Functional restoration of conformational (R175H) and contact (R273H) mutant p53 by Bacillus-derived Hydroxymycotrienin A in non-small cell lung cancer: a computational approach
Hydxymycotrienin A is identified as a promising, bioavailable ‘privileged scaffold’ that offers a non-covalent therapeutic strategy for NSCLC, characterizing Hydroxymycotrienin A as a promising, bioavailable ‘privileged scaffold’ that offers a non-covalent therapeutic strategy.
Abstract
Introduction The TP53 mutations in Non-Small Cell Lung Cancer (NSCLC) remain a formidable clinical challenge. Current strategies, using the covalent binder APR-246, are limited by off-target toxicity and resistance. Methods This study screens 1,580 Bacillus-derived metabolites to identify novel non-covalent pharmacological chaperones for native, conformational (R175H), and contact (R273H) p53 mutants. Virtual screening, ADMET profiling, molecular docking, extended 500 ns Molecular Dynamics (MD) simulations and Principal Component Analysis, against experimental anti-cancer drug APR-246, and top hit compounds. Results Hydroxymycotrienin A emerged as the potential candidate, demonstrating thermodynamic superiority with binding affinities of −6.63 kcal/mol (R175H) and −6.57 kcal/mol (R273H), demonstrated significant superior non covalent docking affinity compared to APR-246 parent scaffold (approximately −3.5 kcal/mol) in the mutated p53 protein, suggesting a direct pharmacological chaperone activity. Unlike the covalent alkylating mechanism of APR-246, Hydroxymycotrienin A utilizes a non-covalent network to chaperone the mutant p53. MD simulations revealed that Hydroxymycotrienin A acted as a structural stabilizer for the conformational mutant R175H by suppressing atomic fluctuations within the L2 loop and reducing overall structural deviations. In the contact mutant R273H, the ligand stabilized the DNA-binding interface while maintaining favorable conformational dynamics without introducing steric clashes. Discussion ADMET profiling predicts high bioavailability and a non-toxic safety profile, characterizing Hydroxymycotrienin A as a promising, bioavailable ‘privileged scaffold’ that offers a non-covalent therapeutic strategy for NSCLC. By restoring p53 function, this study addresses Sustainable Development Goals Target 3.4 to reduce premature mortality from non-communicable diseases. However, future in vitro and in vivo studies are required to validate the efficacy of these compounds.
The 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 nominate FDA-approved drugs as candidate stabilizers of mutant p53 for downstream experimental validation. 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 were consistent with DB09280 acting as a putative conformational clamp on the timescale sampled. The ligand-bound (holo) system exhibited substantially reduced global structural drift (RMSD) and attenuated local residue fluctuation (RMSF) within the core domain compared to the highly unstable apo state. Principal component analysis further indicated that DB09280 restricts the broad conformational sampling of the mutant into a stable, dominant basin within the sampled trajectory. Together, these computational findings nominate DB09280 as a promising candidate structural stabilizer of mutant p53 worthy of experimental follow-up. We emphasize that the in silico stabilization observed here is not equivalent to functional rescue of p53 transcriptional activity; biochemical, biophysical, and cell-based assays will be required to establish whether DB09280 restores wild-type-like DNA binding or tumor-suppressor function in mutant p53 contexts.
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Therapy-induced lineage plasticity is a major mechanism of resistance in advanced prostate cancer, enabling tumor progression despite androgen receptor (AR)-directed therapies and promoting emergence of lethal neuroendocrine prostate cancer (NEPC). Our prior studies identified the pseudokinase Tribbles homolog 2 (TRIB2) as a key regulator of therapy resistance, lineage plasticity, and neuroendocrine (NE) differentiation. Despite strong biological rationale, TRIB2 remains difficult to target because pseudokinases lack catalytic activity and often possess shallow or poorly defined ligand-binding pockets. However, structural analyses indicate that TRIB2 retains a conserved ATP-binding pocket involved in regulating protein stability and scaffolding functions, suggesting a tractable therapeutic vulnerability.
We employed a structure-guided drug discovery workflow integrating computational docking, biochemical validation, thermal shift assay (TSA), cellular thermal shift assay (CETSA), and functional screening to identify compounds targeting the TRIB2 pseudokinase domain. Lead compounds were evaluated for target engagement, selectivity, and biologic activity in enzalutamide-resistant and NEPC models. Mechanistic studies assessed downstream signaling, lineage-plasticity markers, apoptosis, and viability in antiandrogen-resistant and engineered TRIB2-expressing systems.
Structure-guided optimization identified TBI-001 as a bioactive TRIB2-directed small molecule with favorable pharmacologic properties. TSA and CETSA confirmed direct interaction between TBI-001 and TRIB2 in biochemical and cellular contexts. TBI-001 induced TRIB2 destabilization and suppressed downstream survival signaling, including AKT and BCL2 pathways. Treatment reduced expression of TRIB2 regulated lineage-plasticity and NE regulators, including MYCN, EZH2, ASCL1, BRN2 and SOX2. Mechanistically, TRIB2 actively suppresses AR signaling to facilitate resistance; accordingly, TRIB2 inhibition restored AR pathway activity and resensitized resistant cells to enzalutamide. Functionally, TBI-001 selectively reduced viability and induced apoptosis in enzalutamide-resistant and NEPC models while sparing nonmalignant prostate epithelial cells. In xenograft studies, TBI-001 inhibited tumor growth, reduced NE marker expression, and was well tolerated without overt toxicity. Human liver microsomal stability assays demonstrated low intrinsic clearance and prolonged microsomal half-life, supporting favorable drug-like pharmacokinetic properties.
These findings establish TRIB2 as a druggable vulnerability in enzalutamide-resistant and NE prostate tumors despite the inherent challenges of targeting pseudokinases. Pharmacologic disruption of TRIB2 signaling using TBI-001 suppresses oncogenic and lineage-plasticity programs, restores sensitivity to AR-directed therapy, and selectively impairs survival of resistant tumor cells. This work provides a framework for development of first-in-class TRIB2-directed therapeutics for aggressive prostate cancer subtypes.
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