Identification And Pharmacological Characterization Of Two Natural Coumarin Enantiomers For Selective Inhibition Of Heat-activated TRPV2 Channels

Transient receptor potential vanilloid 2?TRPV2?is a non-selective cation channel which broadly expressed in the peripheral and central nervous systems.TRPV2 is activated by extremely noxious heat?>53°C?,membrane stretch,cell swelling as well as a number of chemical ligands.TRPV2 is implicated in the pathology of diseases,such as diabetes and pancreatitis.However,the physiological and pharmacological functions of TRPV2channels haven't been extensively investigated because of the absence of specific modulators.Mammalian body mainly contains brown adipocytes?BAT?and white adipocytes?WAT?.The differentiated brown adipocyte contains numerous mitochondria,the presence of an uncoupling protein 1?Ucp1?in mitochondria uncouples the electron transport chain,and causes the generation of heat allowing for non-shivering thermogenesis.Makoto et.al found that TRPV2 wasimportant for the maintenance of thermogenic function in brown adipose tissue and also for the regulation of brown adipocyte differentiation.Due tolacking of specific inhibitors for TRPV2,the physiological and pharmacological functions of TRPV2 in tissues such as brown adipose tissues remain largely unknown.Therefore,it is necessary to identify TRPV2 specific inhibitors that can be used either as tools for study of TRPV2 function or lead compounds.Objective:To identify TRPV2-specific inhibitors and evaluate their effects on exogenously expressed TRPV2 and endogenously expressed TRPV2,and to determine the binding sites of inhibitors to TRPV2,providing valuable tools for further elucidation of TRPV2 channel function.Methods:Using Flexstation 3 to identify TRPV2 inhibitors;whole-cell path clamp were used to determine the dose-dependent and selective inhibition of TRPV2 by the enantiomers;The expression of TRPV2 in brown adipocytes and the inhibition of endogenous TRPV2 currents by the enantiomers in differentiated mouse brown adipocytes were determined by RT-PCR,Western blot,and whole-cell patch clamp;the binding sites of B304-1 and B304-2 to the TRPV2 channel are determined by using molecular docking and point mutation.Results:We used calcium imaging,Flexstation 3 assay and whole-cell patch clamp recording confirmed that B304-1 and B304-2 inhibited TRPV2 channels;Whole-cell patch clamp recordings further confirmed that B304-1 and B304-2 selectively inhibited TRPV2current activated by 2-APB?2 mM?in dose-dependent manner with an IC₅₀ value of 34.2±7.8?M?B304-1?and 3.7±0.7?M?B304-2?;TRPV2 is highly expressed in brown adipocytes,and the expression of TRPV2 in differentiated brown adipocytes is significantly higher than pre-differentiated adipocytes;B304-1 and B304-2 can also inhibit endogenous TRPV2 current in differentiated brown adipocytes,and reverse TRPV2 agonist-induced inhibition of mouse brown adipocyte differentiation;finally,we performed molecular docking and point mutations found that B304-1 and B304-2 are confined in the sites near upper gate of the ion conductance pathway,and the residue I600 forming van der Waals force with B304-2 or hydrogen bond with B304-1 is critical for their inhibition effects.In this study,we report a pair of natural coumarin derivative enantiomers?-?-murraxocin?B304-1?and?+?-murraxocin?B304-2?from the root of Murraya exoyica for their selective inhibition of TRPV2 channels expressed in HEK293 cells and native TRPV2 currents in differentiated brown adipocytes.And our identification of two coumarin enantiomers B304-1 and B304-2 provides valuable tools and chemical leads for further elucidation of TRPV2 channel function,and pharmacological modulation of thermoTRPV2 in brown adipocytes may represent a new therapeutic strategy for treatment of energy imbalance or metabolic disorders.Conclusions:1.In TRPV2 over-expression HEK293 cells,B304-1 and B304-2 dose-dependent andselective inhibit TRPV2 channels.2.TRPV2 is highly expressed in brown adipocytes,and the expression of TRPV2 indifferentiated brown adipocytes is significantly higher than pre-differentiatedadipocytes,indicating that TRPV2 play an important role in BAT differentiated andthermogenesis.3.B304-1 and B304-2 could inhibit endogenous TRPV2 currents in differentiated mousebrown adipocytes,and reverse TRPV2 agonist-induced inhibition of mouse brownadipocyte differentiation.Indicating that coumarin enantiomers provides essentialtools for further understanding of TRPV2 channel physiology and pharmacology inBAT.4.The binding region of B304-1 and B304-2 to TRPV2 is the selective filter,providing a lead compound for TRPV2 inhibitors with better selectivity.