Abstract

Transient receptor potential melastatin 3 (TRPM3) is a calcium-permeable nonselective cation channel that is expressed in a subset of dorsal root (DRG) and trigeminal ganglia sensory neurons. TRPM3 can be activated by the neurosteroid pregnenolone sulfate (PregS) and heat. TRPM3^−/− mice display an impaired sensation of noxious heat and thermal hyperalgesia. We have previously shown that TRPM3 is blocked by the citrus fruit flavanones hesperetin, naringenin, and eriodictyol as well as by ononetin, a deoxybenzoin from Ononis spinosa. To further improve the tolerability, potency, and selectivity of TRPM3 blockers, we conducted a hit optimization procedure by rescreening a focused library that was composed of chemically related compounds. Within newly identified TRPM3 blockers, isosakuranetin and liquiritigenin displayed favorable properties with respect to their inhibitory potency and a selective mode of action. Isosakuranetin, a flavanone whose glycoside is contained in blood oranges and grapefruits, displayed an IC₅₀ of 50 nM and is to our knowledge the most potent inhibitor of TRPM3 identified so far. Both compounds exhibited a marked specificity for TRPM3 compared with other sensory TRP channels, and blocked PregS-induced intracellular free Ca²⁺ concentration signals and ionic currents in freshly isolated DRG neurons. Furthermore, isosakuranetin and previously identified hesperetin significantly reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Because the physiologic functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo.