Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Abstract

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Natural products as probes in pharmaceutical research

“…2), which was first isolated in 2008 from the bark of the African plant Phyllanthus engleri, it was shown that the block of cell proliferation is correlated with the expression of the TRPC4 cation channel in sensitive cell lines suggesting TRPC4 to be the biologically relevant the target. This could be confirmed by electrophysiology experiments showing that englerin A can induce currents across the membranes of cells in presences of TRPC4, but not in its absence [7]. The same target was found in parallel by Waldmann et al following a similar approach [1].…”

Critical roles of Gi/oproteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

“…TRPC4 is implicated in the regulation of microvascular permeability (5), renal cancer proliferation (6,7), neurotransmitter release (8), intestinal contraction and motility (9), neurite extension (10), epileptiform burst firing, and seizure-induced neurodegeneration (11). The channel mediates Na + and Ca 2+ influx, causing membrane depolarization and intracellular Ca 2+ concentration ([Ca 2+ ] i ) elevation, which in turn alter cell function (12).…”

Englerins: A Comprehensive Review

“…The Waldmann group reported that transfection of TRPC4 into HEK293T cells rendered the otherwise insensitive cells sensitive to englerin A. 65 On the basis of their inability to detect PKC θ in A498 cells or the sensitive Ewing’s sarcoma cell line A673, this group concluded that PKC θ could not be the target of englerin A, but that TRPC4/5 agonism was the relevant effect. 65 A recent report from Beech’s group found that englerin A cytotoxicity was mediated by influx of sodium, rather than of calcium, as had been previously proposed, and that both TRPC4 and TRPC1 must be part of the ion channel complex for cytotoxicity.…”