Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo.

Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo. | 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 Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo . Jeffrey Wong, Steven Kirberger, Ryan Qiu, Arunika Ekanayake, Payam Kelich, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1999287/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Sep, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract In this paper, we report selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine S N Ar chemistry. Modification of phage-displayed libraries SXCX n C-phage, n = 3–5, where X is any amino acid except for cysteine by decafluoro-diphenylsulfone ( DFS ), yields genetically-encoded library of octafluoro-diphenylsulfone-crosslinked macrocycles ( OFS -SXCX n C-phage). Selection from these libraries using albumin as a bait identified a family of significantly enriched perfluoroaryl-macrocycles. Synthesis of perfluoroaryl-macrocycles predicted by phage display and testing their binding properties by 19 F NMR and fluorescent polarization identified OFS -macrocycle with SICRFFC sequence as the most potent albumin binder. We observed that OFS -macrocycles slowly react with biological nucleophiles such as glutathione. Replacing decafluoro-diphenylsulfone by nearly isosteric pentafluorophenyl sulfide yielded perfluorophenylsulfide ( PFS )-crosslinked macrocycles devoid of undesired reactivity. The augmented lead PFS -macrocycle with SICRFFC sequence exhibited K D = 4–6 µM towards human serum albumin and similar affinities towards rat and mouse albumins. When injected in mouse, the PFS -SICRFFCGGG compound was significantly retained in circulation in vivo when compared to control PFS -macrocyclic peptide. The perfluoroaryl-macrocycles with SICRFFC motif are the smallest known peptide macrocycle with significant affinity for human albumin and they are a productive starting point for future development of compact macrocycles with predictable circulation half-life in vivo . Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction There are around 80 peptide drugs on the global market; more than 150 peptides are in clinical development and another 400–600 peptides undergoing preclinical studies. 1 The large surface area of peptides, compared to a typical small molecule drugs allows peptides to interact with expanded binding interfaces commonly found in protein–protein interactions, protein–carbohydrate and protein–DNA interactions. These proteins, classified as “undruggable targets”, have been difficult to target using conventional small molecule therapeutic but many of them have been addressed by peptide, proteins, or antibody therapeutics. Peptides are the smallest among the latter three modalities—2 kDa to 10 kDa for peptides versus 150 kDa for full-sized antibodies—and they possess distinct pharmacokinetic (PK) properties. For example, bio-distribution of peptides and small proteins inside tumors and other non-vascularized tissues is improved when compared to full-size antibodies. Several clinical candidates (TH1902, TH1904, BT5528, BT8009, BT1718, MMP-14) capitalize on such improved biodistribution. 2-4 Unlike antibodies, which remain in circulation for 1–3 weeks due to the association with the neonatal Fc-receptor (FcRn) on the surface of immune cells, 5 peptide therapeutics clear within minutes to hours from plasma by renal filtration. Fast clearance is a beneficial property in several therapeutic applications, such as imaging (e.g., “tumor paint”), radionuclide delivery (e.g., Lurathera TM ), 6 and in administration of short acting peptide hormones. For more widespread adaptation of peptide modalities in diverse therapeutic applications it is desired to tune the circulation life-time of peptides from minutes to hours. Only few peptides exhibit a natural extended circulation lifetime: a therapeutically relevant example is a natural venom, 39-residue peptide ‘exendin 4’, with low renal clearance in humans (5–7 h). 7 This peptide gave rise to FDA-approved drug exenatide for the treatment of type 2 diabetes. 8, 9 Despite favourable circulation half-life, modified derivatives of exenatide—liraglutide, albiglutide, dulaglutide, lixisenatide and semaglutide 1 — have been developed to tune circulation half-life and other PK properties. The majority of peptides and small proteins have to be modified as well to increase their circulation time. Such modification could be divided into several classes: Class 1: increase in size via covalent linkage to polyethylene glycol (PEG), 10, 11 polyglycerol 12 and other synthetic macromolecules. Interestingly, steric hindrance by these size-increasing moieties also protects against proteolytic degradation. 13-15 ; Class 2: increase in size via controlled oligomerization; 16 Class 3: covalent linking to long-living serum protein (e.g., FDA-approved drugs albiglutide and dulaglutide, exanatide that conjugated to albumin and the IfG4 Fc domain) 17-19 and Class 4: incorporation of moieties that bind non-covalently to serum proteins such as albumin, 20-23 immunoglobulin, 24, 25 FcRn, 26 transthyretin, 27 and transferrin. 28, 29 An important example in the last class is lipidation of peptides to allow interaction with serum albumin. Lipidation has been one of the most successful strategies to prolong the half-life of peptides and small proteins such as insulin giving rise to FDA-approved drugs such as Levemir®, Tresiba®, Victoza®, Saxenda®, and Ozempic® with extended serum half-life 30, 31 . The improved properties of these and many other drugs stemming from their association with albumin mandate investigation of albumin as carrier for therapeutic applications. Albumin is the most abundant protein in plasma with an average concentration of 600 µM and has an average half-life of 19 days. 32 The main mechanism leading to the long half-life of albumin and antibody are similar: both proteins interact with FcRn on the surface of immune cells. This binding results in transient endocytosis of these proteins, and as a result, they are frequently sequestered from circulation and protected from clearance. At physiological pH, the binding affinity between albumin and FcRn is low; however, the interaction under acidic conditions in the endosome is strong to avoid lysosomal degradations and recycling of albumin to the extracellular space. 5 Albumin acts as a versatile carrier of essential fatty acids and diverse small organic molecules. 32 Among all the long-circulating serum proteins, albumin is considered to be one of the most important targets because of its ability to interact with hydrophobic small molecule drugs and enhance their pharmacokinetic properties. Recurrent therapeutic success of rationally lipidated peptides and proteins 33 fuels interest in rational development of small molecules as well as non-lipidated proteins and peptides that bind to albumin. Many FDA-approved small molecule drugs have an intrinsic affinity for human serum albumin (HSA). Targeted development of small molecules with high affinity for HSA has been a topic of research over the last 15 years (see recent review 23 ). Anti-HSA antibodies, nanobodies, 34 DARPins, 35 and other protein domains have been also developed. Such proteins can be fused to therapeutic proteins of interest to extend their in vivo circulation. Similarly short peptides that bind to HSA could be used in tandem with therapeutic peptide or protein sequences to dial in predictable half-life for such therapeutics. Such short albumin-binding peptides could empower development of many future therapeutic peptides because they could be built into any genetically encoded peptide library (e.g., displayed on phage, RNA and other platforms) to give rise to billion-scale libraries with predictable in vivo half-life. However, short HSA-binding peptides are scarce. A 31-mer peptide DX-236 (Ac-AEGTGDFWFCDRIAWYPQHLCEFLDPEGGGK-NH 2 ) with a binding affinity of 1.9 µM was identified by Dyax Corp., and used to purify albumin ( Figure 1A ). 36 A 21-mer peptide SA-21 (Ac-RLIEDICLPRWGCLWEDD-NH 2 ) with a binding affinity of 467 nM to HSA was identified at Genentech ( Figure 1B ) 37 and subsequently conjugated to ligands for urokinase-type plasminogen activator, 20, 38 Fab antibody fragments 39, 40 and small proteins 41 to prolong their circulation half-lives. Heinis and co-workers developed a short heptapeptide modified by fluorescein isothiocyanide (FITC) and palmitic acid (FITC-EYEYK palm ESE-NH 2 ) with a binding affinity of 39 nM to HSA ( Figure 1C ) 21 and the presence of both lipid moiety and fluorescein was critical for the binding of this peptide. This FITC-lipopeptide was fused to two different bicyclic peptides to boost the half-lives from minutes to hours. 21 Success of DX-236, SA-21 and FITC-lipopeptide and other examples from the literature demonstrated the possibility of using HSA as a target for genetically-encoded selection to identify HSA-binding peptides with extended circulation half-life. There is a need for development of other albumin binding peptide modalities that have lower molecular weight. 21, 37 Towards this goal, we employ genetically encoded phage-displayed libraries of chemically modified macrocycles to develop new classes of albumin binding mini scaffolds. To hone on shortest possible peptide sequences, we employed a phage-displayed libraries SXCX n C, n =3–5 modified with decafluorodiphenyl sulfone ( DFS ) 42, 43 where X is any amino acid except for cysteine ( Figure 1D ). We hypothesized that a perfluoroaromatic linchpin would serve as useful pharmacophore recognized by one of the binding sites of HSA similarly to the binding of fatty acid in lipidated peptides. Results And Discussion Selection of albumin binders We devised and conducted three discovery campaigns that used different library architecture and selection strategies. In the first discovery campaign, we modified the phage libraries of structure SXCX 4–5 C with DFS following a previously published protocol and confirmed that 85% of the phage library is modified to yield octafluoro-diphenylsulfone-crosslinked macrocycles ( OFS -SXCX 4–5 C-phage) ( Figure 2A , Figure S1A ). 42 We performed three rounds of phage selection using HSA coated to the surface of 96 well polystyrene plates as bait. In parallel, we screened the same library on polystyrene wells coated with Protein A (negative control) to distinguish specific HSA-binding sequences from poly-specific protein binding sequences ( Figure S1A ). In round 3, the phage recovery of the OFS -macrocycle library selection against HSA two-fold increase compared to round 1 and round 2 but only a minor increase compared to selection against unrelated protein ( Figure S1D ). The recovery of unmodified round 3-library panned against HSA was 17-fold lower than the recovery of the OFS -macrocycle library, indicating that the OFS linchpin contributes to protein binding ( Figure S1D ). Differential enrichment (DE) analysis of the next-generation sequencing (NGS) of all test and control experiments (Table S1) identified several families of peptide macrocycles that had statistically significantly higher ( p <0.05) enrichment in binding to HSA when compared to binding to unrelated protein ( Figure S1 B–C ). The analysis yielded three consensus motifs: STCHDITC ( 1a ), STCHYIGC ( 2a ) and STCHANC ( 3a ) ( Figure S1E ). The second discovery campaign employed HSA immobilized on a 96 well plate in rounds 1 and 3, and biotinylated HSA as bait immobilized onto streptavidin beads in round 2 ( Figure S2A ). In round 3, the phage recovery of the OFS -macrocycle library selection against HSA increased by a factor of 200 when compared to round 1 and round 2. The recovery of the unmodified library panned against HSA was insignificant ( Figure S2D ). The binding of the OFS -macrocycle phage library recovered from round 3 to Protein A, ConA and Casein was 2, 14, and 300-fold lower respectively when compared to recovery on HSA-coated wells ( Figure S3 ). These observations suggested that (i) specific albumin-binding sequences had been selected, and (ii) the binding of these sequences to albumin required presence of OFS linchpin ( Figure S3 ). A DE analysis of NGS data ( Figure S4, Table S2 ) identified sequences that were significantly ( p <0.05) enriched in the screen against HSA but not control proteins. The LOGO analysis yielded a consensus motif: STCHTIYC ( 4a ) ( Figure S2E ). Although the original libraries were designed as SXCX n C where n=4 and 5, they contained a small fraction of SXCX 3 C sequences, 44 and we observed the enrichment of such sequences in the selection. To explore the apparent preference for smaller macrocycles, we devised a third selection campaign that employed only SXCX 3 C libraries modified with DFS ( Figure 2A ). The small diversity of the library made it possible to employ a single round of panning and NGS-DE analysis and to identify the binders. To mimic the complex serum environment, the panning was conducted using a mixture of biotinylated HSA (Bio-HSA), His-tag fusion T4-PG protein (His 6 -T4-PG) and unlabelled milk proteins as bait. In a control selection, we used the same mixture with biotinylated ConA (Bio-ConA) in place of Bio-HSA ( Figure 2B ). Proteins were captured with streptavidin or Ni-NTA affinity beads, respectively. The captured phage DNA was liberated from beads by treatment with hexane and the released DNA was amplified by PCR ( Figure S4 ) and sequenced with Illumina deep sequencing ( Figure 2B, Table S3 ). A DE analysis identified a set of 85 sequences that were significantly enriched ( p 3-fold) in the screen against Bio-HSA when compared to the screen against His 6 -T4-GP and Bio-ConA ( Figure 3A–B, Figure S5 ). We applied a pairwise amino acid clustering to identify the 85 hit sequences ( Figure 3C ) and observed 8 motifs: FF, MF, MG, TK, GM, PV, VY and KR associated with these enriched sequences (Figure 3D). Based on this analysis, we nominated sequences SICRFFC ( 5a ), SFCPMFC ( 6a ) and SLCKREC ( 7a ) as hits and STCQGEC ( 8a ) as a negative control for chemical synthesis, and further validation ( Figure 3E ). Validation of albumin binders We observed non-specific reactivity of OFS -macrocyclic peptides with thiol nucleophiles such as glutathione (GSH) over several hours in basic pH ( Figure S6 ). Replacing DFS with a less reactive pentafluorophenyl sulfide ( Figure 1D ) abolished the undesired reactivity: The resulting perfluorophenylsulfide ( PFS )-macrocycles were unreactive to 2-mercaptoethanol over three weeks and unreactive towards free thiol on HSA ( Figure S7 ). Molecular dynamics simulation suggested the OFS -macrocycles and the PFS -macrocycles exhibit similar ground state conformational landscape ( Figure S8 ). Many perfluoro-aryl crosslinked macrocycles were poorly soluble in water, and we synthesized them with either a GGKKK or GGG tag at the C-terminus to increase their solubility; some sequences were synthesized with both tags to check whether these affect HSA binding. The C-terminal tags aided in providing sufficient solubility properties for downstream analyses ( Figure S9 ). The unique fluorine handle in perfluoro-aryl modified peptides made it possible to determine their binding to HSA using 19 F NMR ( Figure 4 ). In a typical experiment, we maintained peptide concentration at 50uM and HSA at 100uM ( Table S4 ). We observed broadening of and disappearance of 19 F signals that correspond to fluoroaromatic groups, which indicated the binding of the peptide to HSA ( Figure 4A , Figure S10 ). We could not fit a definitive K d value to the binding response due to the complex binding behaviour and quality of the NMR signal. However, in an albumin titration series, one can use qualitative estimates such as the concentration of albumin necessary to suppress 50% of the initial fluorine signal. Based on these qualitative analyses, it was apparent that some peptides (e.g., PFS -SICRFFCGGG) have stronger binding to HSA, whereas other macrocycles (e.g., PFS- STCQGECGGG) have weaker binding towards HSA ( Figure 4A , Figure S10 ). By measuring the decrease in the signal at a fixed concentration of peptide and HSA, we evaluated 8 sequences found in all discovery campaigns ( Figure 4B, S11 ) and we nominated PFS -SICRFFCGGG ( 5c ) as the “hit” and PFS -STCQGECGGG ( 8c ) as the negative control for further investigation. Peptides modified at the C-terminus with either GGKKK or GGG solubility tags have similar binding affinity ( Figure 4B, Figure S11 ). We titrated 5c and 8a against rat serum albumin and observed similar binding to rat and human albumin ( Figure S12 ). We attempted to confirm the binding affinity of these sequences by isothermal titration calorimetry (ITC) using SA-21 as a control; 37 however, a complex multi-site binding behaviour for all peptides obscured the accurate evaluation of binding affinity by ITC ( Figure S13–15 ). The 19 F NMR assay, thus, was critically enabling for validation and ranking of the albumin binding leads. A fluorescence polarization binding assay (FP) successfully measured the binding affinities of the macrocycles with the fluorophore BODIPY at the C- or N-terminus. In a typical experiment, we used PFS -SICRFFCGGG (5c) or PFS -SFCPMFCGGG (6c) at 1 µM concentration and titrated HSA from 0.1 µM to 100 µM. The dose-response curve could be fit to a single-state binding model with binding affinity of K d = 4 – 6 µM for 5c and at least 100 times weaker affinity for 6c ( Figure 5A and S16–S19 ). BODIPY alone bound weakly to HSA with > 300 µM binding affinity ( Figure 5A and S16–S19 ). The FP-assay made it possible to measure binding to other proteins or even complex mixtures (serum). A titration of the mouse serum ( Figure S18 ) yielded a similar binding profile to that observed in binding to pure albumin ( Figure 5A ). Replacing HSA with lysozyme and RNAse, the assay detected no binding response, confirming that 5c binding was specific to HSA ( Figure 5B ). Switching location of the fluorescent probe from the N-terminus to C-terminus did not significantly change the affinity of 5c ( K d = 4-6 µM, Figure S19 ). The switching from DFS to PFS also exhibited a minimal effect on the binding of peptides 5b and 5c ( Figure S20 ). The results from FP were in the same order of magnitude as the semi-qualitative estimates acquired for BODIPY-free peptides by the 19 F NMR binding assay, indicating that the presence of a fluorophore did not significantly increase the binding ( Figure S21 ). Heinis and co-workers recently observed that fluorophores could dramatically increase binding affinity for albumin, and removing the fluorophore is detrimental to the binding of the albumin binder. 21 To exclude this possibility, we conducted an NMR-binding assay of N-terminally-labelled PFS -SICRFFCGG and BODIPY-free peptides. We observed that the binding affinity was similar ( Figure S21 ). Elucidation of the binding pocket for perfluoro-macrocycles We evaluated whether binding pockets of 5c are similar to known albumin binders: carbamazepine, diclofenac and ibuprofen ( Figure 6A ). We observed that the binding of PFS -SICRFFCGGG ( 5c ) did not decrease in the presence of any of these drugs; thus, it does not share the same binding pocket as carbamazepine, diclofenac, or ibuprofen ( Figure 6B ). To follow on this observation, we performed a series of docking calculations to seek the most favorable binding locations of PFS -SICRFFCGGG ( 5c ) on the surface of HSA. Nine distinct sites on HSA were previously shown to bind to fatty acids, 45 some of which also bind to other ligands such as ibuprofen and diclofenac 46, 47 ( Figure S22 ). These nine reported fatty acid binding sites on five different initial HSA structures were selected for docking of 5c . Figure 7A shows the HSA protein with some of its bound fatty acids, based on the pdbID 1e7e 45 . Overlaid with this structure is 5c docked to the corresponding fatty acid binding sites on the HSA surface. Figure 7B shows the binding scores for 5c – HSA complexes across different HSA structures, based on the distinct pdbIDs and different binding site locations (complete results summarized in Figure S23 and Table S5 ). Consistently, 5c has the most favorable binding score in binding site 1, with the value of –8.95 ± 1.0 kcal/mol, averaged over all the docking calculations performed. Therefore, the results in Figure 7B suggest that the primary HSA binding site for 5c is binding site 1. The next most favorable binding sites are sites 8, 6, and 7, with the most favorable binding scores of –6.6 ± 1.1 kcal/mol, –6.2 ± 0.7 kcal/mol, and –6.0 ± 1.2 kcal/mol, respectively ( Figure 7 , Table S5 ). We observed binding sites 1 and 8 are near to each other on the HSA surface, with the center of mass distance between fatty acids occupying these sites being 5.3 Å. As such, it is unlikely that binding sites 1 and 8 can be simultaneously occupied by two 5c molecules. Figure 7A shows the four HSA residues that interact with the fatty acid in binding site 1 via charge and nonpolar interactions. In contrast, PFS -SICRFFCGGG ( 5c ) has more interactions with this HSA binding site, including the HSA residues R114, R117, Y138, Y161, I142, L154, S193. Notably, HSA residues R117, Y138, and Y161 in binding site 1 are found to mediate HSA interactions with both the fatty acid and 5c . Combined docking results ( Figure 7 ) and binding observation ( Figure 6 ) suggested that ibuprofen, diclofenac and 5c bind to different locations on the HSA surface. The structural studies 46 demonstrated that ibuprofen binds to the binding sites labeled by 3/4 and 6 (pdbID 2bxg, Figure S22 ), which are distant from the HSA binding site 1. Structure of HSA bound to diclofenac 48 (pdbID 4z69, Figure S22 ) contains two HSA chains in it. One of the HSA chains has a single diclofenac at the binding site 7, while the second HSA chain has three bound diclofenac ligands in total, with two also located at the binding site 7, and the third located near the binding site 1, which is also occupied by a bound fatty acid. The structure locations suggest that diclofenac has the strongest binding to binding site 7, since it is observed there in both HSA chains, and a weaker binding to binding site 1, as only one single HSA chain is observed with diclofenac nearby. Circulation lifetime of albumin peptides in mice To evaluate the half-life circulation of albumin-binding perfluoro-macrocycles, we injected a mixture of peptides PFS -SICRFFCGG ( 5c ), weak binding peptide PFS -STCQGECCGGG ( 8c ) as the negative control and SA-21 as the positive control into mice and monitored the remaining peptide level by LC–MS ( Figure 8A , Figure S24 ). We observed that the negative control 8c disappeared below the limit of detection after 5 min ( Figure 8B ). The concentration of 5c decreased 10-fold and SA-21 concentration decreased 5-fold after 2 hours. The combined results confirm a significant retention of PFS -SICRFFCGG peptide in circulation when compared to unrelated macrocyclic peptides with minimal to no detectable binding to HSA. The single digit micromolar peptide does not rival the mid-nanomolar SA-21 peptide, and the observed differences in half-life likely reflect the relative affinities for albumin. The PFS -SICRFFCGG peptide, thus, provides an attractive minimalistic starting point for further attenuation of binding affinity for albumin and subsequent attenuation of circulation half-life. Conclusion Late-stage modification of peptides and genetically-encoded (GE) libraries of peptides by cross-linkers (linchpins) is one of the common approaches to incorporate beneficial attributes to their properties. 49 Alkylation of cysteine residues in peptides via an S N 2 reaction using bi- or tri-dentate alkyl halides has been use for cyclization of peptides, incorporation of unnatural fragments into the resulting macrocycles, 50 – 52 and late-stage modification of phage- and mRNA-displayed libraries to yield billion-scale GE libraries. Peptide cyclization via S N Ar reaction with perfluoroarenes popularized by the Pentelute group forms alkyl-aryl thioethers; 43 other classes reactions have been developed to form aryl 53 – 55 , alkenyl and alkynyl thioethers 56 in unprotected peptides. Aryl and perfluoroaryl thioethers are more resistant toward oxidation when compared to traditional bis-alkyl thioesters 42 . Decreased conformational mobility or aryl-thioether bond has been proposed to equip the resulting macrocycles with favourable properties such as cell permeability and proteolytic stability. 45 , 57 Our report described the first selection from perfluoro-aryl macrocyclic GE libraries. There exists only one example of GE selection from S N Ar-modified phage libraries: Lu and co-workers recently employed 2,4-Difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB) as a reagent that can modify phage libraries in water. 58 Chen and co-workers also used Pd-catalyzed S N Ar reaction to yield DNA-encoded libraries. 59 Both S N Ar reaction yield macrocycles do not contain any fluorine atoms. On the other hand, fluorine handles present in perfluoroaryl-crosslinked macrocycles offer a unique possibility to use of 19 F NMR to measure protein-macrocycle interactions. Interaction of perfluorinated aryls with proteins is also electronically distinct from non-fluorinated aromatic residues and in some cases it can offer uniquely advantageous interactions 60 . An important observation in selection of GE OFS -macrocycle libraries is mild reactivity of these structures towards thiol nucleophiles. 61 Libraries of mild electrophiles 62 – 64 and phage-displayed libraries with built-in electrophiles 65 have emerged as important starting point for discovery of covalent and reversibly covalent inhibitors. While we do not show it in our report, it is possible that an attenuated reactivity of OFS -macrocycles towards thiols can be used as an advantageous features in discovery or inhibitors that form covalent bonds with thiol residues in proteins. If reactivity of the selected macrocycles is not desired, one can perform late-stage replacement of OFS moiety in the identified hits with nearly isosteric perfluorophenyl-sulfide. The PFS linchpin is not sufficiently reactive for direct modification of phage-displayed libraries in water, but replacement of DFS linchpin by PFS “post discovery” maintains the conformation and binding affinity of the discovered macrocycles while alleviating their undesired electrophilicity. Our report, thus, suggest a general approach for the future utility of perfluoroaryl-modified libraries: Step 1: Select phage-displayed libraries of OFS -macrocycles against the desired target. Step 2: evaluate PFS -modified synthetic macrocycles for their ability to bind to these targets. Human serum albumin (HSA) target used in this publication is a commonly employed model target in screen of phage-displayed or DNA-encoded libraries (DEL) and traditional high-throughput screening (HTS). Albumin is a complex multi-pocket receptor with regions that can bind to fatty acid-like moieties, dicarboxylic acids as well a wide variety of aromatic and heterocyclic compounds and large dye molecules. 46 , 66 Albumin also contains several binding sites for peptides as well as small proteins that have been utilized for half-life extension strategies. 21 , 67 Peptide macrocycles discovered in this report add to a diverse set of known albumin binders and they constitute the first example of small macrocyclic peptide binders for albumin. We note that single digit micromolar affinity of the discovered PFS -SICRFFC motif was not sufficient to retain this macrocycle in circulation as effectively as benchmark SA-21 albumin binding peptide. However, it should be relatively straightforward to optimize this structure to improve the affinity because our docking calculations suggest that a C-terminal extension to this scaffold might be productive avenue for the future optimization. Exploration of such extensions could be done using perfluoro-aryl modified phage-displayed libraries of SICRFFCX n peptides with several randomized C-terminal amino acids. Such optimization should yield a collection of albumin binders with a range of affinities for albumin and, in turn, a range of the circulation half-lives. The small size of such peptide-macrocycle families makes it trivial to make them by solid-phase synthesis or incorporate them a part of another sequence produced by solid phase synthesis. More importantly, the SICRFFC motif or optimized SICRFFCX N motifs emerging from phage display screen can be easily re-introduced into phage-displayed libraries to serve as a constant N-terminal albumin binding motif and giving rise to libraries with predictable circulation half-life. Declarations Supporting information Supporting information document contains Supporting Figures S1–S62, Tables S1–S5, synthetic methods and characterization of compounds, details of phage display selection, next generation sequencing and bioinformatics analysis and all biochemical assays. Supporting data folder contains PDB files produced by docking. Acknowledgements This research was supported by a research contract from the Ferring Research Institute, Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2016-402511 to R. D.), and NSERC Accelerator Supplement (to R. D.). This work was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM124160 (PI: Y.-S.L.) Infrastructure support was provided by CFI New Leader Opportunity (to R. D.). We thank Dr. Randy Whittal for assistance with LCMS, and Mark Miskolzie for assistance with 19 F NMR kinetics. Methods Preparation of SXCX 3 C phage-displayed library The procedures have been adopted and modified as previously described in two publications that produced the M13-displayed SXCXXXC library 44 and M13-SDB vector 68 . In short, the vector SB4 QFT*LHQ was digested with Kpn I HF (NEB cat# R3142S) and Eag I HF (NEB cat# R3505S). A primer/template pair consisting of primer 5’-AT GGC GCC CGG CCG AAC CTC CAC C-3’ and template 5’-CC CGG GTA CCT TTC TAT TCT CAC TCT TCT X TGT XXX TGT GGT GGA GGT TCG GCC GGG CGC TTG ATT-3’ with ‘X’ representing a trinucleotide formed by annealing. The primer/template was then extended using Klenow DNA polymerase (NEB) according to the manufacturer’s instructions. The insert fragment was then digested with Kpn1 HF and Eag1 HF, gel purified, and ligated into the cut vector. The ligation products were then transformed into electrocompetent E. coli cells, and the transformants were grown overnight on E. coli TG1 to allow for phage production. Phage cultures were then centrifuged to remove cells and debris, and then the phage was precipitated by PEG precipitation (5% PEG 0.5 M NaCl). Other SDB vectors have been processed identically. We sequenced the naïve libraries by Illumina sequencing, and the naïve library of SXCX n C ( n =3-5) composition is publicly available at the following link: https://48hd.cloud/file/1470 . SXCX 4 C and SXCX 5 C libraries were prepared as described in previously reported protocols. 69 Panning campaigns Three panning campaigns were conducted to discover binders for Human Serum Albumin (HSA) with protein A, ConA or T4-Gp as the negative controls. The proteins were immobilized on polystyrene plates or magnetic beads and panned against OFS -macrocyclic libraries or unmodified libraries. The details on panning experiments are discussed in supplementary information section. All the analyses were performed by next generation sequencing (NGS) of phage DNA as previously reported. 50, 52, 70 Preparation of Illumina sequencing samples Similar to previous reports, 50, 52, 70 phage eluted from the target was subjected to PCR amplification (see SI for PCR protocols) to append Illumina multiplexing barcodes, and sequencing adapters to randomized library regions. All PCR products were quantified by 2% (w/v) agarose gel in Tris-Borate-EDTA buffer at 100 volts for ~35 min using a low molecular weight DNA ladder as standard (NEB, cat# N3233S). PCR products that contain different indexing barcodes were pooled, allowing 10 ng of each product in the mixture. The mixture was purified by eGel, quantified by quBit and sequenced using the Illumina NextSeq paired-end 500/550 High Output Kit v2.5 (2×75 Cycles). Data were automatically uploaded to BaseSpace™ Sequence Hub. Processing of Illumina data The Gzip compressed FASTQ files were downloaded from BaseSpace™ Sequence Hub. The files were converted into tables of DNA sequences and their counts per experiment. Briefly, FASTQ files were parsed based on unique multiplexing barcodes within the reads discarding any reads that contained a low-quality score. Mapping the forward (F) and reverse (R) barcoding regions, mapping of F and R priming regions allowing no more than one base substitution each and F-R read alignment allowing no mismatches between F and R reads yielded DNA sequences located between the priming regions as described in previous publications. 70 The files with DNA reads, raw counts, and mapped peptide modifications were uploaded to http://48hd.cloud/ server. Each experiment has a unique alphanumeric name and unique static URL in Tables S1-3. General protocol for cyclization with decafluorodiphenylsulfone Procedure was analogous to previously published methods 42, 43 . In short, linear peptide (10 mM) was dissolved in 50% acetonitrile and Tris buffer (50 mM Tris-HCl, pH 8.5), then 2 equivalents of DFS in 50% acetonitrile and Tris buffer (50 mM Tris-HCl, pH 8.5) was added to the mixture. The mixture was vortexed for 30 sec, incubated for 2 h at room temperature, purified by HPLC and further lyophilized to yield product. General protocol for cyclization with pentaflurophenyl-sulfide Procedure was analogous to previously published methods 42, 43 . In short, linear peptide (10 mM) was dissolved in 50 mM Tris in DMF, then 2 equivalents of PFS was added to the mixture. The mixture was vortexed for 30 sec and allow to react for 1 hour at RT. The reaction mixture was purified by HPLC and lyophilized to yield the product. In vivo pharmacokinetic experiment All the procedures and experiments involving animals were carried out using a protocol approved by the Health Sciences Laboratory Animal Services (HSLAS), University of Alberta. The protocol was approved as per the Canadian Council on Animal Care (CCAC) guidelines. All mice were maintained in pathogen-free conditions at the University of Alberta breeding facility. Peptide mixtures of 100 µM were prepared in PBS. Mice were administered with 200 µL of the peptide mixture solution with tail veil injection. A series of 6 blood samples were collected at time points from 2 min up to 240 min. Samples were collected in tubes that contained sodium citrate as an anticoagulant and then centrifuged at 5 min at 2,000×g to collect the blood plasma. 10 µL of plasma portion were transferred into a tube containing 40 µL of 8:2 acetonitrile/water to precipitate proteins. The samples were centrifuged at max speed for 10 min at 4 o C. Supernatants were then transferred to new tubes and subjected to analysis by LC-MS. References Muttenthaler, M.; King, G. F.; Adams, D. J.; Alewood, P. F., Trends in peptide drug discovery. Nat. Rev. Drug Discov.2021, 20 (4), 309–325. Jain, R. 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Sojitra, M.; Sarkar, S.; Maghera, J.; Rodrigues, E.; Carpenter, E. J.; Seth, S.; Ferrer Vinals, D.; Bennett, N. J.; Reddy, R.; Khalil, A.; Xue, X.; Bell, M. R.; Zheng, R. B.; Zhang, P.; Nycholat, C.; Bailey, J. J.; Ling, C. C.; Lowary, T. L.; Paulson, J. C.; Macauley, M. S.; Derda, R., Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage. Nat. Chem. Biol. 2021, 17 (7), 806–816. Additional Declarations There is NO Competing Interest. Supplementary Files DerdaSI.docx Supplementary Information Q12RS.pdf Reporting Summary floatimage1.jpeg Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 13 Sep, 2023 Read the published version in Nature Communications → Version 1 posted 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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Derda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLCCBAYbBgZ2HqLVM4O0pAFpHrBmIrUwMBwmQYt5+/mjGx7uOZ/Y38x78HHlDzsG/vYD+LXInElmu5Hw7HbijMN8yYZnEpIZJM4QsEqCAaTlwO3EhsM8ZpINCcwMBoRcJ8H/GKTlXOL8wzzmPxsS6hkM+B8Q0CIBtuVA4gagLYwNCYcZDCQI2SLx2AyoJdl4I9Avkg1px3kkbhCyhT/x2c0fB+xk5x3vPfixwaZajr+fgC0YgPg0MApGwSgYBaMANwAAzYtDthK0mOgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1365-6570","institution":"University of Alberta","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ratmir","middleName":"","lastName":"Derda","suffix":""}],"badges":[],"createdAt":"2022-08-25 20:45:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1999287/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1999287/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-023-41427-y","type":"published","date":"2023-09-13T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":26188750,"identity":"9313ec3e-30dd-483d-bc68-1b7eb01e8415","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":647566,"visible":true,"origin":"","legend":"\u003cp\u003eDiscovery of albumin binding peptides.\u003c/p\u003e\n\u003cp\u003ePrevious reports of (A) macrocyclic peptide: DX-263,36 (B) macrocyclic peptide: SA-21,37 (C) a linear peptide: FITC-EYEYKpalmESE-NH2.17 (D) This report describes a chemically modified phage-displayed library for discovery of a small macrocyclic albumin binder.\u003c/p\u003e","description":"","filename":"DerdaFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/55df30da9daeeefd99bf7d87.jpg"},{"id":26189398,"identity":"c0eb6080-25f6-4122-a91c-1a3943532928","added_by":"auto","created_at":"2022-09-07 17:01:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":377127,"visible":true,"origin":"","legend":"\u003cp\u003ePhage-displayed peptide modified by \u003cstrong\u003eDFS\u003c/strong\u003e. (A) The modified phage-displayed library panned against two targets (biotinylated HSA and His-tag expressed T4-GP) in solution and captured separately with avidin beads and Ni-NTA beads affinity beads. (B) In the negative control, phage-displayed library modified by \u003cstrong\u003eDFS\u003c/strong\u003e was panned against biotinylated ConA and captured with avidin beads.\u003c/p\u003e","description":"","filename":"DerdaFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/1ac650c69a08ed4fb41569cf.jpg"},{"id":26189399,"identity":"05f91a9f-8fb0-4712-b519-c926750466fd","added_by":"auto","created_at":"2022-09-07 17:01:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":673500,"visible":true,"origin":"","legend":"\u003cp\u003eStudent’s \u003cem\u003et\u003c/em\u003e-test analysis of the third screening campaign\u003c/p\u003e\n\u003cp\u003e: (A, B) A volcano plot and venn diagram visualizing the sequences from the \u003cstrong\u003eOFS\u003c/strong\u003e-SXCX3C phage-displayed library that were significantly enriched in the HSA screen when compared to the naïve library or selection against T4-GP, ConA. (C) A heat map display of the top 25 of 85 hits sequences from differential enrichment results. (D) Dipeptide motif analysis of all 85 hits. (E) Selected sequences for chemical synthesis of macrocycles for validation.\u003c/p\u003e","description":"","filename":"DerdaFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/cbe31ec95c3a11b2741483ce.jpg"},{"id":26188752,"identity":"a16b939f-cbe4-4be7-80f8-28d3e31047b2","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":276799,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the 19F NMR binding measurement. (A) HSA\u003c/p\u003e\n\u003cp\u003etitration spectra of 50 µM of \u003cstrong\u003e1b\u003c/strong\u003e-\u003cstrong\u003e8b\u003c/strong\u003eand \u003cstrong\u003e1c\u003c/strong\u003e-\u003cstrong\u003e8c \u003c/strong\u003eagainst 100 µM of HSA. (B) The percentage represents the remaining peak intensity following the addition of HSA.\u003c/p\u003e","description":"","filename":"DerdaFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/a3d9cffdb2e9846228750a6e.jpg"},{"id":26188757,"identity":"66c8bf8d-84aa-4d04-ae52-ba19f430c7b0","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212521,"visible":true,"origin":"","legend":"\u003cp\u003eBinding assay data\u003c/p\u003e\n\u003cp\u003e: (A) FP assay measured the \u003cem\u003eK\u003c/em\u003ed of the \u003cstrong\u003e5c\u003c/strong\u003e and \u003cstrong\u003e8c\u003c/strong\u003e against various albumins. (B) The FP assay for BODIPY labelled \u003cstrong\u003e5c\u003c/strong\u003e titrated against HSA (black), lysozyme (red), and RNAse A (blue).\u003c/p\u003e","description":"","filename":"DerdaFigure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/035c0123180221101e648762.jpg"},{"id":26189401,"identity":"39902eb1-e18d-4feb-9812-168c77fb1f60","added_by":"auto","created_at":"2022-09-07 17:01:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":483147,"visible":true,"origin":"","legend":"\u003cp\u003eThe 19F NMR HSA binding and competitive binding assay comparison\u003c/p\u003e\n\u003cp\u003e. (A) X-ray crystal structure of diclofenac bound HSA (pbd: 4Z69) and ibuprofen bound HSA (pbd: 2BXG). (B) 19F NMR competitive inhibition assay with \u003cstrong\u003e5c \u003c/strong\u003eagainst carbamazepine, diclofenac and ibuprofen.\u003c/p\u003e","description":"","filename":"DerdaFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/b2e24566058999dc254125e1.jpg"},{"id":26188761,"identity":"ecaac205-a661-4311-be52-805b0f9bf2e1","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":863124,"visible":true,"origin":"","legend":"\u003cp\u003eDocking calculations for \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG (\u003cstrong\u003e5c\u003c/strong\u003e) and HSA protein. (A) HSA protein structure with bound fatty acids (crystal structure-based), overlaid with \u003cstrong\u003e5c\u003c/strong\u003e docked to the respective fatty acid binding sites on the HSA surface. Binding site 1 is circled in red. The bottom left inset shows a fatty acid bound to the binding site 1 in the HSA crystal structure (pdbID 1e7e). The HSA amino acids with direct contact to the fatty acid are highlighted in thin licorice representation. Amino acids shown in blue form hydrogen bonds with the fatty acid, and amino acids shown in red form van der Waals (nonpolar) interactions with the fatty acid. The top inset shows \u003cstrong\u003e5c\u003c/strong\u003e docked in binding site 1. The HSA amino acids with direct contact to \u003cstrong\u003e5c\u003c/strong\u003e are highlighted in thin licorice representation. The bottom right inset shows a magnified \u003cstrong\u003e5c \u003c/strong\u003edocked into the binding site 1 pocket of HSA (grey surface). (B) Plots of \u003cstrong\u003e5c\u003c/strong\u003e–HSA binding scores obtained in one set of docking calculations. Separate docking calculations were performed for different HSA structures extracted from the PDB Databank files with PDB IDs: 1e7e, 1e7f, 1e7g, 1e7h, and 1e7i. \u003cstrong\u003e5c \u003c/strong\u003ewas docked to the binding sites on the HSA surface that were occupied by the fatty acids in the crystal structures.45 Six of the nine HSA binding sites, the corresponding bound fatty acids, and docked \u003cstrong\u003e5c\u003c/strong\u003e are shown in panel A.\u003c/p\u003e","description":"","filename":"DerdaFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/5e69ccec0633b4f43b2b9548.jpg"},{"id":26188758,"identity":"87506c59-35ad-4601-aa16-a6a23211b702","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":169962,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetic studies for \u003cstrong\u003e5c\u003c/strong\u003e, \u003cstrong\u003e8c\u003c/strong\u003e and SA-21. (A) An equimolar (0.1 mM) mixture of macrocycles was injected into mice and blood was drawn at various time points. (B) Blood samples were collected at time points 2, 5, 30, 60, 120, and 240 mins and analyzed by LC-MS (\u003cem\u003en\u003c/em\u003e = 3). The dotted horizontal line represents the limit of detection. (n.d.: not detected).\u003c/p\u003e","description":"","filename":"DerdaFigure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/3b9dc93cd5a8bdbe63931c87.jpg"},{"id":43886676,"identity":"54b5e21f-cad1-43f9-9daf-a3e18de34663","added_by":"auto","created_at":"2023-09-29 16:01:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1394911,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/ddd9cdee-ce83-4723-9fbc-b45d64f6fe27.pdf"},{"id":26188755,"identity":"b15db134-354d-46c7-a20f-ebdbe9ea26ca","added_by":"auto","created_at":"2022-09-07 16:56:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13259808,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"DerdaSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/b77734bed3f0036f1dc41535.docx"},{"id":26190019,"identity":"e876d862-bf1a-4af6-989a-d36bc1c81516","added_by":"auto","created_at":"2022-09-07 17:06:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1771612,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"Q12RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/04eae8263bcde0dbca93304a.pdf"},{"id":26190020,"identity":"d7ad7606-7523-4889-b4a0-f3900eb12633","added_by":"auto","created_at":"2022-09-07 17:06:32","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":79138,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1999287/v1/cf5dc24ebda07e04fd6d657b.jpeg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eGenetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation\u003cem\u003e in-vivo\u003c/em\u003e.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThere are around 80 peptide drugs on the global market; more than 150 peptides are in clinical development and another 400\u0026ndash;600 peptides undergoing preclinical studies.\u003csup\u003e1\u003c/sup\u003e The large surface area of peptides, compared to a typical small molecule drugs allows peptides to interact with expanded binding interfaces commonly found in protein\u0026ndash;protein interactions, protein\u0026ndash;carbohydrate and protein\u0026ndash;DNA interactions. These proteins, classified as \u0026ldquo;undruggable targets\u0026rdquo;, have been difficult to target using conventional small molecule therapeutic but many of them have been addressed by peptide, proteins, or antibody therapeutics. Peptides are the smallest among the latter three modalities\u0026mdash;2 kDa to 10 kDa for peptides versus 150 kDa for full-sized antibodies\u0026mdash;and they possess distinct pharmacokinetic (PK) properties. For example, bio-distribution of peptides and small proteins inside tumors and other non-vascularized tissues is improved when compared to full-size antibodies. Several clinical candidates (TH1902, TH1904, BT5528, BT8009, BT1718, MMP-14) capitalize on such improved biodistribution.\u003csup\u003e2-4\u003c/sup\u003e Unlike antibodies, which remain in circulation for 1\u0026ndash;3 weeks due to the association with the neonatal Fc-receptor (FcRn) on the surface of immune cells,\u003csup\u003e5\u003c/sup\u003e peptide therapeutics clear within minutes to hours from plasma by renal filtration. Fast clearance is a beneficial property in several therapeutic applications, such as imaging (e.g., \u0026ldquo;tumor paint\u0026rdquo;), radionuclide delivery (e.g., Lurathera\u003csup\u003eTM\u003c/sup\u003e),\u003csup\u003e6\u003c/sup\u003e and in administration of short acting peptide hormones. For more widespread adaptation of peptide modalities in diverse therapeutic applications it is desired to tune the circulation life-time of peptides from minutes to hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOnly few peptides exhibit a natural extended circulation lifetime: a therapeutically relevant example is a natural venom, 39-residue peptide \u0026lsquo;exendin 4\u0026rsquo;, with low renal clearance in humans (5\u0026ndash;7\u0026thinsp;h).\u003csup\u003e7\u003c/sup\u003e This peptide gave rise to FDA-approved drug exenatide for the treatment of type 2 diabetes.\u003csup\u003e8, 9\u003c/sup\u003e Despite favourable circulation half-life, modified derivatives of exenatide\u0026mdash;liraglutide, albiglutide, dulaglutide, lixisenatide and semaglutide\u003csup\u003e1\u003c/sup\u003e\u0026mdash; have been developed to tune circulation half-life and other PK properties. The majority of peptides and small proteins have to be modified as well to increase their circulation time. Such modification could be divided into several classes: Class 1: increase in size via covalent linkage to polyethylene glycol (PEG),\u003csup\u003e10, 11\u003c/sup\u003e polyglycerol\u003csup\u003e12\u003c/sup\u003e and other synthetic macromolecules. Interestingly, steric hindrance by these size-increasing moieties also protects against proteolytic degradation.\u003csup\u003e13-15\u003c/sup\u003e; Class 2: increase in size via controlled oligomerization;\u003csup\u003e16\u003c/sup\u003e Class 3: covalent linking to long-living serum protein (e.g., FDA-approved drugs albiglutide and dulaglutide, exanatide that conjugated to albumin and the IfG4 Fc domain)\u003csup\u003e17-19\u003c/sup\u003e and Class 4: incorporation of moieties that bind non-covalently to serum proteins such as albumin,\u003csup\u003e20-23\u003c/sup\u003e immunoglobulin,\u003csup\u003e24, 25\u003c/sup\u003e FcRn,\u003csup\u003e26\u003c/sup\u003e transthyretin,\u003csup\u003e27\u003c/sup\u003e and transferrin.\u003csup\u003e28, 29\u003c/sup\u003e An important example in the last class is lipidation of peptides to allow interaction with serum albumin. Lipidation has been one of the most successful strategies to prolong the half-life of peptides and small proteins such as insulin giving rise to FDA-approved drugs such as Levemir\u0026reg;, Tresiba\u0026reg;, Victoza\u0026reg;, Saxenda\u0026reg;, and Ozempic\u0026reg; with extended serum half-life\u003csup\u003e30, 31\u003c/sup\u003e. The improved properties of these and many other drugs stemming from their association with albumin mandate investigation of albumin as carrier for therapeutic applications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlbumin is the most abundant protein in plasma with an average concentration of 600 \u0026micro;M and has\u0026nbsp;an\u0026nbsp;average half-life of 19 days.\u003csup\u003e32\u003c/sup\u003e The main mechanism leading to the long half-life of albumin and antibody are similar: both proteins interact with FcRn on the surface of immune cells. This binding results in transient endocytosis of these proteins, and as a result, they are frequently sequestered from circulation and protected from clearance. At physiological pH, the binding affinity between albumin and FcRn is low; however, the interaction under acidic conditions in the endosome is strong to avoid lysosomal degradations and recycling of albumin to the extracellular space.\u003csup\u003e5\u003c/sup\u003e Albumin acts as a versatile carrier of essential fatty acids and diverse small organic molecules.\u003csup\u003e32\u003c/sup\u003e Among all the long-circulating serum proteins,\u0026nbsp;albumin is considered to be one of the most important targets because of its ability to interact with hydrophobic small molecule drugs and enhance their pharmacokinetic properties.\u0026nbsp;Recurrent therapeutic success of rationally lipidated peptides and proteins\u003csup\u003e33\u003c/sup\u003e fuels interest in rational development of small molecules as well as non-lipidated proteins and peptides that bind to albumin.\u003c/p\u003e\n\u003cp\u003eMany FDA-approved small molecule drugs have an intrinsic affinity for human serum albumin (HSA). Targeted development of small molecules with high affinity for HSA has been a topic of research over the last 15 years (see recent review\u003csup\u003e23\u003c/sup\u003e). Anti-HSA antibodies, nanobodies,\u003csup\u003e34\u003c/sup\u003e DARPins,\u003csup\u003e35\u003c/sup\u003e and other protein domains have been also developed. Such proteins can be fused to therapeutic proteins of interest to extend their \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003ecirculation. Similarly short peptides that bind to HSA could be used in tandem with therapeutic peptide or protein sequences to dial in predictable half-life for such therapeutics. Such short albumin-binding peptides could empower development of many future therapeutic peptides because they could be built into \u003cem\u003eany\u003c/em\u003e genetically encoded peptide library (e.g., displayed on phage, RNA and other platforms) to give rise to billion-scale libraries with predictable \u003cem\u003ein vivo\u003c/em\u003e half-life. However, short HSA-binding peptides are scarce.\u0026nbsp;A 31-mer peptide\u0026nbsp;DX-236 (Ac-AEGTGDFWFCDRIAWYPQHLCEFLDPEGGGK-NH\u003csub\u003e2\u003c/sub\u003e) with a binding affinity of 1.9 \u0026micro;M was identified by Dyax Corp., and used to purify albumin (\u003cstrong\u003eFigure 1A\u003c/strong\u003e).\u003csup\u003e36\u003c/sup\u003e A 21-mer peptide SA-21 (Ac-RLIEDICLPRWGCLWEDD-NH\u003csub\u003e2\u003c/sub\u003e) with a binding affinity of 467 nM to HSA was identified at Genentech (\u003cstrong\u003eFigure 1B\u003c/strong\u003e)\u003csup\u003e37\u003c/sup\u003e and subsequently conjugated to ligands\u0026nbsp;for urokinase-type plasminogen activator,\u003csup\u003e20, 38\u003c/sup\u003e Fab antibody fragments\u003csup\u003e39, 40\u003c/sup\u003e and small proteins\u003csup\u003e41\u003c/sup\u003e to prolong their circulation half-lives.\u0026nbsp;Heinis and co-workers developed a short heptapeptide modified by fluorescein isothiocyanide (FITC) and palmitic acid (FITC-EYEYK\u003csub\u003epalm\u003c/sub\u003eESE-NH\u003csub\u003e2\u003c/sub\u003e) with a binding affinity of 39 nM to HSA (\u003cstrong\u003eFigure 1C\u003c/strong\u003e)\u003csup\u003e21\u003c/sup\u003e and the presence of both lipid moiety and fluorescein was critical for the binding of this peptide. This FITC-lipopeptide was fused to two different bicyclic peptides to boost the half-lives from minutes to hours.\u003csup\u003e21\u003c/sup\u003e Success of DX-236, SA-21 and FITC-lipopeptide and other examples from the literature demonstrated the possibility of using HSA as a target for genetically-encoded selection to identify HSA-binding peptides with extended circulation half-life. There is a need for development of other albumin binding peptide modalities that have lower molecular weight.\u003csup\u003e21, 37\u003c/sup\u003e Towards this goal, we employ genetically encoded phage-displayed libraries of chemically modified macrocycles to develop new classes of albumin binding mini scaffolds. To hone on shortest possible peptide sequences, we employed a phage-displayed libraries SXCX\u003cem\u003e\u003csub\u003en\u003c/sub\u003e\u003c/em\u003eC, \u003cem\u003en\u003c/em\u003e=3\u0026ndash;5 modified with decafluorodiphenyl sulfone (\u003cstrong\u003eDFS\u003c/strong\u003e)\u003csup\u003e42, 43\u003c/sup\u003e where X is any amino acid except for cysteine (\u003cstrong\u003eFigure 1D\u003c/strong\u003e). We hypothesized that a perfluoroaromatic linchpin would serve as useful pharmacophore recognized by one of the binding sites of HSA similarly to the binding of fatty acid in lipidated peptides.\u0026nbsp;\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003ch2\u003eSelection of albumin binders\u003c/h2\u003e\n\u003cp\u003eWe devised and conducted three discovery campaigns that used different library architecture and selection strategies. In the first discovery campaign, we modified the phage libraries of structure SXCX\u003csub\u003e4\u0026ndash;5\u003c/sub\u003eC with \u003cstrong\u003eDFS\u003c/strong\u003e following a previously published protocol and confirmed that 85% of the phage library is modified to yield octafluoro-diphenylsulfone-crosslinked macrocycles (\u003cstrong\u003eOFS\u003c/strong\u003e-SXCX\u003csub\u003e4\u0026ndash;5\u003c/sub\u003eC-phage) (\u003cstrong\u003eFigure 2A\u003c/strong\u003e, \u003cstrong\u003eFigure S1A\u003c/strong\u003e).\u003csup\u003e42\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe performed three rounds of phage selection using HSA coated to the surface of 96 well polystyrene plates as bait. In parallel, we screened the same library on polystyrene wells coated with Protein A (negative control) to distinguish specific HSA-binding sequences from poly-specific protein binding sequences (\u003cstrong\u003eFigure S1A\u003c/strong\u003e). In round 3, the phage recovery of the \u003cstrong\u003eOFS\u003c/strong\u003e-macrocycle library selection against HSA two-fold increase compared to round 1 and round 2 but only a minor increase compared to selection against unrelated protein (\u003cstrong\u003eFigure S1D\u003c/strong\u003e). The recovery of unmodified round 3-library panned against HSA was 17-fold lower than the recovery of the \u003cstrong\u003eOFS\u003c/strong\u003e-macrocycle library, indicating that the \u003cstrong\u003eOFS\u003c/strong\u003e linchpin contributes to protein binding (\u003cstrong\u003eFigure S1D\u003c/strong\u003e). Differential enrichment (DE) analysis of the next-generation sequencing (NGS) of all test and control experiments (Table S1) identified several families of peptide macrocycles that had statistically significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) enrichment in binding to HSA when compared to binding to unrelated protein (\u003cstrong\u003eFigure S1 B\u0026ndash;C\u003c/strong\u003e). The analysis yielded three consensus motifs: STCHDITC (\u003cstrong\u003e1a\u003c/strong\u003e), STCHYIGC (\u003cstrong\u003e2a\u003c/strong\u003e) and STCHANC (\u003cstrong\u003e3a\u003c/strong\u003e) (\u003cstrong\u003eFigure S1E\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe second discovery campaign employed HSA immobilized on a 96 well plate in rounds 1 and 3, and biotinylated HSA as bait immobilized onto streptavidin beads in round 2 (\u003cstrong\u003eFigure S2A\u003c/strong\u003e). In round 3, the phage recovery of the \u003cstrong\u003eOFS\u003c/strong\u003e-macrocycle library selection against HSA increased by a factor of 200 when compared to round 1 and round 2. The recovery of the unmodified library panned against HSA was insignificant (\u003cstrong\u003eFigure S2D\u003c/strong\u003e). The binding of the \u003cstrong\u003eOFS\u003c/strong\u003e-macrocycle phage library recovered from round 3 to Protein A, ConA and Casein was 2, 14, and 300-fold lower respectively when compared to recovery on HSA-coated wells (\u003cstrong\u003eFigure S3\u003c/strong\u003e). These observations suggested that (i) specific albumin-binding sequences had been selected, and (ii) the binding of these sequences to albumin required presence of \u003cstrong\u003eOFS\u003c/strong\u003e linchpin (\u003cstrong\u003eFigure S3\u003c/strong\u003e). A DE analysis of NGS data (\u003cstrong\u003eFigure S4, Table S2\u003c/strong\u003e) identified sequences that were significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) enriched in the screen against HSA but not control proteins. The LOGO analysis yielded a consensus motif: STCHTIYC (\u003cstrong\u003e4a\u003c/strong\u003e) (\u003cstrong\u003eFigure S2E\u003c/strong\u003e). Although the original libraries were designed as SXCX\u003csub\u003en\u003c/sub\u003eC where n=4 and 5, they contained a small fraction of SXCX\u003csub\u003e3\u003c/sub\u003eC sequences,\u003csup\u003e44\u003c/sup\u003e and we observed the enrichment of such sequences in the selection. To explore the apparent preference for smaller macrocycles, we devised a third selection campaign that employed only SXCX\u003csub\u003e3\u003c/sub\u003eC libraries modified with \u003cstrong\u003eDFS\u003c/strong\u003e (\u003cstrong\u003eFigure 2A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe small diversity of the library made it possible to employ a single round of panning and NGS-DE analysis and to identify the binders. To mimic the complex serum environment, the panning was conducted using a mixture of biotinylated HSA (Bio-HSA), His-tag fusion T4-PG protein (His\u003csub\u003e6\u003c/sub\u003e-T4-PG) and unlabelled milk proteins as bait. In a control selection, we used the same mixture with biotinylated ConA (Bio-ConA) in place of Bio-HSA (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). Proteins were captured with streptavidin or Ni-NTA affinity beads, respectively. The captured phage DNA was liberated from beads by treatment with hexane and the released DNA was amplified by PCR (\u003cstrong\u003eFigure S4\u003c/strong\u003e) and sequenced with Illumina deep sequencing (\u003cstrong\u003eFigure 2B, Table S3\u003c/strong\u003e). A DE analysis identified a set of 85 sequences that were significantly enriched (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u0026gt;3-fold) in the screen against Bio-HSA when compared to the screen against His\u003csub\u003e6\u003c/sub\u003e-T4-GP and Bio-ConA (\u003cstrong\u003eFigure 3A\u0026ndash;B, Figure S5\u003c/strong\u003e). We applied a pairwise amino acid clustering to identify the 85 hit sequences (\u003cstrong\u003eFigure 3C\u003c/strong\u003e) and observed 8 motifs: FF, MF, MG, TK, GM, PV, VY and KR associated with these enriched sequences (Figure 3D). Based on this analysis, we nominated sequences SICRFFC (\u003cstrong\u003e5a\u003c/strong\u003e), SFCPMFC (\u003cstrong\u003e6a\u003c/strong\u003e) and SLCKREC (\u003cstrong\u003e7a\u003c/strong\u003e) as hits and STCQGEC (\u003cstrong\u003e8a\u003c/strong\u003e) as a negative control for chemical synthesis, and further validation (\u003cstrong\u003eFigure 3E\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eValidation of albumin binders\u003c/h2\u003e\n\u003cp\u003eWe observed non-specific reactivity of \u003cstrong\u003eOFS\u003c/strong\u003e-macrocyclic peptides with thiol nucleophiles such as glutathione (GSH) over several hours in basic pH (\u003cstrong\u003eFigure S6\u003c/strong\u003e). Replacing \u003cstrong\u003eDFS\u003c/strong\u003e with a less reactive pentafluorophenyl sulfide (\u003cstrong\u003eFigure 1D\u003c/strong\u003e) abolished the undesired reactivity: The resulting perfluorophenylsulfide (\u003cstrong\u003ePFS\u003c/strong\u003e)-macrocycles were unreactive to 2-mercaptoethanol over three weeks and unreactive towards free thiol on HSA (\u003cstrong\u003eFigure S7\u003c/strong\u003e). Molecular dynamics simulation suggested the \u003cstrong\u003eOFS\u003c/strong\u003e-macrocycles and the \u003cstrong\u003ePFS\u003c/strong\u003e-macrocycles exhibit similar ground state conformational landscape (\u003cstrong\u003eFigure S8\u003c/strong\u003e).\u0026nbsp;Many perfluoro-aryl crosslinked macrocycles were poorly soluble in water, and we synthesized them with either a GGKKK or GGG tag at the C-terminus to increase their solubility; some sequences were synthesized with both tags to check whether these affect HSA binding. The C-terminal tags aided in providing sufficient solubility properties\u0026nbsp;for downstream analyses (\u003cstrong\u003eFigure S9\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe unique fluorine handle in perfluoro-aryl modified peptides made it possible to determine their binding to HSA using \u003csup\u003e19\u003c/sup\u003eF NMR (\u003cstrong\u003eFigure 4\u003c/strong\u003e). In a typical experiment, we maintained peptide concentration at 50uM and HSA at 100uM (\u003cstrong\u003eTable S4\u003c/strong\u003e). We observed broadening of and disappearance of \u003csup\u003e19\u003c/sup\u003eF signals that correspond to fluoroaromatic groups, which indicated the binding of the peptide to HSA (\u003cstrong\u003eFigure 4A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Figure S10\u003c/strong\u003e). We could not fit a definitive \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e value to the binding response due to the complex binding behaviour and quality of the NMR signal. However, in an albumin titration series, one can use qualitative estimates such as the concentration of albumin necessary to suppress 50% of the initial fluorine signal. Based on these qualitative analyses, it was apparent that some peptides (e.g., \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG) have stronger binding to HSA, whereas other macrocycles (e.g., \u003cstrong\u003ePFS-\u003c/strong\u003eSTCQGECGGG) have weaker binding towards HSA (\u003cstrong\u003eFigure 4A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;Figure S10\u003c/strong\u003e). By measuring the decrease in the signal at a fixed concentration of peptide and HSA, we evaluated 8 sequences found in all discovery campaigns (\u003cstrong\u003eFigure 4B, S11\u003c/strong\u003e) and we nominated \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG (\u003cstrong\u003e5c\u003c/strong\u003e) as the \u0026ldquo;hit\u0026rdquo; and \u003cstrong\u003ePFS\u003c/strong\u003e-STCQGECGGG (\u003cstrong\u003e8c\u003c/strong\u003e) as the negative control for further investigation. Peptides modified at the C-terminus with either GGKKK or GGG solubility tags have similar binding affinity (\u003cstrong\u003eFigure 4B, Figure S11\u003c/strong\u003e). We titrated \u003cstrong\u003e5c\u003c/strong\u003e and \u003cstrong\u003e8a\u003c/strong\u003e against rat serum albumin and observed similar binding to rat and human albumin (\u003cstrong\u003eFigure S12\u003c/strong\u003e). We attempted to confirm the binding affinity of these sequences by isothermal titration calorimetry (ITC) using SA-21 as a control;\u003csup\u003e37\u003c/sup\u003e however, a complex multi-site binding behaviour for all peptides obscured the accurate evaluation of binding affinity by ITC (\u003cstrong\u003eFigure S13\u0026ndash;15\u003c/strong\u003e). The \u003csup\u003e19\u003c/sup\u003eF NMR assay, thus, was critically enabling for validation and ranking of the albumin binding leads.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA fluorescence polarization binding assay (FP) successfully measured the binding affinities of the macrocycles with the fluorophore BODIPY at the C- or N-terminus. In a typical experiment, we used \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG\u003cstrong\u003e\u0026nbsp;(5c)\u003c/strong\u003e or \u003cstrong\u003ePFS\u003c/strong\u003e-SFCPMFCGGG\u003cstrong\u003e\u0026nbsp;(6c)\u003c/strong\u003e at 1 \u0026micro;M concentration and titrated HSA from 0.1 \u0026micro;M to 100 \u0026micro;M. The dose-response curve could be fit to a single-state binding model with binding affinity of \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u0026nbsp;\u003c/sub\u003e= 4\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e6 \u0026micro;M for \u003cstrong\u003e5c\u003c/strong\u003e and at least 100 times weaker affinity for \u003cstrong\u003e6c\u003c/strong\u003e (\u003cstrong\u003eFigure 5A\u003c/strong\u003e and \u003cstrong\u003eS16\u0026ndash;S19\u003c/strong\u003e). BODIPY alone bound weakly to HSA with \u0026gt; 300 \u0026micro;M binding affinity (\u003cstrong\u003eFigure 5A\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eS16\u0026ndash;S19\u003c/strong\u003e). The FP-assay made it possible to measure binding to other proteins or even complex mixtures (serum). A titration of the mouse serum (\u003cstrong\u003eFigure S18\u003c/strong\u003e) yielded a similar binding profile to that observed in binding to pure albumin (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). Replacing HSA with lysozyme and RNAse, the assay detected no binding response, confirming that \u003cstrong\u003e5c\u003c/strong\u003e binding was specific to HSA (\u003cstrong\u003eFigure 5B\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSwitching location of the fluorescent probe from the N-terminus to C-terminus did not significantly change the affinity of \u003cstrong\u003e5c\u003c/strong\u003e (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e = 4-6 \u0026micro;M, \u003cstrong\u003eFigure S19\u003c/strong\u003e). The switching from \u003cstrong\u003eDFS\u003c/strong\u003e to \u003cstrong\u003ePFS\u003c/strong\u003e also exhibited a minimal effect on the binding of peptides \u003cstrong\u003e5b\u003c/strong\u003e and \u003cstrong\u003e5c\u003c/strong\u003e (\u003cstrong\u003eFigure S20\u003c/strong\u003e). The results from FP were in the same order of magnitude as the semi-qualitative estimates acquired for BODIPY-free peptides by the \u003csup\u003e19\u003c/sup\u003eF NMR binding assay, indicating that the presence of a fluorophore did not significantly increase the binding (\u003cstrong\u003eFigure S21\u003c/strong\u003e). Heinis and co-workers recently observed that fluorophores could dramatically increase binding affinity for albumin, and removing the fluorophore is detrimental to the binding of the albumin binder.\u003csup\u003e21\u003c/sup\u003e To exclude this possibility, we conducted an NMR-binding assay of N-terminally-labelled \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGG and BODIPY-free peptides. We observed that the binding affinity was similar (\u003cstrong\u003eFigure S21\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eElucidation of the binding pocket for perfluoro-macrocycles\u003c/h2\u003e\n\u003cp\u003eWe evaluated whether binding pockets of \u003cstrong\u003e5c\u003c/strong\u003e are similar to known albumin binders: carbamazepine, diclofenac and ibuprofen (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). We observed that the binding of \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG (\u003cstrong\u003e5c\u003c/strong\u003e) did not decrease in the presence of any of these drugs; thus, it does not share the same binding pocket as carbamazepine, diclofenac, or ibuprofen (\u003cstrong\u003eFigure 6B\u003c/strong\u003e). To follow on this observation, we performed a series of docking calculations to seek the most favorable binding locations of \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG (\u003cstrong\u003e5c\u003c/strong\u003e) on the surface of HSA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNine distinct sites on HSA were previously shown to bind to fatty acids,\u003csup\u003e45\u003c/sup\u003e some of which also bind to other ligands such as ibuprofen and diclofenac\u003csup\u003e46, 47\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(\u003cstrong\u003eFigure S22\u003c/strong\u003e). These nine reported fatty acid binding sites on five different initial HSA structures were selected for docking of \u003cstrong\u003e5c\u003c/strong\u003e. \u003cstrong\u003eFigure 7A\u003c/strong\u003e shows the HSA protein with some of its bound fatty acids, based on the pdbID 1e7e\u003csup\u003e45\u003c/sup\u003e. Overlaid with this structure is \u003cstrong\u003e5c\u0026nbsp;\u003c/strong\u003edocked to the corresponding fatty acid binding sites on the HSA surface. \u003cstrong\u003eFigure 7B\u0026nbsp;\u003c/strong\u003eshows the binding scores for \u003cstrong\u003e5c\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003eHSA complexes across different HSA structures, based on the distinct pdbIDs and different binding site locations (complete results summarized in \u003cstrong\u003eFigure S23\u003c/strong\u003e and \u003cstrong\u003eTable S5\u003c/strong\u003e). Consistently, \u003cstrong\u003e5c\u003c/strong\u003e has the most favorable binding score in binding site 1, with the value of \u0026ndash;8.95 \u0026plusmn; 1.0 kcal/mol, averaged over all the docking calculations performed. Therefore, the results in \u003cstrong\u003eFigure 7B\u003c/strong\u003e suggest that the primary HSA binding site for \u003cstrong\u003e5c\u003c/strong\u003e is binding site 1. The next most favorable binding sites are sites 8, 6, and 7, with the most favorable binding scores of \u0026ndash;6.6 \u0026plusmn; 1.1 kcal/mol, \u0026ndash;6.2 \u0026plusmn; 0.7 kcal/mol, and \u0026ndash;6.0 \u0026plusmn; 1.2 kcal/mol, respectively (\u003cstrong\u003eFigure 7\u003c/strong\u003e, \u003cstrong\u003eTable S5\u003c/strong\u003e). We observed binding sites 1 and 8 are near to each other on the HSA surface, with the center of mass distance between fatty acids occupying these sites being 5.3 \u0026Aring;. As such, it is unlikely that binding sites 1 and 8 can be simultaneously occupied by two \u003cstrong\u003e5c\u0026nbsp;\u003c/strong\u003emolecules. \u003cstrong\u003eFigure 7A\u003c/strong\u003e shows the four HSA residues that interact with the fatty acid in binding site 1 via charge and nonpolar interactions. In contrast, \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGGG (\u003cstrong\u003e5c\u003c/strong\u003e) has more interactions with this HSA binding site, including the HSA residues R114, R117, Y138, Y161, I142, L154, S193. Notably, HSA residues R117, Y138, and Y161 in binding site 1 are found to mediate HSA interactions with both the fatty acid and \u003cstrong\u003e5c\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCombined docking results (\u003cstrong\u003eFigure 7\u003c/strong\u003e)\u0026nbsp;and binding observation (\u003cstrong\u003eFigure 6\u003c/strong\u003e) suggested that ibuprofen, diclofenac and \u003cstrong\u003e5c\u003c/strong\u003e bind to different locations on the HSA surface. The structural studies\u003csup\u003e46\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003edemonstrated that ibuprofen binds to the binding sites labeled by 3/4 and 6 (pdbID 2bxg, \u003cstrong\u003eFigure S22\u003c/strong\u003e), which are distant from the HSA binding site 1. Structure of HSA bound to diclofenac\u003csup\u003e48\u003c/sup\u003e (pdbID 4z69, \u003cstrong\u003eFigure S22\u003c/strong\u003e) contains two HSA chains in it. One of the HSA chains has a single diclofenac at the binding site 7, while the second HSA chain has three bound diclofenac ligands in total, with two also located at the binding site 7, and the third located near the binding site 1, which is also occupied by a bound fatty acid. The structure locations suggest that diclofenac has the strongest binding to binding site 7, since it is observed there in both HSA chains, and a weaker binding to binding site 1, as only one single HSA chain is observed with diclofenac nearby. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCirculation lifetime of albumin peptides in mice\u003c/h2\u003e\n\u003cp\u003eTo evaluate the half-life circulation of albumin-binding perfluoro-macrocycles, we injected a mixture of peptides \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGG (\u003cstrong\u003e5c\u003c/strong\u003e), weak binding peptide \u003cstrong\u003ePFS\u003c/strong\u003e-STCQGECCGGG (\u003cstrong\u003e8c\u003c/strong\u003e) as the negative control and SA-21 as the positive control into mice and monitored the remaining peptide level by LC\u0026ndash;MS (\u003cstrong\u003eFigure 8A\u003c/strong\u003e, \u003cstrong\u003eFigure S24\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe observed that the negative control \u003cstrong\u003e8c\u003c/strong\u003e disappeared below the limit of detection after 5 min (\u003cstrong\u003eFigure 8B\u003c/strong\u003e). The concentration of \u003cstrong\u003e5c\u003c/strong\u003e decreased 10-fold and SA-21 concentration decreased 5-fold after 2 hours. The combined results confirm a significant retention of \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGG peptide in circulation when compared to unrelated macrocyclic peptides with minimal to no detectable binding to HSA. The single digit micromolar peptide does not rival the mid-nanomolar SA-21 peptide, and the observed differences in half-life likely reflect the relative affinities for albumin. The \u003cstrong\u003ePFS\u003c/strong\u003e-SICRFFCGG peptide, thus, provides an attractive minimalistic starting point for further attenuation of binding affinity for albumin and subsequent attenuation of circulation half-life. \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLate-stage modification of peptides and genetically-encoded (GE) libraries of peptides by cross-linkers (linchpins) is one of the common approaches to incorporate beneficial attributes to their properties.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Alkylation of cysteine residues in peptides via an S\u003csub\u003eN\u003c/sub\u003e2 reaction using bi- or tri-dentate alkyl halides has been use for cyclization of peptides, incorporation of unnatural fragments into the resulting macrocycles,\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and late-stage modification of phage- and mRNA-displayed libraries to yield billion-scale GE libraries. Peptide cyclization via S\u003csub\u003eN\u003c/sub\u003eAr reaction with perfluoroarenes popularized by the Pentelute group forms alkyl-aryl thioethers; \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e other classes reactions have been developed to form aryl\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, alkenyl and alkynyl thioethers\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e in unprotected peptides. Aryl and perfluoroaryl thioethers are more resistant toward oxidation when compared to traditional bis-alkyl thioesters\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Decreased conformational mobility or aryl-thioether bond has been proposed to equip the resulting macrocycles with favourable properties such as cell permeability and proteolytic stability.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e Our report described the first selection from perfluoro-aryl macrocyclic GE libraries. There exists only one example of GE selection from S\u003csub\u003eN\u003c/sub\u003eAr-modified phage libraries: Lu and co-workers recently employed 2,4-Difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB) as a reagent that can modify phage libraries in water.\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e Chen and co-workers also used Pd-catalyzed S\u003csub\u003eN\u003c/sub\u003eAr reaction to yield DNA-encoded libraries.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e Both S\u003csub\u003eN\u003c/sub\u003eAr reaction yield macrocycles do not contain any fluorine atoms. On the other hand, fluorine handles present in perfluoroaryl-crosslinked macrocycles offer a unique possibility to use of \u003csup\u003e19\u003c/sup\u003eF NMR to measure protein-macrocycle interactions. Interaction of perfluorinated aryls with proteins is also electronically distinct from non-fluorinated aromatic residues and in some cases it can offer uniquely advantageous interactions\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn important observation in selection of GE \u003cb\u003eOFS\u003c/b\u003e-macrocycle libraries is mild reactivity of these structures towards thiol nucleophiles.\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Libraries of mild electrophiles\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e and phage-displayed libraries with built-in electrophiles\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e have emerged as important starting point for discovery of covalent and reversibly covalent inhibitors. While we do not show it in our report, it is possible that an attenuated reactivity of \u003cb\u003eOFS\u003c/b\u003e-macrocycles towards thiols can be used as an advantageous features in discovery or inhibitors that form covalent bonds with thiol residues in proteins. If reactivity of the selected macrocycles is not desired, one can perform late-stage replacement of \u003cb\u003eOFS\u003c/b\u003e moiety in the identified hits with nearly isosteric perfluorophenyl-sulfide. The \u003cb\u003ePFS\u003c/b\u003e linchpin is not sufficiently reactive for direct modification of phage-displayed libraries in water, but replacement of \u003cb\u003eDFS\u003c/b\u003e linchpin by \u003cb\u003ePFS\u003c/b\u003e \u0026ldquo;post discovery\u0026rdquo; maintains the conformation and binding affinity of the discovered macrocycles while alleviating their undesired electrophilicity. Our report, thus, suggest a general approach for the future utility of perfluoroaryl-modified libraries: Step 1: Select phage-displayed libraries of \u003cb\u003eOFS\u003c/b\u003e-macrocycles against the desired target. Step 2: evaluate \u003cb\u003ePFS\u003c/b\u003e-modified synthetic macrocycles for their ability to bind to these targets.\u003c/p\u003e \u003cp\u003eHuman serum albumin (HSA) target used in this publication is a commonly employed model target in screen of phage-displayed or DNA-encoded libraries (DEL) and traditional high-throughput screening (HTS). Albumin is a complex multi-pocket receptor with regions that can bind to fatty acid-like moieties, dicarboxylic acids as well a wide variety of aromatic and heterocyclic compounds and large dye molecules.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e Albumin also contains several binding sites for peptides as well as small proteins that have been utilized for half-life extension strategies.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e Peptide macrocycles discovered in this report add to a diverse set of known albumin binders and they constitute the first example of small macrocyclic peptide binders for albumin. We note that single digit micromolar affinity of the discovered \u003cb\u003ePFS\u003c/b\u003e-SICRFFC motif was not sufficient to retain this macrocycle in circulation as effectively as benchmark SA-21 albumin binding peptide. However, it should be relatively straightforward to optimize this structure to improve the affinity because our docking calculations suggest that a C-terminal extension to this scaffold might be productive avenue for the future optimization. Exploration of such extensions could be done using perfluoro-aryl modified phage-displayed libraries of SICRFFCX\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e peptides with several randomized C-terminal amino acids. Such optimization should yield a collection of albumin binders with a range of affinities for albumin and, in turn, a range of the circulation half-lives. The small size of such peptide-macrocycle families makes it trivial to make them by solid-phase synthesis or incorporate them a part of another sequence produced by solid phase synthesis. More importantly, the SICRFFC motif or optimized SICRFFCX\u003csub\u003e\u003cem\u003eN\u003c/em\u003e\u003c/sub\u003e motifs emerging from phage display screen can be easily re-introduced into phage-displayed libraries to serve as a constant N-terminal albumin binding motif and giving rise to libraries with predictable circulation half-life.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting information document contains Supporting Figures S1\u0026ndash;S62, Tables S1\u0026ndash;S5, synthetic methods and characterization of compounds, details of phage display selection, next generation sequencing and bioinformatics analysis and all biochemical assays. Supporting data folder contains PDB files produced by docking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a research contract from the Ferring Research Institute, Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2016-402511 to R. D.), and NSERC Accelerator Supplement (to R. D.).\u0026nbsp;This work was also supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM124160 (PI: Y.-S.L.)\u0026nbsp;Infrastructure support was provided by CFI New Leader Opportunity (to R. D.). We thank Dr. Randy Whittal for assistance with LCMS, and Mark Miskolzie for assistance with \u003csup\u003e19\u003c/sup\u003eF NMR kinetics.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePreparation of SXCX\u003csub\u003e3\u003c/sub\u003eC phage-displayed library\u003c/p\u003e\n\u003cp\u003eThe procedures have been adopted and modified as previously described in two publications that produced the M13-displayed SXCXXXC library\u003csup\u003e44\u003c/sup\u003e and M13-SDB vector\u003csup\u003e68\u003c/sup\u003e. In short, the vector SB4 QFT*LHQ was digested with Kpn I HF (NEB cat# R3142S) and Eag I HF (NEB cat# R3505S). A primer/template pair consisting of primer 5\u0026rsquo;-AT GGC GCC CGG CCG AAC CTC CAC C-3\u0026rsquo; and template 5\u0026rsquo;-CC CGG GTA CCT TTC TAT TCT CAC TCT TCT X TGT XXX TGT GGT GGA GGT TCG GCC GGG CGC TTG ATT-3\u0026rsquo; with \u0026lsquo;X\u0026rsquo; representing a trinucleotide formed by annealing. The primer/template was then extended using Klenow DNA polymerase (NEB) according to the manufacturer\u0026rsquo;s instructions. The insert fragment was then digested with Kpn1 HF and Eag1 HF, gel purified, and ligated into the cut vector. The ligation products were then transformed into electrocompetent \u003cem\u003eE. coli\u003c/em\u003e cells, and the transformants were grown overnight on \u003cem\u003eE. coli\u003c/em\u003e TG1 to allow for phage production. Phage cultures were then centrifuged to remove cells and debris, and then the phage was precipitated by PEG precipitation (5% PEG 0.5 M NaCl). Other SDB vectors have been processed identically. We sequenced the na\u0026iuml;ve libraries by Illumina sequencing, and the na\u0026iuml;ve library of SXCX\u003cem\u003e\u003csub\u003en\u003c/sub\u003e\u003c/em\u003eC (\u003cem\u003en\u003c/em\u003e=3-5) composition is publicly available at the following link: \u003ca href=\"https://48hd.cloud/file/1470\"\u003ehttps://48hd.cloud/file/1470\u003c/a\u003e.\u0026nbsp;SXCX\u003csub\u003e4\u003c/sub\u003eC and SXCX\u003csub\u003e5\u003c/sub\u003eC libraries were prepared as described in previously reported protocols.\u003csup\u003e69\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePanning\u0026nbsp;campaigns\u003c/p\u003e\n\u003cp\u003eThree panning campaigns were conducted to discover binders for Human Serum Albumin (HSA) with protein A, ConA or T4-Gp as the negative controls. The proteins were immobilized on polystyrene plates or magnetic beads and panned against \u003cstrong\u003eOFS\u003c/strong\u003e-macrocyclic libraries or unmodified libraries. \u0026nbsp;The details on panning experiments are discussed in supplementary information section. All the analyses were performed by next generation sequencing (NGS) of phage DNA as previously reported.\u003csup\u003e50, 52, 70\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePreparation of Illumina sequencing samples\u003c/p\u003e\n\u003cp\u003eSimilar to previous reports,\u003csup\u003e50, 52, 70\u003c/sup\u003e phage eluted from the target was subjected to PCR amplification (see SI for PCR protocols) to append Illumina multiplexing barcodes, and sequencing adapters to randomized library regions. All PCR products were quantified by 2% (w/v) agarose gel in Tris-Borate-EDTA buffer at 100 volts for ~35 min using a low molecular weight DNA ladder as standard (NEB, cat# N3233S). PCR products that contain different indexing barcodes were pooled, allowing 10 ng of each product in the mixture. The mixture was purified by eGel, quantified by quBit and sequenced using the Illumina NextSeq paired-end 500/550 High Output Kit v2.5 (2\u0026times;75 Cycles). Data were automatically uploaded to\u0026nbsp;BaseSpace\u0026trade; Sequence Hub.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcessing of Illumina data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Gzip compressed FASTQ files were downloaded from\u0026nbsp;BaseSpace\u0026trade; Sequence Hub. The files\u0026nbsp;were converted into tables of DNA sequences and their counts per experiment. Briefly, FASTQ files were parsed based on unique multiplexing barcodes within the reads discarding any reads that contained a low-quality score. Mapping the forward (F) and reverse (R) barcoding regions, mapping of F and R priming regions allowing no more than one base substitution each and F-R read alignment allowing no mismatches between F and R reads yielded DNA sequences located between the priming regions as described in previous publications.\u003csup\u003e70\u003c/sup\u003e The files with DNA reads, raw counts, and mapped peptide modifications were uploaded to \u003ca href=\"http://48hd.cloud/\"\u003ehttp://48hd.cloud/\u003c/a\u003e server. Each experiment has a unique alphanumeric name and unique static URL in Tables S1-3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral protocol for cyclization with decafluorodiphenylsulfone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProcedure was analogous to previously published methods\u003csup\u003e42, 43\u003c/sup\u003e. In short, linear peptide (10 mM) was dissolved in 50% acetonitrile and Tris buffer (50 mM Tris-HCl, pH 8.5), then 2 equivalents of \u003cstrong\u003eDFS\u003c/strong\u003e in 50% acetonitrile and Tris buffer (50 mM Tris-HCl, pH 8.5) was added to the mixture. The mixture was vortexed for 30 sec, incubated for 2 h at room temperature,\u0026nbsp;\u0026nbsp;purified by HPLC and further lyophilized to yield product.\u003c/p\u003e\n\u003cp\u003eGeneral protocol for cyclization with pentaflurophenyl-sulfide\u003c/p\u003e\n\u003cp\u003eProcedure was analogous to previously published methods\u003csup\u003e42, 43\u003c/sup\u003e. In short, linear peptide (10 mM) was\u0026nbsp;dissolved\u0026nbsp;in 50 mM Tris in DMF, then 2 equivalents of \u003cstrong\u003ePFS\u003c/strong\u003e was added to the mixture. The mixture was vortexed for 30 sec and allow to react for 1 hour at RT.\u0026nbsp;The reaction mixture was\u0026nbsp;purified by HPLC and lyophilized to yield the product.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e pharmacokinetic experiment\u003c/p\u003e\n\u003cp\u003eAll the procedures and experiments involving animals were carried out using a protocol approved by the Health Sciences Laboratory Animal Services (HSLAS), University of Alberta. The protocol was approved as per the Canadian Council on Animal Care (CCAC) guidelines. All mice were maintained in pathogen-free conditions at the University of Alberta breeding facility. Peptide mixtures of 100 \u0026micro;M were prepared in PBS. Mice were administered with 200 \u0026micro;L of the peptide mixture solution with tail veil injection. A series of 6 blood samples were collected at time points from 2 min up to 240 min. Samples were collected in tubes that contained sodium citrate as an anticoagulant and then centrifuged at 5 min at 2,000\u0026times;g to collect the blood plasma. 10 \u0026micro;L of plasma portion were transferred into a tube containing 40 \u0026micro;L of 8:2 acetonitrile/water to precipitate proteins. The samples were centrifuged at max speed for 10 min at 4 \u003csup\u003eo\u003c/sup\u003eC. Supernatants were then transferred to new tubes and subjected to analysis by LC-MS.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMuttenthaler, M.; King, G. F.; Adams, D. J.; Alewood, P. F., Trends in peptide drug discovery. Nat. Rev. Drug Discov.2021, \u003cem\u003e20\u003c/em\u003e (4), 309\u0026ndash;325.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, R. K.; Stylianopoulos, T., Delivering nanomedicine to solid tumors. Nat. Revi. 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Biol. 2021, \u003cem\u003e17\u003c/em\u003e (7), 806\u0026ndash;816.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1999287/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1999287/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this paper, we report selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine S\u003csub\u003eN\u003c/sub\u003eAr chemistry. Modification of phage-displayed libraries SXCX\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003eC-phage, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u0026ndash;5, where X is any amino acid except for cysteine by decafluoro-diphenylsulfone (\u003cb\u003eDFS\u003c/b\u003e), yields genetically-encoded library of octafluoro-diphenylsulfone-crosslinked macrocycles (\u003cb\u003eOFS\u003c/b\u003e-SXCX\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003eC-phage). Selection from these libraries using albumin as a bait identified a family of significantly enriched perfluoroaryl-macrocycles. Synthesis of perfluoroaryl-macrocycles predicted by phage display and testing their binding properties by \u003csup\u003e19\u003c/sup\u003eF NMR and fluorescent polarization identified \u003cb\u003eOFS\u003c/b\u003e-macrocycle with SICRFFC sequence as the most potent albumin binder. We observed that \u003cb\u003eOFS\u003c/b\u003e-macrocycles slowly react with biological nucleophiles such as glutathione. Replacing decafluoro-diphenylsulfone by nearly isosteric pentafluorophenyl sulfide yielded perfluorophenylsulfide (\u003cb\u003ePFS\u003c/b\u003e)-crosslinked macrocycles devoid of undesired reactivity. The augmented lead \u003cb\u003ePFS\u003c/b\u003e-macrocycle with SICRFFC sequence exhibited \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e = 4\u0026ndash;6 \u0026micro;M towards human serum albumin and similar affinities towards rat and mouse albumins. When injected in mouse, the \u003cb\u003ePFS\u003c/b\u003e-SICRFFCGGG compound was significantly retained in circulation \u003cem\u003ein vivo\u003c/em\u003e when compared to control \u003cb\u003ePFS\u003c/b\u003e-macrocyclic peptide. The perfluoroaryl-macrocycles with SICRFFC motif are the smallest known peptide macrocycle with significant affinity for human albumin and they are a productive starting point for future development of compact macrocycles with predictable circulation half-life \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-07 16:56:30","doi":"10.21203/rs.3.rs-1999287/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"21499db2-308d-48e0-9a06-06729713312a","owner":[],"postedDate":"September 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-09-29T16:01:09+00:00","versionOfRecord":{"articleIdentity":"rs-1999287","link":"https://doi.org/10.1038/s41467-023-41427-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-09-13 04:00:00","publishedOnDateReadable":"September 13th, 2023"},"versionCreatedAt":"2022-09-07 16:56:30","video":"","vorDoi":"10.1038/s41467-023-41427-y","vorDoiUrl":"https://doi.org/10.1038/s41467-023-41427-y","workflowStages":[]},"version":"v1","identity":"rs-1999287","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1999287","identity":"rs-1999287","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}