FDA-approved drug repurposing as p53 mutants rescue candidates using structure-based virtual screening and molecular simulations

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This study presents a structure-based virtual screening and molecular dynamics approach to repurpose FDA-approved drugs as p53 rescue candidates. A virtual screening library of FDA-approved compounds was docked against three representative p53 mutants (7DHY, 7DHZ, and 7V97) to evaluate their binding potential. The prioritized candidates demonstrated consistent, multi-conformer binding affinities. Protein-ligand interaction profiling revealed that the candidate DB09280 possesses a highly dense interaction network, particularly against the V272M and R249S variants. Residue-level analysis of the G245S structural mutant showed that DB09280 uniquely engages His19, a crucial residue for zinc coordination, and forms stabilizing contacts with adjacent flexible loop residues, including ASN35 and PRO32. Subsequent 500 ns molecular dynamics simulations demonstrated that DB09280 acts as a conformational clamp. The ligand-bound (holo) system exhibited substantially reduced global structural drift (RMSD) and attenuated local residue fluctuations (RMSF) within the core domain compared to the highly unstable apo state. Principal component analysis further confirmed that DB09280 restricts the broad conformational sampling of the mutant into a stable, dominant energetic basin. These findings highlight DB09280 as a robust p53 stabilizer and provide a compelling mechanistic foundation for its repurposing in oncology. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Structural biology p53 mutant Drug repurposing Molecular dynamics simulations Virtual screening Structural stabilization Allosteric modulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The tumor suppressor protein p53 is a central guardian of genomic integrity, orchestrating cell-cycle arrest, apoptosis, and DNA damage responses in stressed cells. In human cancers, TP53 is the most frequently mutated gene, and these alterations often disable p53’s transcriptional activity, promote malignant progression, and correlate with poor clinical outcomes. Many oncogenic TP53 variants arise within the DNA-binding domain and can be broadly divided into “DNA-contact” mutations that directly impair sequence recognition and “structural” mutations that destabilize the loop-sheet-helix motif and the underlying β-sandwich scaffold. The latter class, which includes cavity-creating substitutions such as G245S, R249S, and V272M, compromises zinc coordination and reduces thermodynamic stability, predisposing p53 to partial unfolding and aggregation [ 1 ], [ 2 ], [ 3 ].​ Reactivating mutant p53 has therefore emerged as an attractive yet challenging therapeutic strategy. Several approaches have been pursued, including small molecules that restore native-like conformation of structural mutants, compounds that refold misfolded p53, and agents that target auxiliary regulators such as MDM2 to indirectly enhance p53 signaling. However, most candidate reactivators show a narrow mutation spectrum, limited potency, or suboptimal pharmacokinetic properties, which has hindered their broad clinical translation. Recent work has revealed a cryptic allosteric site within the DNA-binding domain that can be targeted by cysteine-reactive small molecules such as arsenic trioxide (ATO), which stabilizes the loop-sheet-helix motif and β-sandwich fold of multiple structural p53 mutants. These studies provide a mechanistic blueprint for allosteric rescue, but the repertoire of clinically actionable ligands that exploit this pocket remains sparse [ 3 ], [ 4 ]. Drug repurposing offers a complementary route to expand the pool of p53-stabilizing compounds by leveraging the safety and pharmacology of existing FDA-approved agents. Structure-based virtual screening combined with molecular dynamics (MD) simulations is particularly well suited to this task, because it can account for the pronounced conformational plasticity of mutant p53 and evaluate ligand binding across multiple disease-relevant variants. In the context of the arsenic-defined allosteric pocket, such an integrative in silico pipeline enables the identification of molecules that not only bind with favorable affinity but also act as dynamic stabilizers that clamp flexible loops and reinforce zinc-associated structural motifs [ 5 ].​ In this study, we employed a structure-based virtual screening workflow to interrogate a curated library of FDA-approved drugs against three representative mutant p53 DNA-binding domains - G245S, R249S, and V272M - whose crystal structures capture distinct structural, DNA-contact, and hydrophobic perturbations. Top-ranked candidates were subjected to detailed protein-ligand interaction profiling at the arsenic-defined allosteric site, followed by long-timescale MD simulations to assess their ability to stabilize the mutant core domain. Our results highlight the compound DB09280 as a promising p53 rescue candidate that engages key residues involved in zinc coordination and loop flexibility, and functions as a conformational clamp that restricts aberrant motions of the G245S structural mutant. By integrating virtual screening, interaction fingerprinting, and MD-based conformational analysis, this work establishes a mechanistic framework for repurposing FDA-approved drugs as broad-spectrum stabilizers of mutant p53. Materials and methods p53 mutants preparation Three relevant p53 mutants namely, G245S, R249S, and V272M (PDB IDs: 7DHY, 7DHZ, and 7V97, respectively) were retrieved from the RCSB Protein Data Bank (PDB) [ 6 ]. Structures were selected based on the availability of high-resolution transmembrane domains, the presence of resolved or inferred fenestration pathways, and their relevance to clinically validated channelopathies. Prior to molecular docking, all protein structures were subjected to standardized preprocessing and optimization. Protonation states of titratable residues were assigned using the H + + server ( http://newbiophysics.cs.vt.edu/H++/ ) at physiological pH [ 7 ]. Crystallographic water molecules were removed. Atom types and Gasteiger partial charges were then assigned using ForliLab’s Meeko package, and the prepared structures were converted to PDBQT format for subsequent docking simulations [ 8 ]. Table 1 p53 Mutants Used for Virtual Screening and Molecular Dynamics p53 Variant PDB ID Mutation Class Structural Consequence G245S 7DHY Structural Destabilizes local secondary structure elements and perturbs critical zinc coordination loops. R249S 7DHZ DNA-contact Disrupts direct interactions with consensus DNA sequences without completely unfolding the core. V272M 7V97 Hydrophobic Alters internal hydrophobic packing within the core beta-sandwich, inducing thermodynamic instability. Ligand library preparation FDA-approved compounds were extracted from DrugBank to construct the virtual screening library. Ligand structures were initially provided in SDF format and subjected to salt removal using RDKit’s SaltRemover module [ 9 ]. Reactive electrophilic substructures were filtered based on predefined SMARTS patterns, followed by elimination of pan-assay interference compounds (PAINS) using RDKit’s FilterCatalog to reduce false-positive risk. For each retained molecule, explicit hydrogens were added, and three-dimensional conformers were generated. Geometry optimization was performed using the MMFF94 force field to obtain energetically reasonable ligand conformations prior to docking [ 10 ]. The optimized ligands were then processed with Meeko (mk_prepare_ligand.py), which assigned Gasteiger partial charges, detected rotatable bonds, preserved stereochemistry, and converted structures into PDBQT format compatible with AutoDock Vina v1.2.7 [ 8 ], [ 11 ]. Molecular docking Molecular docking was performed using AutoDock Vina to evaluate binding of FDA-approved compounds to the allosteric pocket of mutant p53 corresponding to the arsenic-binding rescue site. The docking region was defined based on the experimentally characterized allosteric cavity, previously reported as a targetable rescue hotspot. The center of the docking grid was determined in UCSF Chimera using the “measure center” command applied to the arsenic-binding site, yielding coordinates of x = − 34.188, y = 3.782, and z = − 7.713 [ 12 ]. This center was used to construct a cubic grid box large enough to fully encompass the allosteric pocket and surrounding flexible regions of the DNA-binding domain, ensuring adequate sampling of ligand conformations. Prepared ligands in PDBQT format were docked against the prepared mutant p53 structures using AutoDock Vina with default exhaustiveness parameters. For each ligand, multiple binding poses were generated, and the top-ranked pose based on predicted binding affinity was retained for downstream analysis. Docking results were subsequently processed to extract best-scoring complexes, which were converted to PDB format for protein-ligand interaction profiling and molecular dynamics simulations [ 13 ], [ 14 ]. Molecular dynamics simulations The top-ranked compound identified from molecular docking were selected for molecular dynamics simulations to evaluate the stability and dynamic behavior of their complexes with mutant p53. Protein-ligand systems were constructed using the CHARMM-GUI web interface, applying the CHARMM36m force field for the protein and associated parameters for the ligands [ 15 ], [ 16 ]. The complex was embedded in a rectangular TIP3P water box with a minimum padding of 10 Å from the solute to the box boundary. Sodium and chloride ions were added to neutralize the systems and adjust ionic strength to 0.15 M. Periodic boundary conditions were applied, and long-range electrostatics were treated using the Particle Mesh Ewald method with automatically generated FFT grid dimensions. Energy minimization was performed to remove unfavorable contacts, followed by stepwise equilibration under constant volume (NVT) and constant pressure (NPT) conditions at 300 K and 1 bar, respectively. Temperature regulation was achieved using a Langevin thermostat, while pressure coupling was maintained with a Monte Carlo barostat. Production simulations were subsequently carried out for 500 ns using OpenMM [ 13 ]. Trajectories were saved at regular intervals for downstream analysis. Post-simulation analyses, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), principal component analysis (PCA), and ligand stability assessment, were conducted using MDTraj and MDAnalysis, as implemented in the analysis notebook [ 17 ], [ 18 ]. Ethics Statement This study did not involve human participants, human data, or animal subjects. All analyses were performed using publicly available datasets and computational modeling approaches. Therefore, ethical approval and informed consent were not required for this work. Results and discussion Structure-based virtual screening results Molecular docking was performed against three mutant p53 crystal structures (7DHY, 7DHZ, and 7V97) to evaluate the binding potential of the top ten rescue candidates. Overall, the compounds exhibited consistently favorable binding affinities across all conformations, with docking scores ranging from − 6.8 to − 8.3 kcal/mol, indicating stable interactions within the mutant p53 binding pocket. Among the screened molecules, DB11363 emerged as the strongest binder to the 7DHY structure (− 8.3 kcal/mol), while DB00762 and DB08896 showed the highest affinity toward 7V97 (both − 8.1 kcal/mol). Several candidates, including DB06589, DB01586, DB15133, and DB09280, demonstrated reproducible binding across all three structures, suggesting robustness against p53 conformational variability. Notably, DB00619 displayed balanced affinities across the panel, with improved binding to 7V97 (− 7.8 kcal/mol), whereas DB15233 and DB04868 showed slightly reduced interaction energies for 7DHZ, reflecting possible structural sensitivity of this mutant form. Interestingly, DB08896 exhibited a marked increase in affinity toward 7V97 despite weaker binding to 7DHZ, highlighting potential mutation-specific stabilization effects. These results indicate that multiple candidates possess consistent multi-conformer binding capability, a critical property for rescuing structurally heterogeneous mutant p53. In particular, DB11363, DB00762, DB08896, and DB00619 were prioritized based on their superior or balanced docking profiles and were selected for subsequent interaction analysis and dynamic stability assessment. The observed binding consistency across multiple mutant p53 conformations is particularly relevant given the structural plasticity associated with oncogenic p53 variants. Compounds exhibiting stable affinity across 7DHY, 7DHZ, and 7V97 are more likely to tolerate mutation-induced pocket rearrangements and may function as structural stabilizers rather than mutation-specific binders. The enhanced affinity of DB11363 and DB00762 suggests stronger stabilization potential, whereas DB08896’s mutation-dependent variability may indicate selective conformational preference. These findings support a mechanism in which small molecules bind to destabilized regions of mutant p53, potentially restoring partial structural integrity and functional activity (Fig. 1 ). Protein-ligand interactions analysis To complement docking scores and evaluate the structural stabilization potential of selected candidates, protein-ligand interaction profiles were analyzed using PLIP across three representative mutant p53 classes: V272M (hydrophobic), R249S (DNA-contact), and G245S (structural). Distinct interaction patterns were observed among the compounds, reflecting mutation-dependent binding behavior. DB09280 exhibited the highest overall interaction density, particularly against V272M (11 interactions) and R249S (10 interactions), suggesting strong anchoring within the destabilized pocket. DB06589 and DB00762 also demonstrated elevated interaction counts across multiple variants, indicating consistent engagement with key binding residues. Several compounds, including DB15133 and DB08896, showed preferential interaction with specific mutants, with DB08896 displaying enhanced binding to R249S despite reduced contacts with V272M. In contrast, DB11363 and DB15233 exhibited comparatively weaker interaction profiles, consistent with their moderate docking performance. Importantly, multiple candidates displayed balanced interaction distributions across all three mutant classes, highlighting their potential to act as general p53 stabilizers rather than mutation-specific ligands (Fig. 2 ). To gain mechanistic insight into ligand-mediated stabilization of mutant p53, residue-level interaction fingerprints were generated for the G245S structural mutant. This analysis revealed recurrent engagement of key residues within the DNA-binding core domain, including THR6, ARG14, LEU15, PHE17, TYR30, PRO32, ASN35, LYS68, LEU56, ASN72, and GLU75. DB09280 displayed the most extensive interaction network, contacting multiple hydrophobic and polar residues and uniquely engaging His19, a residue implicated in zinc coordination and structural stabilization. This interaction is highlighted as a potential rescue hotspot, suggesting a direct contribution to restoring local folding integrity. Several candidates, including DB06589, DB00619, and DB00762, showed consistent contacts with ASN35, LEU56, and ASN72, residues located within flexible regions surrounding the mutation site. Such multi-point anchoring may counteract the destabilizing effect of G245S by reinforcing local secondary structure elements. In contrast, DB11363 and DB15233 exhibited fewer residue contacts, consistent with their weaker interaction density observed in earlier PLIP analyses (Fig. 3 ). The binding fingerprints indicate that effective rescue candidates preferentially interact with residues surrounding the destabilized β-sandwich of mutant p53. In particular, engagement of His19 by DB09280 suggests a potential mechanism involving reinforcement of zinc-associated structural motifs, a known strategy for restoring mutant p53 stability. The presence of dense interaction clusters around ASN35, LEU56, and ASN72 further supports a model in which ligands stabilize flexible loops adjacent to the mutation site, thereby promoting partial refolding toward a native-like conformation. These residue-level interactions provide molecular evidence that top candidates act as structural stabilizers rather than simple pocket binders, supporting their advancement to molecular dynamics simulations for validation of long-timescale rescue effects. To visualize the molecular basis of mutant p53 rescue, the binding pose of the top-ranked candidate DB09280 was examined in detail within the G245S structure. As shown in Fig. 4 (A) , DB09280 occupies a hydrophobic pocket adjacent to the mutation site, engaging residues distributed across flexible loop regions and β-sheet elements of the DNA-binding domain. The ligand establishes a dense interaction network involving ASN35, PRO32, TYR30, LEU34, PHE17, and HIS19, forming a stabilizing scaffold around the destabilized core (Fig. 4 (B) ). Multiple hydrogen bonds anchor the ligand to ASN35 and PRO32, while π-π and π-alkyl interactions with TYR30 and surrounding hydrophobic residues further reinforce binding. The structural binding mode of DB09280 reveals a multi-point stabilization strategy, in which the ligand bridges flexible loops and β-sheet elements surrounding the G245S mutation. Rather than acting as a simple affinity-driven binder, DB09280 appears to function as a conformational clamp, reinforcing local structural elements and potentially restoring native-like folding. The interaction with HIS19 is particularly noteworthy, as perturbation of zinc coordination is a hallmark of structural p53 mutants. By engaging this residue, DB09280 may partially reconstitute the metal-supported architecture of the DNA-binding domain, providing a plausible molecular basis for rescue activity. (A) Three-dimensional representation of the ligand bound within the p53 cavity, shown in surface and cartoon view, highlighting key interacting residues surrounding the mutation site. (B) Two-dimensional interaction map illustrating hydrogen bonds, van der Waals contacts, and π-mediated interactions between DB09280 and critical p53 residues. Molecular dynamics trajectory analysis Root-mean square deviation To evaluate the structural impact of ligand binding on mutant p53 stability, 500 ns molecular dynamics simulations were performed for both apo and holo systems. The Cα RMSD profile (Fig. 5 (A) ) reveals pronounced structural fluctuations in the apo system, with deviations frequently exceeding 8–10 Å during the early and mid simulation phases. In contrast, the holo complex exhibits comparatively moderated fluctuations, particularly after ~ 350 ns, where RMSD values stabilize around 3–5 Å. Notably, between 380–430 ns, the holo system maintains a lower and more stable RMSD relative to the apo form, suggesting ligand-induced conformational stabilization. Although both systems display intrinsic flexibility, consistent with the destabilized nature of structural p53 mutants, the ligand-bound complex demonstrates reduced amplitude and shorter-lived excursions from equilibrium, indicating partial structural constraint. Ligand RMSD analysis (Fig. 5 (B) ) further supports stable complex formation. The ligand maintains deviations primarily within 1.5–2.5 Å throughout the 500 ns trajectory, without evidence of dissociation or major reorientation. Transient increases are observed but rapidly re-equilibrate, indicating persistent pocket occupancy and sustained interactions. The MD results reinforce the docking and interaction fingerprint analyses by demonstrating dynamic stabilization of mutant p53 upon ligand binding. While the apo structure exhibits large conformational excursions characteristic of destabilized mutants, the holo system shows comparatively restrained fluctuations, particularly during the later stages of the simulation. The stable ligand RMSD confirms that the compound remains firmly anchored within the binding pocket, enabling continuous multi-point interactions that likely reinforce local secondary structure elements. Importantly, the reduction in large-scale structural drift in the holo system supports a rescue-like mechanism driven by conformational constraint rather than global rigidification. This behaviour is consistent with small-molecule-mediated stabilization of partially unfolded p53 variants. (A) Cα RMSD of the protein backbone for apo (blue) and ligand-bound (holo, orange) systems. (B) Ligand RMSD relative to the initial docked conformation during the simulation. Root-mean square fluctuation Residue-wise root mean square fluctuation (RMSF) analysis was performed to assess local backbone dynamics of mutant p53 in the presence and absence of the bound ligand. Overall, the holo system exhibits reduced fluctuations across multiple regions of the protein compared to the apo form (Fig. 6 ), indicating ligand-mediated dampening of local flexibility. The most pronounced differences are observed in the central core domain, particularly around residues ~ 80–100, where the apo structure displays elevated mobility reaching ~ 7 Å, whereas the holo complex shows substantially attenuated fluctuations (~ 4 Å). This region encompasses structural elements proximal to the G245S mutation and is critical for maintaining the integrity of the DNA-binding domain. Additional stabilization is observed in loop regions near residues ~ 20–30 and ~ 120–140, consistent with earlier interaction fingerprint analyses showing ligand engagement with residues in these flexible segments. Although terminal regions retain higher mobility, as expected for solvent-exposed tails, the core domain demonstrates clear rigidity upon ligand binding. The reduction in residue-level fluctuations within the p53 core domain suggests that ligand binding restricts excessive conformational freedom induced by the G245S mutation. By stabilizing flexible loops and adjacent secondary structure elements, the ligand likely promotes a more compact and native-like fold. This localized rigidity complements the global RMSD stabilization and supports a rescue mechanism driven by reinforcement of structurally compromised regions rather than uniform protein rigidification. Principal component analysis Principal component analysis was performed to characterize large-scale collective motions of mutant p53 in apo and holo states. Projection of the trajectories onto the first two principal components reveals markedly different conformational sampling between the two systems (Fig. 7 ). The apo protein explores a broad and highly dispersed conformational space, indicative of pronounced structural heterogeneity and dynamic instability. Multiple clusters are observed, reflecting frequent transitions between distinct conformational substates throughout the simulation. In contrast, the holo complex exhibits a more compact and structured distribution along PC1 and PC2, with trajectory points concentrating within fewer dominant regions. This reduced dispersion suggests ligand-induced restriction of global motions and stabilization of preferred conformational states. Time-colored projections further indicate progressive convergence toward a stable basin in the holo system, whereas the apo form continues to sample diverse conformations over the entire trajectory. The PCA results demonstrate that ligand binding reshapes the conformational energy landscape of mutant p53 by narrowing accessible structural states and promoting population of a dominant basin. This behavior is characteristic of small-molecule-mediated conformational rescue, where flexible and partially unfolded regions become constrained into energetically favorable arrangements. Together with RMSD and RMSF analyses, these findings support a mechanism in which the ligand acts as a dynamic stabilizer, suppressing aberrant motions associated with the G245S mutation and facilitating adoption of more native-like conformations. Conclusion The therapeutic reactivation of mutant p53 requires small molecules capable of counteracting mutation-induced thermodynamic instability. Through an integrated computational pipeline comprising molecular docking, interaction fingerprinting, and 500 ns molecular dynamics simulations, we evaluated FDA-approved drugs as prospective p53 rescue agents. Our virtual screening identified several compounds with robust, cross-mutant binding profiles, highlighting DB09280 as a standout candidate capable of tolerating structural plasticity. Structural interaction analyses revealed that DB09280 functions not merely as an affinity-driven pocket binder, but as a conformational clamp that reinforces local secondary structure elements. By strategically anchoring to flexible loops and directly engaging His19, a residue vital for zinc coordination, the ligand addresses the core structural deficits characteristic of the G245S mutation. Long-time-scale dynamic analyses strongly corroborated this stabilization mechanism. The holo complex exhibited profound reductions in global RMSD and localized RMSF within the compromised central core domain. Furthermore, principal component analysis demonstrated that ligand binding effectively limits aberrant large-scale motions, forcing the destabilized mutant to converge into a stabilized, native-like conformational state. These in silico findings provide strong molecular evidence that DB09280 acts as a potent structural stabilizer of mutant p53. This study establishes a rigorous mechanistic rationale for advancing DB09280 and structurally related FDA-approved scaffolds into in vitro and in vivo models to validate their efficacy in restoring p53 tumor suppressor function. Declaration of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declarations Funding This research received no external funding. Author Contribution M.A. conceived and designed the study. Y.B. performed the molecular docking, molecular dynamics simulations, and data analysis. M.A. interpreted the results and wrote the main manuscript text. Y.B. contributed to methodological development, data interpretation, and critical revision of the manuscript. All authors reviewed and approved the final manuscript. Data Availability The datasets generated and/or analyzed during the current study, including molecular docking results, molecular dynamics trajectories, and associated analyses (RMSD, RMSF, and PCA), are available from the corresponding author upon reasonable request. Publicly available data used in this study include protein structures obtained from the RCSB Protein Data Bank (PDB IDs: 7DHY, 7DHZ, and 7V97) and FDA-approved compounds retrieved from DrugBank. All computational methods and workflows are described in detail in the Methods section to ensure reproducibility. References Chen, S. et al. ‘Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site’. Cancer Cell , 39 , 2, pp. 225–239 .e8, Feb. 2021, 10.1016/j.ccell.2020.11.013 Balourdas, D. I., Markl, A. M., Krämer, A., Settanni, G. & Joerger, A. C. Structural basis of p53 inactivation by cavity-creating cancer mutations and its implications for the development of mutant p53 reactivators. Cell. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9203880","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":617659968,"identity":"c386a40b-327c-4fff-aaad-4c40cc361a3a","order_by":0,"name":"Mena Abdelsayed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCQYDhgdgVgLjA4aCA0RqSYBoYTZgMCBRC5sEUVr4ZzdvfJDwxy6xnz07reqGwR17Bvbexy/wWnLnWLFBYlty4syet9tu5xg8S2zgOW5mgdeaGzlmEokNzIkbbuSCtBxOYJBIYzPAp0P+Ro75j4Q/9Yn7gVqKgVrsCWoxANoC9PjhxA0SuduYgVoYGyTSmB/g02II9ItEYttx4xln3m6WBmpJbOM5xobXK3K3mzd++PCnWra/PXfj55yKw/b87G3MH/DqgQLHBhgLaAUwgogA9sgc4mwZBaNgFIyCEQMAGrlSlgwKqAwAAAAASUVORK5CYII=","orcid":"","institution":"Lankenau Institute for Medical Research","correspondingAuthor":true,"prefix":"","firstName":"Mena","middleName":"","lastName":"Abdelsayed","suffix":""},{"id":617659970,"identity":"54674c8f-6365-4970-8bd1-ba9fd732236d","order_by":1,"name":"Yassir Boulaamane","email":"","orcid":"","institution":"National School of Applied Sciences of Tangier, Abdelmalek Essaadi University","correspondingAuthor":false,"prefix":"","firstName":"Yassir","middleName":"","lastName":"Boulaamane","suffix":""}],"badges":[],"createdAt":"2026-03-23 18:53:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9203880/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9203880/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106227949,"identity":"a6f0f966-0b9c-421f-a01c-63c4f9cf1096","added_by":"auto","created_at":"2026-04-06 11:50:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70600,"visible":true,"origin":"","legend":"\u003cp\u003eDocking scores (kcal/mol) of the top ten rescue candidates against three mutant p53 crystal structures (7DHY, 7DHZ, and 7V97). Lower (more negative) values indicate stronger predicted binding affinity. The heatmap highlights consistency and mutation-specific differences in binding profiles across conformational variants.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/465569158ced5b86ec41a784.png"},{"id":106227954,"identity":"687dca3f-f8dc-4f01-8f6a-368d848375b5","added_by":"auto","created_at":"2026-04-06 11:50:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62736,"visible":true,"origin":"","legend":"\u003cp\u003eNon-covalent interaction profiles of prioritized FDA-approved candidates across three mutant p53 classes (V272M, R249S, and G245S), calculated using PLIP. Bars represent the total number of stabilizing interactions, including hydrogen bonds, hydrophobic contacts, salt bridges, and π-stacking interactions.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/9bdf2d3182259785dcc26beb.png"},{"id":106227955,"identity":"52254ff9-5c41-44bf-89a7-12773d7f009c","added_by":"auto","created_at":"2026-04-06 11:50:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70519,"visible":true,"origin":"","legend":"\u003cp\u003eBinding fingerprint of prioritized rescue candidates within the G245S mutant p53 core domain. Dots indicate ligand-residue contacts identified by PLIP. Gray circles represent standard non-covalent interactions, while red diamonds highlight direct engagement with rescue-relevant residues (Cys/His). Residues are shown along the x-axis and compounds along the y-axis.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/88b447c595398cb4d0cb4263.png"},{"id":106403986,"identity":"4e2dd1ad-e334-4ef2-be36-d44b24500428","added_by":"auto","created_at":"2026-04-08 09:15:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":745478,"visible":true,"origin":"","legend":"\u003cp\u003eStructural binding mode of DB09280 within the G245S mutant p53 core domain.\u003cbr\u003e\n(A) Three-dimensional representation of the ligand bound within the p53 cavity, shown in surface and cartoon view, highlighting key interacting residues surrounding the mutation site. (B) Two-dimensional interaction map illustrating hydrogen bonds, van der Waals contacts, and π-mediated interactions between DB09280 and critical p53 residues.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/d6e05cf40556d12162a58ab0.png"},{"id":106402427,"identity":"6c35a9bd-5561-4a7f-82ad-1eedc3a3ca01","added_by":"auto","created_at":"2026-04-08 09:12:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":324253,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dynamics analysis of mutant p53 in apo and holo forms over 500 ns.\u003cbr\u003e\n(A) Cα RMSD of the protein backbone for apo (blue) and ligand-bound (holo, orange) systems.\u003cbr\u003e\n(B) Ligand RMSD relative to the initial docked conformation during the simulation.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/6163fc22e517c10f9f001027.png"},{"id":106227951,"identity":"bcf5d3b2-965e-4bea-9a7a-7fdfabccb300","added_by":"auto","created_at":"2026-04-06 11:50:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61985,"visible":true,"origin":"","legend":"\u003cp\u003eCα RMSF profiles of mutant p53 in apo (blue) and ligand-bound (holo, orange) states over 500 ns MD simulation. Lower RMSF values indicate reduced residue-level flexibility upon ligand binding.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/f5eb6d1a90600e2ee3a65b0b.png"},{"id":106403253,"identity":"a9a3ffd5-c37c-4e14-a5a5-2fc49aa13a7e","added_by":"auto","created_at":"2026-04-08 09:13:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":318309,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of mutant p53 dynamics over 500 ns MD simulations in apo (left) and ligand-bound (holo, right) states. Projections along the first two principal components (PC1 and PC2) illustrate the conformational space sampled during the trajectories, colored by simulation time.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/7a6ac929f62867478251be76.png"},{"id":108492529,"identity":"9af35ff0-8c00-46ce-87cc-3349f069fabb","added_by":"auto","created_at":"2026-05-05 09:58:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1930883,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9203880/v1/410ea876-f4e2-45d7-a9d9-61c024472eb1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FDA-approved drug repurposing as p53 mutants rescue candidates using structure-based virtual screening and molecular simulations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tumor suppressor protein p53 is a central guardian of genomic integrity, orchestrating cell-cycle arrest, apoptosis, and DNA damage responses in stressed cells. In human cancers, TP53 is the most frequently mutated gene, and these alterations often disable p53\u0026rsquo;s transcriptional activity, promote malignant progression, and correlate with poor clinical outcomes. Many oncogenic TP53 variants arise within the DNA-binding domain and can be broadly divided into \u0026ldquo;DNA-contact\u0026rdquo; mutations that directly impair sequence recognition and \u0026ldquo;structural\u0026rdquo; mutations that destabilize the loop-sheet-helix motif and the underlying β-sandwich scaffold. The latter class, which includes cavity-creating substitutions such as G245S, R249S, and V272M, compromises zinc coordination and reduces thermodynamic stability, predisposing p53 to partial unfolding and aggregation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].​\u003c/p\u003e \u003cp\u003eReactivating mutant p53 has therefore emerged as an attractive yet challenging therapeutic strategy. Several approaches have been pursued, including small molecules that restore native-like conformation of structural mutants, compounds that refold misfolded p53, and agents that target auxiliary regulators such as MDM2 to indirectly enhance p53 signaling. However, most candidate reactivators show a narrow mutation spectrum, limited potency, or suboptimal pharmacokinetic properties, which has hindered their broad clinical translation. Recent work has revealed a cryptic allosteric site within the DNA-binding domain that can be targeted by cysteine-reactive small molecules such as arsenic trioxide (ATO), which stabilizes the loop-sheet-helix motif and β-sandwich fold of multiple structural p53 mutants. These studies provide a mechanistic blueprint for allosteric rescue, but the repertoire of clinically actionable ligands that exploit this pocket remains sparse [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrug repurposing offers a complementary route to expand the pool of p53-stabilizing compounds by leveraging the safety and pharmacology of existing FDA-approved agents. Structure-based virtual screening combined with molecular dynamics (MD) simulations is particularly well suited to this task, because it can account for the pronounced conformational plasticity of mutant p53 and evaluate ligand binding across multiple disease-relevant variants. In the context of the arsenic-defined allosteric pocket, such an integrative \u003cem\u003ein silico\u003c/em\u003e pipeline enables the identification of molecules that not only bind with favorable affinity but also act as dynamic stabilizers that clamp flexible loops and reinforce zinc-associated structural motifs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].​\u003c/p\u003e \u003cp\u003eIn this study, we employed a structure-based virtual screening workflow to interrogate a curated library of FDA-approved drugs against three representative mutant p53 DNA-binding domains - G245S, R249S, and V272M - whose crystal structures capture distinct structural, DNA-contact, and hydrophobic perturbations. Top-ranked candidates were subjected to detailed protein-ligand interaction profiling at the arsenic-defined allosteric site, followed by long-timescale MD simulations to assess their ability to stabilize the mutant core domain. Our results highlight the compound DB09280 as a promising p53 rescue candidate that engages key residues involved in zinc coordination and loop flexibility, and functions as a conformational clamp that restricts aberrant motions of the G245S structural mutant. By integrating virtual screening, interaction fingerprinting, and MD-based conformational analysis, this work establishes a mechanistic framework for repurposing FDA-approved drugs as broad-spectrum stabilizers of mutant p53.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ep53 mutants preparation\u003c/h2\u003e \u003cp\u003eThree relevant p53 mutants namely, G245S, R249S, and V272M (PDB IDs: 7DHY, 7DHZ, and 7V97, respectively) were retrieved from the RCSB Protein Data Bank (PDB) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Structures were selected based on the availability of high-resolution transmembrane domains, the presence of resolved or inferred fenestration pathways, and their relevance to clinically validated channelopathies. Prior to molecular docking, all protein structures were subjected to standardized preprocessing and optimization. Protonation states of titratable residues were assigned using the H\u0026thinsp;+\u0026thinsp;+\u0026thinsp;server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://newbiophysics.cs.vt.edu/H++/\u003c/span\u003e\u003cspan address=\"http://newbiophysics.cs.vt.edu/H++/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) at physiological pH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Crystallographic water molecules were removed. Atom types and Gasteiger partial charges were then assigned using ForliLab\u0026rsquo;s Meeko package, and the prepared structures were converted to PDBQT format for subsequent docking simulations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ep53 Mutants Used for Virtual Screening and Molecular Dynamics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep53 Variant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePDB ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMutation Class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStructural Consequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG245S\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7DHY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStructural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDestabilizes local secondary structure elements and perturbs critical zinc coordination loops.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR249S\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7DHZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA-contact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDisrupts direct interactions with consensus DNA sequences without completely unfolding the core.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV272M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7V97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHydrophobic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlters internal hydrophobic packing within the core beta-sandwich, inducing thermodynamic instability.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLigand library preparation\u003c/h3\u003e\n\u003cp\u003eFDA-approved compounds were extracted from DrugBank to construct the virtual screening library. Ligand structures were initially provided in SDF format and subjected to salt removal using RDKit\u0026rsquo;s SaltRemover module [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Reactive electrophilic substructures were filtered based on predefined SMARTS patterns, followed by elimination of pan-assay interference compounds (PAINS) using RDKit\u0026rsquo;s FilterCatalog to reduce false-positive risk. For each retained molecule, explicit hydrogens were added, and three-dimensional conformers were generated. Geometry optimization was performed using the MMFF94 force field to obtain energetically reasonable ligand conformations prior to docking [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The optimized ligands were then processed with Meeko (mk_prepare_ligand.py), which assigned Gasteiger partial charges, detected rotatable bonds, preserved stereochemistry, and converted structures into PDBQT format compatible with AutoDock Vina v1.2.7 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMolecular docking\u003c/h3\u003e\n\u003cp\u003eMolecular docking was performed using AutoDock Vina to evaluate binding of FDA-approved compounds to the allosteric pocket of mutant p53 corresponding to the arsenic-binding rescue site. The docking region was defined based on the experimentally characterized allosteric cavity, previously reported as a targetable rescue hotspot. The center of the docking grid was determined in UCSF Chimera using the \u0026ldquo;measure center\u0026rdquo; command applied to the arsenic-binding site, yielding coordinates of x\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;34.188, y\u0026thinsp;=\u0026thinsp;3.782, and z\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;7.713 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This center was used to construct a cubic grid box large enough to fully encompass the allosteric pocket and surrounding flexible regions of the DNA-binding domain, ensuring adequate sampling of ligand conformations. Prepared ligands in PDBQT format were docked against the prepared mutant p53 structures using AutoDock Vina with default exhaustiveness parameters. For each ligand, multiple binding poses were generated, and the top-ranked pose based on predicted binding affinity was retained for downstream analysis. Docking results were subsequently processed to extract best-scoring complexes, which were converted to PDB format for protein-ligand interaction profiling and molecular dynamics simulations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMolecular dynamics simulations\u003c/h3\u003e\n\u003cp\u003eThe top-ranked compound identified from molecular docking were selected for molecular dynamics simulations to evaluate the stability and dynamic behavior of their complexes with mutant p53. Protein-ligand systems were constructed using the CHARMM-GUI web interface, applying the CHARMM36m force field for the protein and associated parameters for the ligands [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The complex was embedded in a rectangular TIP3P water box with a minimum padding of 10 \u0026Aring; from the solute to the box boundary. Sodium and chloride ions were added to neutralize the systems and adjust ionic strength to 0.15 M. Periodic boundary conditions were applied, and long-range electrostatics were treated using the Particle Mesh Ewald method with automatically generated FFT grid dimensions. Energy minimization was performed to remove unfavorable contacts, followed by stepwise equilibration under constant volume (NVT) and constant pressure (NPT) conditions at 300 K and 1 bar, respectively. Temperature regulation was achieved using a Langevin thermostat, while pressure coupling was maintained with a Monte Carlo barostat. Production simulations were subsequently carried out for 500 ns using OpenMM [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Trajectories were saved at regular intervals for downstream analysis. Post-simulation analyses, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), principal component analysis (PCA), and ligand stability assessment, were conducted using MDTraj and MDAnalysis, as implemented in the analysis notebook [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEthics Statement\u003c/h3\u003e\n\u003cp\u003eThis study did not involve human participants, human data, or animal subjects. All analyses were performed using publicly available datasets and computational modeling approaches. Therefore, ethical approval and informed consent were not required for this work.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStructure-based virtual screening results\u003c/h2\u003e \u003cp\u003eMolecular docking was performed against three mutant p53 crystal structures (7DHY, 7DHZ, and 7V97) to evaluate the binding potential of the top ten rescue candidates. Overall, the compounds exhibited consistently favorable binding affinities across all conformations, with docking scores ranging from \u0026minus;\u0026thinsp;6.8 to \u0026minus;\u0026thinsp;8.3 kcal/mol, indicating stable interactions within the mutant p53 binding pocket. Among the screened molecules, DB11363 emerged as the strongest binder to the 7DHY structure (\u0026minus;\u0026thinsp;8.3 kcal/mol), while DB00762 and DB08896 showed the highest affinity toward 7V97 (both \u0026minus;\u0026thinsp;8.1 kcal/mol). Several candidates, including DB06589, DB01586, DB15133, and DB09280, demonstrated reproducible binding across all three structures, suggesting robustness against p53 conformational variability. Notably, DB00619 displayed balanced affinities across the panel, with improved binding to 7V97 (\u0026minus;\u0026thinsp;7.8 kcal/mol), whereas DB15233 and DB04868 showed slightly reduced interaction energies for 7DHZ, reflecting possible structural sensitivity of this mutant form. Interestingly, DB08896 exhibited a marked increase in affinity toward 7V97 despite weaker binding to 7DHZ, highlighting potential mutation-specific stabilization effects. These results indicate that multiple candidates possess consistent multi-conformer binding capability, a critical property for rescuing structurally heterogeneous mutant p53. In particular, DB11363, DB00762, DB08896, and DB00619 were prioritized based on their superior or balanced docking profiles and were selected for subsequent interaction analysis and dynamic stability assessment.\u003c/p\u003e \u003cp\u003eThe observed binding consistency across multiple mutant p53 conformations is particularly relevant given the structural plasticity associated with oncogenic p53 variants. Compounds exhibiting stable affinity across 7DHY, 7DHZ, and 7V97 are more likely to tolerate mutation-induced pocket rearrangements and may function as structural stabilizers rather than mutation-specific binders. The enhanced affinity of DB11363 and DB00762 suggests stronger stabilization potential, whereas DB08896\u0026rsquo;s mutation-dependent variability may indicate selective conformational preference. These findings support a mechanism in which small molecules bind to destabilized regions of mutant p53, potentially restoring partial structural integrity and functional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein-ligand interactions analysis\u003c/h3\u003e\n\u003cp\u003eTo complement docking scores and evaluate the structural stabilization potential of selected candidates, protein-ligand interaction profiles were analyzed using PLIP across three representative mutant p53 classes: V272M (hydrophobic), R249S (DNA-contact), and G245S (structural). Distinct interaction patterns were observed among the compounds, reflecting mutation-dependent binding behavior. DB09280 exhibited the highest overall interaction density, particularly against V272M (11 interactions) and R249S (10 interactions), suggesting strong anchoring within the destabilized pocket. DB06589 and DB00762 also demonstrated elevated interaction counts across multiple variants, indicating consistent engagement with key binding residues. Several compounds, including DB15133 and DB08896, showed preferential interaction with specific mutants, with DB08896 displaying enhanced binding to R249S despite reduced contacts with V272M. In contrast, DB11363 and DB15233 exhibited comparatively weaker interaction profiles, consistent with their moderate docking performance. Importantly, multiple candidates displayed balanced interaction distributions across all three mutant classes, highlighting their potential to act as general p53 stabilizers rather than mutation-specific ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain mechanistic insight into ligand-mediated stabilization of mutant p53, residue-level interaction fingerprints were generated for the G245S structural mutant. This analysis revealed recurrent engagement of key residues within the DNA-binding core domain, including THR6, ARG14, LEU15, PHE17, TYR30, PRO32, ASN35, LYS68, LEU56, ASN72, and GLU75.\u003c/p\u003e \u003cp\u003eDB09280 displayed the most extensive interaction network, contacting multiple hydrophobic and polar residues and uniquely engaging His19, a residue implicated in zinc coordination and structural stabilization. This interaction is highlighted as a potential rescue hotspot, suggesting a direct contribution to restoring local folding integrity. Several candidates, including DB06589, DB00619, and DB00762, showed consistent contacts with ASN35, LEU56, and ASN72, residues located within flexible regions surrounding the mutation site. Such multi-point anchoring may counteract the destabilizing effect of G245S by reinforcing local secondary structure elements. In contrast, DB11363 and DB15233 exhibited fewer residue contacts, consistent with their weaker interaction density observed in earlier PLIP analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe binding fingerprints indicate that effective rescue candidates preferentially interact with residues surrounding the destabilized β-sandwich of mutant p53. In particular, engagement of His19 by DB09280 suggests a potential mechanism involving reinforcement of zinc-associated structural motifs, a known strategy for restoring mutant p53 stability. The presence of dense interaction clusters around ASN35, LEU56, and ASN72 further supports a model in which ligands stabilize flexible loops adjacent to the mutation site, thereby promoting partial refolding toward a native-like conformation. These residue-level interactions provide molecular evidence that top candidates act as structural stabilizers rather than simple pocket binders, supporting their advancement to molecular dynamics simulations for validation of long-timescale rescue effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo visualize the molecular basis of mutant p53 rescue, the binding pose of the top-ranked candidate DB09280 was examined in detail within the G245S structure. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(A)\u003c/b\u003e, DB09280 occupies a hydrophobic pocket adjacent to the mutation site, engaging residues distributed across flexible loop regions and β-sheet elements of the DNA-binding domain. The ligand establishes a dense interaction network involving ASN35, PRO32, TYR30, LEU34, PHE17, and HIS19, forming a stabilizing scaffold around the destabilized core (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e(B)\u003c/b\u003e). Multiple hydrogen bonds anchor the ligand to ASN35 and PRO32, while π-π and π-alkyl interactions with TYR30 and surrounding hydrophobic residues further reinforce binding.\u003c/p\u003e \u003cp\u003eThe structural binding mode of DB09280 reveals a multi-point stabilization strategy, in which the ligand bridges flexible loops and β-sheet elements surrounding the G245S mutation. Rather than acting as a simple affinity-driven binder, DB09280 appears to function as a conformational clamp, reinforcing local structural elements and potentially restoring native-like folding. The interaction with HIS19 is particularly noteworthy, as perturbation of zinc coordination is a hallmark of structural p53 mutants. By engaging this residue, DB09280 may partially reconstitute the metal-supported architecture of the DNA-binding domain, providing a plausible molecular basis for rescue activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Three-dimensional representation of the ligand bound within the p53 cavity, shown in surface and cartoon view, highlighting key interacting residues surrounding the mutation site. (B) Two-dimensional interaction map illustrating hydrogen bonds, van der Waals contacts, and π-mediated interactions between DB09280 and critical p53 residues.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics trajectory analysis\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eRoot-mean square deviation\u003c/h2\u003e \u003cp\u003eTo evaluate the structural impact of ligand binding on mutant p53 stability, 500 ns molecular dynamics simulations were performed for both apo and holo systems.\u003c/p\u003e \u003cp\u003eThe Cα RMSD profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(A)\u003c/b\u003e) reveals pronounced structural fluctuations in the apo system, with deviations frequently exceeding 8\u0026ndash;10 \u0026Aring; during the early and mid simulation phases. In contrast, the holo complex exhibits comparatively moderated fluctuations, particularly after ~\u0026thinsp;350 ns, where RMSD values stabilize around 3\u0026ndash;5 \u0026Aring;. Notably, between 380\u0026ndash;430 ns, the holo system maintains a lower and more stable RMSD relative to the apo form, suggesting ligand-induced conformational stabilization. Although both systems display intrinsic flexibility, consistent with the destabilized nature of structural p53 mutants, the ligand-bound complex demonstrates reduced amplitude and shorter-lived excursions from equilibrium, indicating partial structural constraint. Ligand RMSD analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e(B)\u003c/b\u003e) further supports stable complex formation. The ligand maintains deviations primarily within 1.5\u0026ndash;2.5 \u0026Aring; throughout the 500 ns trajectory, without evidence of dissociation or major reorientation. Transient increases are observed but rapidly re-equilibrate, indicating persistent pocket occupancy and sustained interactions. The MD results reinforce the docking and interaction fingerprint analyses by demonstrating dynamic stabilization of mutant p53 upon ligand binding. While the apo structure exhibits large conformational excursions characteristic of destabilized mutants, the holo system shows comparatively restrained fluctuations, particularly during the later stages of the simulation. The stable ligand RMSD confirms that the compound remains firmly anchored within the binding pocket, enabling continuous multi-point interactions that likely reinforce local secondary structure elements. Importantly, the reduction in large-scale structural drift in the holo system supports a rescue-like mechanism driven by conformational constraint rather than global rigidification. This behaviour is consistent with small-molecule-mediated stabilization of partially unfolded p53 variants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Cα RMSD of the protein backbone for apo (blue) and ligand-bound (holo, orange) systems.\u003c/p\u003e \u003cp\u003e(B) Ligand RMSD relative to the initial docked conformation during the simulation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRoot-mean square fluctuation\u003c/h2\u003e \u003cp\u003eResidue-wise root mean square fluctuation (RMSF) analysis was performed to assess local backbone dynamics of mutant p53 in the presence and absence of the bound ligand. Overall, the holo system exhibits reduced fluctuations across multiple regions of the protein compared to the apo form (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating ligand-mediated dampening of local flexibility. The most pronounced differences are observed in the central core domain, particularly around residues\u0026thinsp;~\u0026thinsp;80\u0026ndash;100, where the apo structure displays elevated mobility reaching\u0026thinsp;~\u0026thinsp;7 \u0026Aring;, whereas the holo complex shows substantially attenuated fluctuations (~\u0026thinsp;4 \u0026Aring;). This region encompasses structural elements proximal to the G245S mutation and is critical for maintaining the integrity of the DNA-binding domain. Additional stabilization is observed in loop regions near residues\u0026thinsp;~\u0026thinsp;20\u0026ndash;30 and ~\u0026thinsp;120\u0026ndash;140, consistent with earlier interaction fingerprint analyses showing ligand engagement with residues in these flexible segments. Although terminal regions retain higher mobility, as expected for solvent-exposed tails, the core domain demonstrates clear rigidity upon ligand binding. The reduction in residue-level fluctuations within the p53 core domain suggests that ligand binding restricts excessive conformational freedom induced by the G245S mutation. By stabilizing flexible loops and adjacent secondary structure elements, the ligand likely promotes a more compact and native-like fold. This localized rigidity complements the global RMSD stabilization and supports a rescue mechanism driven by reinforcement of structurally compromised regions rather than uniform protein rigidification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis\u003c/h2\u003e \u003cp\u003ePrincipal component analysis was performed to characterize large-scale collective motions of mutant p53 in apo and holo states. Projection of the trajectories onto the first two principal components reveals markedly different conformational sampling between the two systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe apo protein explores a broad and highly dispersed conformational space, indicative of pronounced structural heterogeneity and dynamic instability. Multiple clusters are observed, reflecting frequent transitions between distinct conformational substates throughout the simulation.\u003c/p\u003e \u003cp\u003eIn contrast, the holo complex exhibits a more compact and structured distribution along PC1 and PC2, with trajectory points concentrating within fewer dominant regions. This reduced dispersion suggests ligand-induced restriction of global motions and stabilization of preferred conformational states. Time-colored projections further indicate progressive convergence toward a stable basin in the holo system, whereas the apo form continues to sample diverse conformations over the entire trajectory. The PCA results demonstrate that ligand binding reshapes the conformational energy landscape of mutant p53 by narrowing accessible structural states and promoting population of a dominant basin. This behavior is characteristic of small-molecule-mediated conformational rescue, where flexible and partially unfolded regions become constrained into energetically favorable arrangements. Together with RMSD and RMSF analyses, these findings support a mechanism in which the ligand acts as a dynamic stabilizer, suppressing aberrant motions associated with the G245S mutation and facilitating adoption of more native-like conformations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe therapeutic reactivation of mutant p53 requires small molecules capable of counteracting mutation-induced thermodynamic instability. Through an integrated computational pipeline comprising molecular docking, interaction fingerprinting, and 500 ns molecular dynamics simulations, we evaluated FDA-approved drugs as prospective p53 rescue agents. Our virtual screening identified several compounds with robust, cross-mutant binding profiles, highlighting \u003cb\u003eDB09280\u003c/b\u003e as a standout candidate capable of tolerating structural plasticity. Structural interaction analyses revealed that \u003cb\u003eDB09280\u003c/b\u003e functions not merely as an affinity-driven pocket binder, but as a conformational clamp that reinforces local secondary structure elements. By strategically anchoring to flexible loops and directly engaging His19, a residue vital for zinc coordination, the ligand addresses the core structural deficits characteristic of the G245S mutation. Long-time-scale dynamic analyses strongly corroborated this stabilization mechanism. The holo complex exhibited profound reductions in global RMSD and localized RMSF within the compromised central core domain. Furthermore, principal component analysis demonstrated that ligand binding effectively limits aberrant large-scale motions, forcing the destabilized mutant to converge into a stabilized, native-like conformational state. These \u003cem\u003ein silico\u003c/em\u003e findings provide strong molecular evidence that DB09280 acts as a potent structural stabilizer of mutant p53. This study establishes a rigorous mechanistic rationale for advancing DB09280 and structurally related FDA-approved scaffolds into \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models to validate their efficacy in restoring p53 tumor suppressor function.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDeclaration of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.A. conceived and designed the study. Y.B. performed the molecular docking, molecular dynamics simulations, and data analysis. M.A. interpreted the results and wrote the main manuscript text. Y.B. contributed to methodological development, data interpretation, and critical revision of the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study, including molecular docking results, molecular dynamics trajectories, and associated analyses (RMSD, RMSF, and PCA), are available from the corresponding author upon reasonable request. Publicly available data used in this study include protein structures obtained from the RCSB Protein Data Bank (PDB IDs: 7DHY, 7DHZ, and 7V97) and FDA-approved compounds retrieved from DrugBank. All computational methods and workflows are described in detail in the Methods section to ensure reproducibility.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen, S. et al. \u0026lsquo;Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site\u0026rsquo;. \u003cem\u003eCancer Cell\u003c/em\u003e, \u003cb\u003e39\u003c/b\u003e, 2, pp. 225\u0026ndash;239 .e8, Feb. 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccell.2020.11.013\u003c/span\u003e\u003cspan address=\"10.1016/j.ccell.2020.11.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalourdas, D. I., Markl, A. M., Kr\u0026auml;mer, A., Settanni, G. \u0026amp; Joerger, A. C. 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J.\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e (3), 272a\u0026ndash;273a. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bpj.2021.11.1368\u003c/span\u003e\u003cspan address=\"10.1016/j.bpj.2021.11.1368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Feb. 2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"p53 mutant, Drug repurposing, Molecular dynamics simulations, Virtual screening, Structural stabilization, Allosteric modulation","lastPublishedDoi":"10.21203/rs.3.rs-9203880/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9203880/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe restoration of mutant p53 stability is a highly sought-after strategy in targeted cancer therapy. This study presents a structure-based virtual screening and molecular dynamics approach to repurpose FDA-approved drugs as p53 rescue candidates. A virtual screening library of FDA-approved compounds was docked against three representative p53 mutants (7DHY, 7DHZ, and 7V97) to evaluate their binding potential. The prioritized candidates demonstrated consistent, multi-conformer binding affinities. Protein-ligand interaction profiling revealed that the candidate DB09280 possesses a highly dense interaction network, particularly against the V272M and R249S variants. Residue-level analysis of the G245S structural mutant showed that DB09280 uniquely engages His19, a crucial residue for zinc coordination, and forms stabilizing contacts with adjacent flexible loop residues, including ASN35 and PRO32. Subsequent 500 ns molecular dynamics simulations demonstrated that DB09280 acts as a conformational clamp. The ligand-bound (holo) system exhibited substantially reduced global structural drift (RMSD) and attenuated local residue fluctuations (RMSF) within the core domain compared to the highly unstable apo state. Principal component analysis further confirmed that DB09280 restricts the broad conformational sampling of the mutant into a stable, dominant energetic basin. These findings highlight DB09280 as a robust p53 stabilizer and provide a compelling mechanistic foundation for its repurposing in oncology.\u003c/p\u003e","manuscriptTitle":"FDA-approved drug repurposing as p53 mutants rescue candidates using structure-based virtual screening and molecular simulations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 11:50:14","doi":"10.21203/rs.3.rs-9203880/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aceafec4-53b0-4b0d-b0bb-14ac3f32ed8a","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-03T17:01:26+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65733981,"name":"Biological sciences/Biochemistry"},{"id":65733982,"name":"Biological sciences/Cancer"},{"id":65733983,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":65733984,"name":"Biological sciences/Drug discovery"},{"id":65733985,"name":"Biological sciences/Structural biology"}],"tags":[],"updatedAt":"2026-05-03T17:10:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 11:50:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9203880","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9203880","identity":"rs-9203880","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}