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bioRxiv · 10.64898/2026.02.25.707690

Transcriptome-based lead generation, ligand- and structure-based prioritization and experimental validation of TLR5-activating molecules

Jain, A.·Hungharla, H.·Subbarao, N.·Tandon, V.·Ahmad, S.
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Abstract

Current in silico drug discovery protocols ubiquitously depend on lead generation using a ligand-based approach in which novel leads are generated by fragment-signature matching or by a structure-based search involving molecular docking and conformational dynamics. None of them incorporates cellular contexts in which these drugs ultimately operate, leaving the task to a later stage of optimization leading to a high failure rate. Incorporating systems-level responses of drugs in an early stage of lead generation can significantly address this concern but has not been sufficiently explored. In this work, we employ a systems-level approach using connectivity map (CMAP) library to generate leads against a challenging system of a TLR pathway. Starting with gene expression data of TLR5 activation by its natural ligand, we generated molecular leads using CMAP and rigorously analyzed their validity using ligand and structure-based approaches, and helping to prioritize top hits. Experimental validation using ELISA-based antibody assay confirmed the activation of TLR5 by each of the top nine prioritized leads with their dose-dependent patterns suggesting that some of them may actually interact with the TLR signaling pathway in a complex manner. Although, demonstrated on TLR5, the proposed framework is intuitively scalable to other lead generation and optimization tasks.

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Jain, A., Hungharla, H., Subbarao, N., Tandon, V., Ahmad, S.. 2026-02-26. Transcriptome-based lead generation, ligand- and structure-based prioritization and experimental validation of TLR5-activating molecules. https://doi.org/10.64898/2026.02.25.707690

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