Molecular Mechanism Of Capsaicin Analogs Regulating TRPV1 Channel Activation

Objectives:The TRPV1（transient receptor potential vanillic acid 1）channel acts as a non-selective cation channel that can be activated by a variety of harmful stimuli such as ligands,acids,hyperthermia,and animal toxins.TRPV1 is widely distributed in afferent neurons detecting environmental stimuli and is therefore an important receptor for sensing and regulating pain.Previous studies have shown that inhibiting TRPV1 channels can produce analgesic effects.Therefore,the development of new analgesics targeting the TRPV1 channel has become a focus for many multinational pharmaceutical companies.However,TRPV1channel inhibitors can cause side effects of raising body temperature,thus resulting in failures in their clinical trials stage.This is also one of the most important reasons that development of TRPV1 channels modulators research has been on hold.Recent studies have demonstrated that TRPV1 responds differently to different stimuli,suggesting that it is feasible to exert analgesic effects by selectively inhibiting TRPV1 channels binding to its ligands without interfering with temperature regulation.Although the binding mode and molecular interaction between TRPV1 and the specific TRPV1 channel agonist capsaicin have been well described,the molecular mechanism of TRPV1 channel inhibitors in binding and gating process is still unclear.In this study,we selected AMG9810,a TRPV1 inhibitor with a similar structure to capsaicin and affecting body temperature,as a representative of TRPV1 channel inhibitor to study the molecular mechanism underlying the interaction between AMG9810 and TRPV1channel,and then based on these mechanisms carrying out rational structure optimization of AMG9810 and functional verification of structural modification compound,finally obtain lead molecules with highly effective and little effect on temperature targeting TRPV1 with modality-specificity.The proposed project will provide reliable theoretical guidance for the development of novel analgesic drugs targeting TRPV1.Methods:Firstly,the molecular docking software was used to simulate the mode of AMG9810 binding to the TRPV1 channel.The key residues forming hydrogen bonds in the pair interface cave were predicted before,single or double site-directed mutations were made using molecular biology methods.The human and murine-derived wild-type TRPV1or mutants were transfected onto HEK293T cells and TRPV1 currents were recorded using whole-cell or inside-out mode patch-clamp assays.Finally,AMG9810 was structurally modified and its derivatives were tested on inhibition of TRPV1 channel function.Results:1.AMG9810 concentration-dependently inhibited the capsaicin activation on murine TRPV1 with an IC₅₀of 1.65±0.56μM（n=3）.2.AMG9810 inhibited the ligand AEA activation on murine wild-type TRPV1,mutant T551V and S513A with IC₅₀s of 0.53±0.03μM（n=3）,8.65±2.78μM（n=3）,and 1.52±0.33μM（n=3）respectively.Compared with wild-type,the application of AMG9810 caused a significant rightward shift of IC₅₀values of mutants for mutants T551V and S513A,while T551V showed a more significant increase of IC₅₀relative to S513A.3.Using whole-cell patch-clamp recording,we observed that the IC₅₀values of AMG9810on ligand 2-APB activation for murine wild-type TRPV1 and different mutants were as below:wild-type TRPV1:IC₅₀=0.05±0.01μM（n=3）;S513A:IC₅₀=0.07±0.01μM（n=4）;Relative to wild-type TRPV1,the IC₅₀curve of mutant S513A did not shift significantly to the right;Y512A:IC₅₀>1μM（n=3）,and the IC₅₀values for mutant T551V and double mutant T551V-Y512A:IC₅₀>100μM（n=3）.The IC₅₀values of mutants T551V,Y512A and double mutant T551V-Y512A changed significantly relative to wild-type TRPV1 and mutant S513A.4.AMG9810 inhibited the p H activation on murine wild-type TRPV1,mutant S513A,T551V,Y512A and double mutant T551V-Y512A,the IC₅₀of AMG9810 inhibiting wild-type TRPV1 was 1.42±0.04μM（n=6）,for the S513A was 2.31±0.24μM（n=3）,and for the T551V,Y512A and double mutant T551V-Y512A were greater than 150μM（n=3 to 4）.Compared with wild-type TRPV1 and mutant S513A,the IC₅₀values of T551V,Y512A and double mutant T551V-Y512A all showed a significant rightward shift.5.For temperature activation studies,we used 2-APB（a non-selective TRPV1 agonist）to shift the entire temperature recording results to the left by lowering the temperature threshold of wild-type TRPV1,so we can easily observe the temperature activation process of AMG9810.The inside-out patch clamp records of AMG9810 on 2-APB co-temperature-activated human wild-type TRPV1,mutants S512A,Y511A and T550V showed that the thermal activation threshold of 2-APB-activated wild-type TRPV1 occur significant left-shift to 31.1±1.8°C（n=5）.The application of low concentration AMG9810 reversed this left-shift to 37.7±2.1°C（n=9）.In addition,the thermal activation threshold for that high concentration increased to 38.1±2.1°C（n=9）;AMG9810 also inhibited the thermal activation threshold of 2-APB co-temperature-activated mutant S512A at 37.4±1.6°C（n=6）.As for mutant T550V,the thermal activation threshold was 33.5±1.9°C（n=4）and that of mutant Y511A was 43.2±0.9°C（n=5）.Compared with wild-type TRPV1 and mutant S512A,the mutations T550 and Y511 significantly changed thermal activation threshold of AMG9810.6.Compared with AMG9810,the structural modification compounds AMG9810-1 and AMG9810-2 did not significantly change the IC₅₀values.But the inhibition of TRPV1channel activation by AMG9810-3 was significantly more effective than AMG9810.Conclusions:1.Similar with capsaicin and AEA,AMG9810 binds to the VBP（vanilloid binding pocket）in TRPV1.2.AMG9810 likely binds to TRPV1 channel in two different modes in VBP,（1）the"head-up,tail-down"binding mode:hydrogen bonding with the residue T551 in the S4 fragment through the"neck"binds to VBP;（2）the combination of"head down,tail up":through the interaction of"head"with residue S513,"neck"with residue Y512 to form hydrogen bonds.3.Through binding to T551 or Y512/S513,AMG9810 not only modulates TRPV1 channel activation by agonists but proton or temperature,and is therefore not a competitive antagonist of TRPV1.4.Compared with“tail”,the structural modification of AMG9810"head"and"neck"had no significant change for ligand-induced TRPV1activation.Therefore,our findings suggest that the modification of the"tail"of TRPV1inhibitor AMG9810 may guide potential development of the channel regulators without side effects affecting body temperature.