Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Probing the therapeutic potential of TRPC6 for Alzheimer's disease in live neurons from patient-specific iPSCs.

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer's disease (AD) under patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher levels of AD pathological markers Aß and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neurons with TRPC6 protein fragment or agonist inhibited the elevation of Aß and phospho-tau. Our results in live AD neurons manifest that the compromised expression of TRPC6 substantially contributed to Aß pathology of sporadic AD, suggesting that targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.

Structural basis for pharmacological modulation of the TRPC6 channel.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

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.

TRPC6 contributes to LPS-induced inflammation through ERK1/2 and p38 pathways in bronchial epithelial cells.

receptor potential canonical (TRPC) channels are presently an emerging target for airway disorders. Recent evidence has indicated that TRPC6 as a member of the TRPC family plays an important role in airway inflammation, but its precise function in bronchial epithelial cells remains unclear. The aim of this study was to investigate the role of TRPC6 in Toll-like receptor 4 (TLR4)-mediated inflammation in human bronchial epithelial cells stimulated by endotoxin [lipopolysaccharide (LPS)]. Hyp9 is a simplified phloroglucinol derivative of hyperforin that highly selectively activates TRPC6 channels. The results show that the activation of TRPC6 by Hyp9 induced the production of interleukin (IL)-8 and IL-6. LPS was also able to induce the release of IL-8 and IL-6, which was significantly aggravated by Hyp9 and reduced by knockdown of TRPC6. Treatment with LPS not only chronically induced the expression of TRPC6 mRNA and protein in a TLR4-dependent manner but also acutely increased Ca2+ influx through TRPC6 channels. In addition, LPS-induced overexpression of TRPC6 and Ca2+ influx were associated with the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt. Importantly, TRPC6 was required for the activation of ERK1/2, p38, and NF-κB. In conclusion, these data reveal that LPS induced the overexpression of TRPC6 and TRPC6-dependent Ca2+ influx via the TLR4/PI3K/Akt pathway resulting in Ca2+ mobilization, which subsequently promoted the activation of ERK1/2, p38, and NF-κB and the inflammatory response in bronchial epithelial cells.