Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a β–lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

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Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic β–lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3’-phenyl-2’-dibenzylamino)prop-1’-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2’R > 3S,4S,2’R ≅ 3R,4R,2’S > 3S,4S,2’S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these β-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.

Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug–protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC50 1.25 ± 0.26 μM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 μM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.

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