Molecular Mechanism Of TRPM8S2Specific Recognition For Menthol Optical Isomers

Over the several thousand years, the mint has been used for reducing inflammation, sterilization, anti-virus, anti-cancer, tranquillization, spasmolysis, and exciting of central nervous system. Now it also finds wide applications in medical, flavor and fragrance, and food industries. The major active agent menthol, which was listed by FDA as a safe, non-toxic drug, is one of the most widely used cooling agents. The menthol molecular has three chiral carbon atoms and exists as eight optical (optically active) isomers. As a cooling agent, the eight optical isomers take on different cooling effects. It was found that the menthol’s cooling acting sites was on the Tyr745at helix S2of transient receptor potential cation channel subfamily M member8(TRPM8S2), yet the experimental structure of TRPM8remains unknown and how cooling agent acts on TRP in terms of molecular mechanism is not so clearly. The considerable differences displayed in the cooling effects of the eight optical isomers are not yet identified. The study of TRPM S2recognizing menthol molecules will be of vital importance to indentifying the TRPM8structure, understanding how cooling agent acts as well as to developing new cooling agents and targeted medicines.Firstly, we used Gaussian98(a very powerful electronic structure calculation package) to calculate out the structural data of the eight optical isomers, then used Spss18.0software to analyze the differences of the said structural data and their relevance to the cooling effects. Results showed that1) there are considerable differences in bond lengths, bond angles and dihedral angles in the structure of the eight isomers; that2) the coplanarity of the menthol carbon molecules is good for the cooling effect; and that3) the dipole moment thereof also adds to the cooling effect.By using Discovery Studio software, we homology-modeled a TRPM8S2, then optimized its initial structure and analyzed its kinetics, electrostatic potential and pharmacophore, finding the major acting sites of the molecular docking in which H-bond donor/receptor, surface electrostatic potential and hydrophobic-/hydrophilic groups are involved.With Discovery Studio software, we conducted the semi-flexible, accurate molecular docking between TRPM8S2part and the eight optical isomers, and also calculated the bonding energy between donor and receptor. The results showed:1) the bonding energy of the eight optical isomers was closely related (Relevance R2=0.6922) with the menthol’s cooling activity;2) Van der Waals force was the largest contributor to the interactions between donor and receptor, followed by electrostatic force;3) there were no considerable H-bond interactions between TRPM8S2part and menthol; and4) the acting sites of menthol onto TRPM8S2were the active cavities formed by such amino acids (as Asn741, Val742, Val743, Tyr745, Ile746, Leu749and Leu750) in S2helix chains.Given the presence of merely eight isomers for menthol, we thought it unreasonable to conduct a Structure-Activity Analysis (SAP). Instead, by using enaminone with the very cooling effect similar to menthol, we conducted a2D and3D quantitative SAP to build up a model of the relations between cooling intensity and structure. In the2D SAP, we calculated more than1000molecular descriptors (in20classes) of those enaminone cooling agents using molecular connectivity index method, finding six indices descriptive of the cooling activity. The said indexes are all related to their topology and electric property, proving that the indexes (e.g., molecular volume) have a considerable effect on the cooling activity. In the3D SAP, we obtained all molecularly superimposed conformations using common substructure method and then developed a Comparative Molecular Field Analysis (CoMFA) model and a Comparative Molecular Similarity Indices Analysis (CoMSIA) model. Results showed that1) in the CoMFA analysis, the cooling activity was most affected by stereo field (51.6%of the molecular force field) and electrostatic field (48.4%of the molecular force field), showing that the stereo structure has a relatively large effect on the activity and that the cooling receptor’s acting sites are highly stereo selective;2) in the CoMSIA analysis, the cooling activity was most affected by hydrophobic field (33.4%of the molecular force field) and electrostatic field (31.8%of the molecular force field), showing that both hydrophobic property and electrostatic field have a relatively large effect on the activity. The conclusions above are in consistent with the results of molecular docking between TRPM8S2and the eight optical isomers.To conclude, we proposed a molecular mechanism of TRPM8S2’s specific recognition of menthol optical isomers. This explains why there are differences in the cooling effects of the eight menthol optical isomers. The results provide insight in unraveling how the cooling agents act on TRPM8in terms of molecular basis and mechanism, identifying the TRPM8structure and developing new cooling agents and targeted medicines.