Title : Therapeutic repositioning of Carglumic acid and Mesalazine against mycobacterium tuberculosis by targeting its essential enzyme ThyX: An overview of the biophysical strategies toward novel drug repurposing framework
Abstract:
Tuberculosis (TB) is a deadly disease that is difficult to treat. Existing drugs are not always effective, and new drugs are needed. Drug repurposing has become a popular approach for drug discovery due to the advancements in high throughput computational screening methods. Structurebased drug discovery, which involves computational screening of compounds followed by in-vitro testing, is a common method for drug repurposing. However, this method does not provide mechanistic insights into how these compounds interact with and affect their targets. This study addresses this gap by integrating highly sensitive biophysical methods into existing computational repurposing methods. The study also validates the computational and biophysical findings on H37Rv for the anti-mycobacterial activity of selected drugs in-vitro and ex-vivo conditions. Carglumic acid and Mesalazine were identified as high-affinity binders to flavin-dependent thymidylate synthase (FDTS), also known as ThyX through multi-stage docking and were further validated using biophysical studies. The binding affinities of these drugs (K~ 106 M-1) were determined using fluorescence quenching experiments. Differential Scanning Fluorimetry (DSF) and urea-based chemical denaturation studies demonstrated a destabilizing effect of these drugs on the target, which was further confirmed by MD simulations. Conformational changes in secondary structures were characterized using CD spectra. Additionally, Carglumic acid and Mesalazine inhibited M.tb growth in-vitro and ex-vivo while exhibiting no toxicity to mice peritoneal macrophages. Hence our findings suggest that these drugs could be taken forward as a potential anti-TB drug and can be further validated in animal and clinical models that could be repurposed to treat TB.
