Compound Property Optimization in Drug Discovery Using Quantitative Surface Sampling Micro Liquid Chromatography with Tandem Mass Spectrometry.

Surface sampling micro liquid chromatography tandem mass spectrometry (SSµLC-MS/MS) was explored as a quantitative tissue distribution technique for probing compound properties in drug discovery. A method was developed for creating standard curves using surrogate tissue sections from blank tissue homogenate spiked with compounds. The resulting standard curves showed good linearity and high sensitivity. The accuracy and precision of standards met acceptance criteria of ±30%. A new approach was proposed based on an experimental and mathematical method for tissue extraction efficiency evaluation by means of consecutively sampling a location on tissue twice by SSµLC-MS/MS. The observed extraction efficiency ranged from 69% to 82% with acceptable variation for the test compounds. Good agreement in extraction efficiency was observed between surrogate tissue sections and incurred tissue sections. This method was successfully applied to two case studies in which tissue distribution was instrumental in advancing project teams' understanding of compound properties.

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

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.