DHFR inhibitors(Vyera Pharmaceuticals LLC)

Ectopic pregnancy (EP) remains a major cause of maternal morbidity and mortality during the first trimester, affecting approximately 1-3 % of all pregnancies and often necessitating invasive surgery when medical therapy fails. Methotrexate (MTX), a dihydrofolate reductase (DHFR) inhibitor, is the current gold standard for medical management, yet its limitations include treatment failure rates of 10-20 % and significant systemic toxicity. This study aimed to design and evaluate novel AI-fragmented MTX derivatives as improved DHFR inhibitors for EP. The three-dimensional structure of DHFR was retrieved from the AlphaFold Protein Structure Database and validated with a pLDDT score of 96.15, a Ramachandran plot showing 93.1 % residues in most favored regions, and an ERRAT quality factor of 98.85, confirming its suitability for computational studies. Protein-protein interaction mapping confirmed DHFR's central role in folate metabolism. Using an AI-driven fragmentation approach, 20 MTX analogues were generated, of which Derivative 8 (Replace) emerged as the most promising. Docking simulations demonstrated a binding affinity of -10.6 kcal/mol compared to MTX (-8.6 kcal/mol), with detailed interaction analysis revealing extensive hydrogen bonding, electrostatic stabilization, and hydrophobic packing within the DHFR active site. Molecular dynamics (500 ns) confirmed the stability of the DHFR-Derivative 8 complex, with backbone RMSD stabilizing at 0.15-0.20 nm, radius of gyration averaging 1.61 nm, consistent SASA (∼970-980 Ų), and stable hydrogen bonding throughout. Free energy calculations further supported binding, with MM-GBSA yielding a favorable ΔG_total of -45.54 kcal/mol. Pharmacophore characterization showed Derivative 8 possessed enhanced features (3 aromatic rings, 7 acceptors, 5 hydrophobic centers), while DFT analysis revealed a narrower HOMO-LUMO gap, indicating higher electronic reactivity. ADMET analysis predicted reduced toxicity for Derivative 8 (LD50 ≈ 135 mg/kg, class III) compared to MTX (LD50 ≈ 3 mg/kg, class I). Collectively, these results suggest that Derivative 8 is a more potent and safer DHFR inhibitor than MTX. Limitations include reliance on in-silico methods without experimental validation; thus, future directions involve synthesis, in vitro assays, and reproductive toxicity evaluation.