Challenges and Opportunities in Enabling High-Throughput, Miniaturized High Content Screening.

Within the Drug Discovery industry, there is a growing recognition of the value of high content screening (HCS), particularly as researchers aim to screen compounds and identify hits using more physiologically relevant in vitro cell-based assays. Image-based high content screening, with its combined ability to yield multiparametric data, provide subcellular resolution, and enable cell population analysis, is well suited to this challenge. While HCS has been in routine use for over a decade, a number of hurdles have historically prohibited very large, miniaturized high-throughput screening efforts with this platform.

Identification and optimization of a novel series of indoleamine 2,3-dioxygenase inhibitors.

The discovery of a series of structurally-novel biaryl urea IDO inhibitors is described. Optimization of a micromolar hit through iterative cycles of synthesis and screening in an assay measuring IDO-mediated intracellular conversion of tryptophan to kynurenine led to potent inhibitors with favorable selectivity and metabolic stability profiles.

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.

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain.

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach.

Development of an LC-MS/MS method for high throughput quantification of metformin uptake in transporter inhibition assays.

A high throughput LC-MS/MS method for quantification of metformin substrate uptake enables conversion of radiometric transporter inhibition assays for multidrug and toxin extrusion transporters (MATE 1 and 2) and organic cation transporter 2 (OCT2) to a nonradioactive format. Such conversion greatly simplifies assay complexity and reduces assay costs. The development of a quantitative LC-MS/MS method for metformin in support of the high throughput transporter inhibition assays faced specific challenges of achieving both adequate chromatographic retention and rapid analytical turnaround.

Application of an in vitro OAT assay in drug design and optimization of renal clearance.

1. Optimization of renal clearance is a complex balance between passive and active processes mediated by renal transporters. This work aimed to characterize the interaction of a series of compounds with rat and human organic anion transporters (OATs) and develop quantitative structure-activity relationships (QSARs) to optimize renal clearance.

Drug-induced perturbations of the bile acid pool, cholestasis, and hepatotoxicity: mechanistic considerations beyond the direct inhibition of the bile salt export pump.

The bile salt export pump (BSEP) is located on the canalicular plasma membrane of hepatocytes and plays an important role in the biliary clearance of bile acids (BAs). Therefore, any drug or new chemical entity that inhibits BSEP has the potential to cause cholestasis and possibly liver injury. In reality, however, one must consider the complexity of the BA pool, BA enterohepatic recirculation (EHR), extrahepatic (renal) BA clearance, and the interplay of multiple participant transporters and enzymes (e.

Imidazo[1,2-a]pyrazine diaryl ureas: inhibitors of the receptor tyrosine kinase EphB4.

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy.

Inhibitor scaffolds as new allele specific kinase substrates.

The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl)-ADP bound to c-Src (T338G).