The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function.

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca2+ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O2 levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O2-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O2 and show less mitochondrial Ca2+ uptake in response to bradykinin-induced ER Ca2+ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O2 levels.

Development of a high-throughput mass spectrometry based analytical method to support an in vitro OATP1B1 inhibition screening assay.

RATIONALE: It is well known that the organic anion transporting polypeptide 1B1 (OATP1B1) plays a major role in the hepatic uptake of a range of drugs. To this end, it is pivotal that the potential for new molecular entities (NMEs) to inhibit OATP1B1 activity be assessed during early drug discovery. The work reported herein describes the development of a high-throughput analytical method to measure the clinically relevant probe substrate, pitavastatin, for the in vitro assessment of OATP1B1 inhibition. METHODS: Development of an analytical method capable of very fast throughput was crucial for the success of this assay and was accomplished using a system which combines direct, on-line solid-phase extraction (SPE) with highly sensitive, label-free tandem mass spectrometry (MS/MS)-based detection. Mass spectrometry analysis of pitavastatin, along with the stable isotopically labeled internal standard d5-pitavastatin, was conducted using positive electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. RESULTS: The on-line SPE-MS/MS platform demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness to existing methodologies while achieving analytical cycle times of 10.4 seconds per well. Sensitivity exceeded what was necessary for our assay conditions, with a determined lower limit of quantification (LLOQ) for pitavastatin of 10 pM (picomolar) in assay matrix. Furthermore, the potency of multiple reference compounds was shown to be within 2-fold of IC50 values generated from liquid chromatography (LC)/MS/MS-based literature values. CONCLUSIONS: A very fast and robust analytical method was successfully developed for the measurement of the clinically relevant OATP1B1 substrate, pitavastatin. The successful development and implementation of this very important early liability screen has helped to facilitate judicious lead candidate progression and will ultimately help build a greater understanding of OATP1B1-NME interactions, in general. Copyright © 2016 John Wiley & Sons, Ltd.