Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein

Abstract

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4–S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

Effect of Tempeh and Daidzein on Calcium Status, Calcium Transporters, and Bone Metabolism Biomarkers in Ovariectomized Rats

“…Similarly, the alterations in bone biomarkers, such as increased bone alkaline phosphatase [ 61 ] and procollagen type I N-terminal propeptide [ 62 ] levels, are consistent with studies highlighting the bone-forming properties of these phytoestrogens. Although our research did not show the expected impact on TRPV5 and TRPV6 mRNA expression, but these results confirmed several studies [ 63 , 64 ]. Thus, our results contribute valuable insights into the complex mechanisms underlying the bone-protective effects of daidzein and tempeh, emphasizing the need for further investigation to elucidate their full therapeutic potential in managing bone health, particularly in menopausal osteoporosis.…”

“…By targeting these estrogenic pathways, isoflavones contribute to the enhancement of bone metabolism and a concurrent decrease in bone resorption, ultimately promoting bone health. Furthermore, their ability to modulate other pathways involved in calcium homeostasis [ 56 ], such as vitamin D metabolism [ 75 ] and calcium transport [ 64 ], further underscores their potential as therapeutic agents for osteoporosis.…”

Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6

“…Of note, in all our analysis, levels of inhibition were considerably higher when derived from electrophysiological measurements compared to Ca 2+ imaging experiments, what is interestingly a common observation when comparing inhibitory levels yielded from different methods [ 47 , 49 , 62 ]. In this regard, it needs to be emphasized that the patch clamp technique directly monitors Ca 2+ influx via TRPV6 channels, whereas in Ca 2+ imaging experiments, resultant changes in intracellular Ca 2+ levels are yielded from a fluorescent read-out, which are impacted by various cellular processes, as well as dependent on the experimental conditions.…”

“…The channel periphery is formed by TM1-TM4, which associate with the pore domain in a domain-swapped arrangement, i.e., residing in closer proximity to TM5-TM6 of a neighboring TRPV6 protomer rather than the one of the same subunit [ 45 , 46 ]. The interfaces between the individual subunits of the channel serve as association sites for lipids [ 46 , 47 , 48 , 49 , 50 ]. For channels including those composed of closely related TRPV5 proteins, cryogenic electron microscopy (cryo-EM) structural investigations succeeded in resolving presumptive PIP 2 binding sites [ 8 , 10 , 51 , 52 , 53 , 54 ].…”

“…For channels including those composed of closely related TRPV5 proteins, cryogenic electron microscopy (cryo-EM) structural investigations succeeded in resolving presumptive PIP 2 binding sites [ 8 , 10 , 51 , 52 , 53 , 54 ]. Yet, for TRPV6, none of the four different resolved non-protein densities matches PIP 2 in shape [ 46 , 47 , 48 , 49 , 55 ]. Still, the presence of positively charged residues within the binding pocket intercalated between TM3, TM4, and the TM4-TM5 linker of one subunit and TM5 as well as TM6 of the neighboring protomer would predestine this site, termed as lipid binding site 2 (LBS-2, see Supplementary Figure S1A ), for interactions with PIP 2 [ 46 ].…”