Exploiting the Achilles' heel of cancer through a structure-based drug-repurposing approach and experimental validation of top drugs using the TRAP assay.

Telomerase, a reverse transcriptase implicated in replicative immortality of cancers, remains a challenging target for therapeutic intervention due to its structural complexity and the absence of clinically approved small-molecule inhibitors. In this study, we explored drug repurposing as a pragmatic approach to address this gap, leveraging FDA-approved drugs to accelerate the identification of potential telomerase inhibitors. Using a structure-based drug discovery framework, we screened the DrugBank database through a previously validated pharmacophore model for the FVYL pocket in the hTERT thumb domain, the established binding site of BIBR1532. This was followed by molecular docking, pharmacokinetic filtering, and molecular dynamics (MD) simulations to evaluate the stability of protein-ligand complexes. Binding free energy calculations (MM-PBSA and MM-GBSA) were employed for cross-validation, identifying five promising candidates. Experimental validation using the Telomerase Repeat Amplification Protocol (TRAP) assay confirmed the inhibitory potential of Raltitrexed, showing significant inhibition with IC₅₀ 8.899 µM in comparison to control. Decomposition analysis and Structure-Activity Relationship (SAR) studies further offered insights into the binding mechanism, reinforcing the utility of the FVYL pocket as a druggable site. Raltitrexed's dual mechanism of action, targeting both telomerase and thymidylate synthase, underscores its potential as a versatile anticancer agent, suitable for combination therapies or standalone treatment. As the top lead, Raltitrexed demonstrates the potential of repurposed drugs in telomerase-targeted therapies, offering a time and cost-effective strategy for advancing its clinical development. The study also provides a robust framework for future drug development, addressing challenges in targeting telomerase for anticancer therapy.