Nebivolol as a Potent TRPM8 Channel Blocker: A Drug-Screening Approach through Automated Patch Clamping and Ligand-Based Virtual Screening

Transient Receptor Potential Melastatin 8 (TRPM8) from the melastatin TRP channel subfamily is a non-selective Ca2+-permeable ion channel with multimodal gating which can be activated by low temperatures and cooling compounds, such as menthol and icilin. Different conditions such as neuropathic pain, cancer, overactive bladder syndrome, migraine, and chronic cough have been linked to the TRPM8 mode of action. Despite the several potent natural and synthetic inhibitors of TRPM8 that have been identified, none of them have been approved for clinical use. The aim of this study was to discover novel blocking TRPM8 agents using automated patch clamp electrophysiology combined with a ligand-based virtual screening based on the SwissSimilarity platform. Among the compounds we have tested, nebivolol and carvedilol exhibited the greatest inhibitory effect, with an IC50 of 0.97 ± 0.15 µM and 9.1 ± 0.6 µM, respectively. This study therefore provides possible candidates for future drug repurposing and suggests promising lead compounds for further optimization as inhibitors of the TRPM8 ion channel

Identifying antagonist drugs for TRPM8 ion channel as candidates for repurposing

Even though it is confirmed that ion channels are at the centre of many diseases, approved drugs are only available for small percentage of these proteins, and yet many pathologically important ion channels like transient receptor potential (TRP) cation channels remain without approved drugs. One reason could be the time-consuming and expensive process in drug discovery. Which has high possibility of failure in any step even after approval and marketing. Therefore, repurposing approved drugs might be considered as a solution and may offer an accelerated procedure in finding new treatments for patients. For the present research we selected TRPM8 ion channel as a neglected target despite growing number of studies regarding its association with numerous diseases. In this project we have first identified potent antagonists for TRPM8 ion channel among approved drugs, by using mainly the automated patch clamp device IonFlux 16. Such device allowed us to screen blocking potency of drugs against TRPM8 ion channel in time efficient way. Our approach consisted of using ligand-based virtual screening method, to optimize our screening by identifying candidates for further screening. We also studied possible interactions of identified drugs with antagonist binding site on TRPM8 channel by molecular docking. Furthermore, we have evaluated the effects of identified antagonists against different types of pancreatic ductal adenocarcinoma (PDAC) cells. We were able to identify four drugs with IC50 lower than 50 µM including propranolol, propafenone, carvedilol and nebivolol. Among them nebivolol with IC50 = 0.97± 0.15 µM and carvedilol with IC50 = 9.1 ± 0.6 µM were the most potent blockers. Studying the interactions of identified drugs with known binding site of TRPM8 by molecular docking, revealed high possibility of direct binding of nebivolol to binding site of TRPM8. Nebivolol was the most cytotoxic drug against PDACs, but it was also toxic against non-cancerous HEK-293 cells. While carvedilol had cytotoxic against PDACs, interestingly it wasn’t cytotoxic against HEK-293 cells. Result of these study will provide promising candidates for drug repurposing and will propose promising lead compound in drug discovery for new antagonists of TRPM8 ion channel. Also, our method of approach for identifying candidate drugs as agonist or antagonist could be applied for other ion channels