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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.

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.

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. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.

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. Suitable hardware and consumables for conducting 1536-well HCS have only recently become available, and developing a reliable informatics framework to accommodate the scale of high-throughput HCS data remains a considerable challenge. Additionally, innovative approaches are needed to interpret the large volumes of content-rich information generated. Despite these hurdles, there has been a growing interest in screening large compound inventories using this platform. Here, we outline the infrastructure developed and applied at Bristol-Myers Squibb for 1536-well high content screening and discuss key lessons learned.

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. Suitable hardware and consumables for conducting 1536-well HCS have only recently become available, and developing a reliable informatics framework to accommodate the scale of high-throughput HCS data remains a considerable challenge. Additionally, innovative approaches are needed to interpret the large volumes of content-rich information generated. Despite these hurdles, there has been a growing interest in screening large compound inventories using this platform. Here, we outline the infrastructure developed and applied at Bristol-Myers Squibb for 1536-well high content screening and discuss key lessons learned.

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. Here we report a method that circumvents both challenges. The utilization of a porous graphitic carbon column (Hypercarb) ensured adequate retention of highly polar metformin in biological samples. The combined employment of a ballistic gradient on a 3 mm × 30 mm, 5 μm Hypercarb column, and dual staggered chromatography coupled with multiple injection chromatography acquisition, yielded a fast injection-to-injection cycle time of 30 s. The method demonstrated good accuracy, precision and excellent robustness for high throughput applications, and has been successfully implemented in the development and validation of the nonradioactive transporter inhibition assays for MATEs and OCT2.

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.g., sulfotransferase 2A1, multidrug resistance-associated protein 2, 3, and 4). Moreover, BAs undergo extensive enzyme-catalyzed amidation and are subjected to metabolism by enterobacteria during EHR. Expression of the various enzymes and transporters described above is governed by nuclear hormone receptors (NHRs) that mount an adaptive response when intracellular levels of BAs are increased. The intracellular trafficking of transporters, and their ability to mediate the vectorial transport of BAs, is governed by specific kinases also. Finally, bile flow, micelle formation, canalicular membrane integrity, and BA clearance can be influenced by the inhibition of multidrug resistant protein 3- or ATPase-aminophospholipid transporter-mediated phospholipid flux. Consequently, when screening compounds in a discovery setting or conducting mechanistic studies to address clinical findings, one has to consider the direct (inhibitory) effect of the parent drug and metabolites on multiple BA transporters, as well as inhibition of BA sulfation and amidation and NHR function. Vectorial BA transport, in addition to BA EHR and homoeostasis, could also be impacted by drug-dependent modulation of kinases and enterobacteria.

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.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.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.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 WileySons, Ltd.

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. 2. In vitro inhibition assays were established for human OAT1 and rat Oat3 and rat in vivo renal clearance was obtained. Statistically significant quantitative relationships were explored between the compounds' physical properties, their affinity for OAT1 and oat3 and the inter-relationship with unbound renal clearance (URC) in rat. 3. Many of the compounds were actively secreted and in vitro analysis demonstrated that these were ligands for rat and human OAT transporters (IC50 values ranging from1 to100 µM). Application of resultant QSAR models reduced renal clearance in the rat from 24 to0.1 ml/min/kg. Data analysis indicated that the properties associated with increasing affinity at OATs are the same as those associated with reducing URC but orthogonal in nature. 4. This study has demonstrated that OAT inhibition data and QSAR models can be successfully used to optimize rat renal clearance in vivo and provide confidence of translation to humans.

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. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.

Introduction JI-101 is a highly selective and potent angiogenesis inhibitor with unique EphB4 activity that distinguishes it from other agents in clinical development. We report the preclinical pharmacology and early clinical experience with this compound. Procedures Lead optimization was used to identify cgi1842 (now JI-101) with a targeted activity profile that is selective and unique. JI-101 was tested in binding, enzymatic, and cell-based assays for activity against kinase and non-kinase targets. Preclinical in vivo testing for PK, PD, efficacy, and safety pharmacology was performed in mouse, rat, guinea pig, and dog. A Phase I clinical trial is underway: 12 patients have been treated thus far with 28-day oral daily dosing at 100 mg, 200 mg, 400 mg QD or at 200 mg BID. PK, PD, and imaging analyses have been performed. Data JI-101 has a MW of 466D and high ( Conclusions JI-101 potently inhibits 3 critical angiogenic kinases (VEGFR2, EphB4, and PDGFR) and is the only such “triple” angiogenesis inhibitor currently in development. JI-101 shows robust preclinical and clinical pharmacology and is well-tolerated to date in human trials. In tumors where EphB4 may play an oncogenic role, such as head and neck cancer, JI-101 may have anti-proliferative effects in addition to anti-angiogenic effects and should be amenable to combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 17.

Introduction: JI-101 is a highly selective and potent angiogenesis inhibitor with unique EphB4 activity that distinguishes it from other agents in clinical development. We report the preclinical pharmacology and early clinical experience with this compound. Procedures: Lead optimization was used to identify cgi1842 (now JI-101) with a targeted activity profile that is selective and unique. JI-101 was tested in binding, enzymatic, and cell-based assays for activity against kinase and non-kinase targets. Preclinical in vivo testing for PK, PD, efficacy, and safety pharmacology was performed in mouse, rat, guinea pig, and dog. A Phase I clinical trial is underway: 9 patients, 3/cohort, are being treated with 28-day oral daily dosing at 100 mg, 200 mg, and 400 mg. PK, PD, and imaging analyses are being performed. Data: JI-101 has a MW of 466D and high ( Conclusions: JI-101 potently inhibits 3 critical angiogenic kinases (VEGFR2, EphB4, and PDGFR) and is the only such “triple” angiogenesis inhibitor currently in development. JI-101 shows robust preclinical and clinical pharmacology and is well-tolerated to date in human trials. In tumors where EphB4 may play an oncogenic role, such as head and neck cancer, JI-101 may have anti-proliferative effects in addition to anti-angiogenic effects and should be amenable to combination therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B11.