e and explore the proximity of the warhead to Cys13, each docked complex was submitted to molecular dynamics (MD) simulations using GROMACS\u003csup\u003e\u003cspan\u003e37\u003c/span\u003e\u003c/sup\u003e and CHARMM36 force field.\u003csup\u003e\u003cspan\u003e38\u003c/span\u003e\u003c/sup\u003e Each complex was prepared using the Solution Builder tool available on the CHARMM-GUI web server,\u003csup\u003e\u003cspan\u003e39\u003c/span\u003e\u003c/sup\u003e inserted in cubic boxes with a minimum distance of 10 \u0026Aring; between the solute and the box edges, solvated with TIP3P water molecules, and neutralized with a 0.15 M NaCl concentration. Simulations were carried out at physiological pH and temperature (298 K) using an integration step of 2 fs. The LINCS algorithm was used to constrain the lengths of hydrogen bonds, while the PME method was used to calculate the long-range electrostatic interactions. The V-rescale thermostat with two coupling groups was used to maintain the temperature of the system, and the Parrinello-Rahman barostat was used to maintain the pressure of the system. The systems were initially equilibrated for 1ns for a canonic ensemble (NVT) with the atomic positions restrained using a 5,000 kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e force on both protein and ligand atoms. After this step, five 1 ns simulations under the isothermal\u0026ndash;isobaric ensemble (NPT) were performed, progressively lowering the above-mentioned force in steps of 1,000 kJ.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After this careful equilibration process, production runs were performed for 1 \u0026micro;s, each complex being simulated with three replicas.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATP, adenosinetriphosphate; CADD, computer aided drug design; CDI, carbonyldiimidazole; DIPEA, \u003cem\u003eN,N\u003c/em\u003e-diisopropylethylamine; DMF, dimethylformamide; GDP, guanosinediphosphate; GN, guanosine nucleotide; GppNHp, guanosine-5\u0026apos;-[(\u0026beta;,\u0026gamma;)-imido]triphosphate; GST, glutathione-S-transferase; GTP, guanosinetriphosphate; KRAS, Kirsten rat sarcoma; MD, molecular dynamics; MS, mass spectrometry; RBD, ras binding domain; SOS, son of sevenless; SWIIP, switch-II-pocket.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was co-funded by the German Research Foundation (DFG), the State of North Rhine-Westphalia (NRW), the European Union (European Regional Development Fund: Investing In Your Future) (EFRE-800400), DDHD (Drug Discovery Hub Dortmund), the German Federal Ministry of Education and Research (InCa, 01ZX2201B), the Mercator Research Center Ruhr (MERCUR, IGNITE (Ex-2021-0033)), the German Cancer Aid ((Deutsche Krebshilfe), Targeting Transcriptional Addiction in Cancer (TACTIC)), the \u0026quot;Netzwerke 2021\u0026quot; program, an initiative of the Ministry of Culture and Science of the State of North Rhine-Westphalia (CANcer TARgeting, NW21-062C) and, supported by the European Synchrotron Radiation Facility (ESRF, Grenoble, France, proposal MX-2580, beamline ID30B; DOI 10.15151/ESRF-ES-1581727707). We thank Andreas Arndt for his support with protein expression and purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Supporting Information is available online free of charge. The authors will release the atomic coordinates upon article publication.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available in the paper and its Supplementary Information. The crystal structure data generated in this study have been deposited in the PDB database under accession code 9I7Y (DOI: https://doi.org/10.2210/pdb9I7Y/pdb).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.R., M.P.M., H.V. are responsible for initiating and supervising the project. T.K. designed and synthesized the compounds and performed biological and biochemical experiments including protein MS studies. J.N. performed co-crystallization experiments, data processing, structure building and structural analysis. J.R. and E.G.M. performed computational experiments including MD simulations. The manuscript was written with contributions from all authors. All authors approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eScheffzek, K.\u003cem\u003e et al.\u003c/em\u003e The Ras-RasGAP Complex: Structural Basis for GTPase Activation and Its Loss in Oncogenic Ras Mutants. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 333-339, doi:10.1126/science.277.5324.333 (1997).\u003c/li\u003e\n\u003cli\u003eSimanshu, D. K., Nissley, D. V. \u0026amp; McCormick, F. RAS Proteins and Their Regulators in Human Disease. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e170\u003c/strong\u003e, 17-33, doi:https://doi.org/10.1016/j.cell.2017.06.009 (2017).\u003c/li\u003e\n\u003cli\u003eGoody, R. S., M\u0026uuml;ller, M. P. \u0026amp; Rauh, D. Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules. \u003cem\u003eCell Chemical Biology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1338-1348, doi:https://doi.org/10.1016/j.chembiol.2019.07.005 (2019).\u003c/li\u003e\n\u003cli\u003eKarnoub, A. E. \u0026amp; Weinberg, R. A. Ras oncogenes: split personalities. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 517-531, doi:10.1038/nrm2438 (2008).\u003c/li\u003e\n\u003cli\u003eOstrem, J. M., Peters, U., Sos, M. L., Wells, J. A. \u0026amp; Shokat, K. M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e503\u003c/strong\u003e, 548-551, doi:10.1038/nature12796 (2013).\u003c/li\u003e\n\u003cli\u003eGoebel, L., M\u0026uuml;ller, M. P., Goody, R. S. \u0026amp; Rauh, D. KRasG12C inhibitors in clinical trials: a short historical perspective. \u003cem\u003eRSC Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 760-770, doi:10.1039/D0MD00096E (2020).\u003c/li\u003e\n\u003cli\u003eCanon, J.\u003cem\u003e et al.\u003c/em\u003e The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e575\u003c/strong\u003e, 217-223, doi:10.1038/s41586-019-1694-1 (2019).\u003c/li\u003e\n\u003cli\u003eFell, J. B.\u003cem\u003e et al.\u003c/em\u003e Identification of the Clinical Development Candidate MRTX849, a Covalent KRASG12C Inhibitor for the Treatment of Cancer. \u003cem\u003eJournal of Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 6679-6693, doi:10.1021/acs.jmedchem.9b02052 (2020).\u003c/li\u003e\n\u003cli\u003eHallin, J.\u003cem\u003e et al.\u003c/em\u003e The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. \u003cem\u003eCancer Discovery\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 54-71, doi:10.1158/2159-8290.CD-19-1167 (2020).\u003c/li\u003e\n\u003cli\u003eWang, X.\u003cem\u003e et al.\u003c/em\u003e Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor. \u003cem\u003eJournal of medicinal chemistry\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 3123-3133, doi:10.1021/acs.jmedchem.1c01688 (2022).\u003c/li\u003e\n\u003cli\u003eYu, Z.\u003cem\u003e et al.\u003c/em\u003e Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles. \u003cem\u003eJournal of the American Chemical Society\u003c/em\u003e \u003cstrong\u003e145\u003c/strong\u003e, 20403-20411, doi:10.1021/jacs.3c05899 (2023).\u003c/li\u003e\n\u003cli\u003eZheng, Q., Zhang, Z., Guiley, K. Z. \u0026amp; Shokat, K. M. Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D. \u003cem\u003eNature Chemical Biology\u003c/em\u003e, doi:10.1038/s41589-024-01565-w (2024).\u003c/li\u003e\n\u003cli\u003eZhang, Z., Guiley, K. Z. \u0026amp; Shokat, K. M. Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S). \u003cem\u003eNature Chemical Biology\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 1177-1183, doi:10.1038/s41589-022-01065-9 (2022).\u003c/li\u003e\n\u003cli\u003eZhang, Z., Morstein, J., Ecker, A. K., Guiley, K. Z. \u0026amp; Shokat, K. M. Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile. \u003cem\u003eJournal of the American Chemical Society\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 15916-15921, doi:10.1021/jacs.2c05377 (2022).\u003c/li\u003e\n\u003cli\u003eKirschner, T., M\u0026uuml;ller, M. P. \u0026amp; Rauh, D. Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy. \u003cem\u003eJournal of Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 6044-6051, doi:10.1021/acs.jmedchem.3c02403 (2024).\u003c/li\u003e\n\u003cli\u003eForbes, S. A.\u003cem\u003e et al.\u003c/em\u003e COSMIC: exploring the world\u0026apos;s knowledge of somatic mutations in human cancer. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, D805-D811, doi:10.1093/nar/gku1075 (2015).\u003c/li\u003e\n\u003cli\u003eVisscher, M., Arkin, M. R. \u0026amp; Dansen, T. B. Covalent targeting of acquired cysteines in cancer. \u003cem\u003eCurrent Opinion in Chemical Biology\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 61-67, doi:https://doi.org/10.1016/j.cbpa.2015.11.004 (2016).\u003c/li\u003e\n\u003cli\u003eZhang, Z. \u0026amp; Shokat, K. M. Bifunctional Small-Molecule Ligands of K-Ras Induce Its Association with Immunophilin Proteins. \u003cem\u003eAngewandte Chemie International Edition\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 16314-16319, doi:https://doi.org/10.1002/anie.201910124 (2019).\u003c/li\u003e\n\u003cli\u003eSchulze, C. J.\u003cem\u003e et al.\u003c/em\u003e Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 794-799, doi:10.1126/science.adg9652 (2023).\u003c/li\u003e\n\u003cli\u003eTraut, T. W. Physiological concentrations of purines and pyrimidines. \u003cem\u003eMolecular and Cellular Biochemistry\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 1-22, doi:10.1007/BF00928361 (1994).\u003c/li\u003e\n\u003cli\u003eGoody, R. S., Frech, M. \u0026amp; Wittinghofer, A. Affinity of guanine nucleotide binding proteins for their ligands: facts and artefacts. \u003cem\u003eTrends in Biochemical Sciences\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 327-328, doi:https://doi.org/10.1016/0968-0004(91)90134-H (1991).\u003c/li\u003e\n\u003cli\u003eThaimattam, R., Banerjee, R., Miglani, R. \u0026amp; Iqbal, J. Protein Kinase Inhibitors: Structural Insights Into Selectivity. \u003cem\u003eCurrent Pharmaceutical Design\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2751-2765, doi:http://dx.doi.org/10.2174/138161207781757042 (2007).\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller, M. P., Jeganathan, S., Heidrich, A., Campos, J. \u0026amp; Goody, R. S. Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 3687, doi:10.1038/s41598-017-03973-6 (2017).\u003c/li\u003e\n\u003cli\u003eLim, S. M.\u003cem\u003e et al.\u003c/em\u003e Therapeutic Targeting of Oncogenic K-Ras by a Covalent Catalytic Site Inhibitor. \u003cem\u003eAngewandte Chemie International Edition\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 199-204, doi:https://doi.org/10.1002/anie.201307387 (2014).\u003c/li\u003e\n\u003cli\u003eHunter, J. C.\u003cem\u003e et al.\u003c/em\u003e In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 8895-8900, doi:10.1073/pnas.1404639111 (2014).\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller, M. P., Jeganathan, S., Heidrich, A., Campos, J. \u0026amp; Goody, R. S. Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 3687, doi:10.1038/s41598-017-03973-6 (2017).\u003c/li\u003e\n\u003cli\u003eGoebel, L.\u003cem\u003e et al.\u003c/em\u003e Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e82184, doi:10.7554/eLife.82184 (2023).\u003c/li\u003e\n\u003cli\u003eLategahn, J., Keul, M. \u0026amp; Rauh, D. Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer. \u003cem\u003eAngewandte Chemie International Edition\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 2307-2313, doi:https://doi.org/10.1002/anie.201710398 (2018).\u003c/li\u003e\n\u003cli\u003eForbes, S. A.\u003cem\u003e et al.\u003c/em\u003e COSMIC: exploring the world\u0026apos;s knowledge of somatic mutations in human cancer. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, D805-811, doi:10.1093/nar/gku1075 (2015).\u003c/li\u003e\n\u003cli\u003eWolber, G. \u0026amp; Langer, T. LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. \u003cem\u003eJ Chem Inf Model\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 160-169, doi:10.1021/ci049885e (2005).\u003c/li\u003e\n\u003cli\u003eYan, X.\u003cem\u003e et al.\u003c/em\u003e Multiscale calculations reveal new insights into the reaction mechanism between KRAS\u0026lt;sup\u0026gt;G12C\u0026lt;/sup\u0026gt; and \u0026amp;#x3b1;, \u0026amp;#x3b2;-unsaturated carbonyl of covalent inhibitors. \u003cem\u003eComputational and Structural Biotechnology Journal\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1408-1417, doi:10.1016/j.csbj.2024.03.027 (2024).\u003c/li\u003e\n\u003cli\u003eRead, R. Pushing the boundaries of molecular replacement with maximum likelihood. \u003cem\u003eActa Crystallographica Section D\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 1373-1382, doi:doi:10.1107/S0907444901012471 (2001).\u003c/li\u003e\n\u003cli\u003eEmsley, P. \u0026amp; Cowtan, K. Coot: model-building tools for molecular graphics. \u003cem\u003eActa Crystallographica Section D\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2126-2132, doi:doi:10.1107/S0907444904019158 (2004).\u003c/li\u003e\n\u003cli\u003eAdams, P. D.\u003cem\u003e et al.\u003c/em\u003e PHENIX: a comprehensive Python-based system for macromolecular structure solution. \u003cem\u003eActa Crystallographica Section D\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 213-221, doi:doi:10.1107/S0907444909052925 (2010).\u003c/li\u003e\n\u003cli\u003eVerli, H. \u0026amp; Oostenbrink, C. Status and Prospects of Molecular Simulations for Drug Discovery. \u003cem\u003eJournal of the Brazilian Chemical Society\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, doi:10.21577/0103-5053.20240119 (2024).\u003c/li\u003e\n\u003cli\u003eGuedes, I. A.\u003cem\u003e et al.\u003c/em\u003e New machine learning and physics-based scoring functions for drug discovery. \u003cem\u003eSci Rep-Uk\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 3198, doi:10.1038/s41598-021-82410-1 (2021).\u003c/li\u003e\n\u003cli\u003eAbraham, M. J.\u003cem\u003e et al.\u003c/em\u003e GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. \u003cem\u003eSoftwareX\u003c/em\u003e \u003cstrong\u003e1-2\u003c/strong\u003e, 19-25, doi:https://doi.org/10.1016/j.softx.2015.06.001 (2015).\u003c/li\u003e\n\u003cli\u003eHuang, J. \u0026amp; MacKerell Jr, A. D. CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data. \u003cem\u003eJ Comput Chem\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 2135-2145, doi:https://doi.org/10.1002/jcc.23354 (2013).\u003c/li\u003e\n\u003cli\u003eLee, J.\u003cem\u003e et al.\u003c/em\u003e CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. \u003cem\u003eJ Chem Theory Comput\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 405-413, doi:10.1021/acs.jctc.5b00935 (2016).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 and 2","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"KRAS, G13C, Nucleotide-based inhibitors, Computer-aided drug design","lastPublishedDoi":"10.21203/rs.3.rs-6028840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6028840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe small GTPase KRAS is a key driver of carcinogenesis when mutated, and significant progress has been made in targeting KRAS\u003csup\u003eG\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003eC\u003c/sup\u003e and other oncogenic variants. Building on our previous work demonstrating the potential of nucleotide-based inhibitors with an acrylamide warhead to target KRAS\u003csup\u003eG\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003eC\u003c/sup\u003e, we designed and synthesized a library of nucleotide-based compounds with cyclic linkers to explore the effect of warhead orientation on reactivity toward Cys13. Using mass spectrometry, kinetic studies, and protein X-ray crystallography, we validated the binding and reactivity of these modulators. In addition, computational predictions of the conformational space of the linkers and warheads provided insights into their reactivity, which agreed well with the experimental data. These findings advance our understanding of the structure-reactivity relationship in these nucleotide-based KRAS inhibitors and will be the basis for further optimization.\u003c/p\u003e","manuscriptTitle":"Targeting KRASG13C: Exploring Warhead Orientation with Cyclic Linker-Based Inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-17 06:30:47","doi":"10.21203/rs.3.rs-6028840/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-17T18:17:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-10T12:17:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-28T21:53:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-27T21:44:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127216707974619812902935269675256978699","date":"2025-02-19T09:11:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210474520742208068225129407891891472042","date":"2025-02-17T14:53:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175702735853284129435987154142810969647","date":"2025-02-17T12:19:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-17T12:15:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-17T12:11:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-17T11:58:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-14T10:32:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-14T08:34:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cc1492b-db3e-466a-a193-3e897057f64e","owner":[],"postedDate":"February 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44337970,"name":"Biological sciences/Cancer"},{"id":44337971,"name":"Biological sciences/Chemical biology"},{"id":44337972,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":44337973,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2025-11-03T16:00:48+00:00","versionOfRecord":{"articleIdentity":"rs-6028840","link":"https://doi.org/10.1038/s41598-025-22145-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-31 15:57:29","publishedOnDateReadable":"October 31st, 2025"},"versionCreatedAt":"2025-02-17 06:30:47","video":"","vorDoi":"10.1038/s41598-025-22145-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22145-5","workflowStages":[]},"version":"v1","identity":"rs-6028840","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6028840","identity":"rs-6028840","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}