Discovery and characterization of a positive allosteric modulator of transient receptor potential canonical 6 (TRPC6) channels.

The non-selective second messenger-gated cation channel TRPC6 (transient receptor potential canonical 6) is activated by diacylglycerols (DAG) in a PKC-independent manner and plays important roles in a variety of physiological processes and diseases. In order to facilitate novel therapies, the development of potent inhibitors as well as channel-activating agents is of great interest. The screening of a chemical library, comprising about 17,000 small molecule compounds, revealed an agent, which induced increases in intracellular Ca2+ concentrations ([Ca2+ ]i ) in a concentration-dependent manner (EC50  = 2.37 ± 0.25 μM) in stably TRPC6-expressing HEK293 cells. This new compound (C20) selectively acts on TRPC6, unlike OAG (1-oleoyl-1-acetyl-sn-glycerol), which also activates PKC and does not discriminate between TRPC6 and the closely related channels TRPC3 and TRPC7. Further evaluation by Ca2+ assays and electrophysiological studies revealed that C20 rather operated as an enhancer of channel activation than as an activator by itself and led to the assumption that the compound C20 is an allosteric modulator of TRPC6, enabling low basal concentrations of DAG to induce activation of the ion channel. Furthermore, C20 was tested in human platelets that express TRPC6. A combined activation of TRPC6 with C20 and OAG elicited a robust increase in [Ca2+ ]i in human platelets. This potentiated channel activation was sensitive to TRPC6 channel blockers. To achieve sufficient amounts of C20 for biological studies, we applied a one-pot synthesis strategy. With regard to studies in native systems, the sensitizing ability of C20 can be a valuable pharmacological tool to selectively exaggerate TRPC6-dependent signals.