Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1.

Structure-Based Design of Potent, Selective, and Orally Bioavailable VPS34 Kinase Inhibitors.

VPS34 is a class III phosphoinositide 3-kinase involved in endosomal trafficking and autophagosome formation. Inhibitors of VPS34 were believed to have value as anticancer agents, but genetic and pharmacological data suggest that sustained inhibition of VPS34 kinase activity may not be well tolerated. Here we disclose the identification of a novel series of dihydropyrazolopyrazinone compounds represented by compound as potent, selective, and orally bioavailable VPS34 inhibitors through a structure-based design strategy.

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma.

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy.

Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies.

A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'.

MK-2461, a novel multitargeted kinase inhibitor, preferentially inhibits the activated c-Met receptor.

The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC(50) values of 0.

High-throughput analysis of HGF-stimulated cell scattering.

Historically, only relatively low-throughput or expensive methods have been available to measure cell migration. Hepatocyte growth factor (HGF) is a ligand for the tyrosine kinase receptor Met that, in addition to mediating proliferation and survival, increases cell motility and metastasis. The authors have developed a high-throughput imaging assay for measuring inhibition of HGF-induced scattering in human HPAF-II pancreatic adenocarcinoma cells.

Automated cell plating and sample treatments for fixed cells in high content assays.

Robust and reliable methods for the manipulation of neural cell lines, by passaging, plating, dye labeling, imaging, fixation, and immunocytochemistry, are required to enable consistent, reproducible screens to be performed. We describe herein procedures and processes we have established to maximize the level of consistency of cell plating, fixation, and dye or antibody labeling, to ensure that assays which we are running on a routine basis remain consistent across long periods of time. These procedures involve a variety of fully or semiautomated steps, using high-quality commercially available liquid handling and dispensing technology.

A morphology- and kinetics-based cascade for human neural cell high content screening.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds.

High content kinetic assays of neuronal signaling implemented on BD pathway HT.

A great deal of information can be gained from kinetic fluorescence-based measurement of cellular responses; however, until recently the use of such approaches has been limited by the manual nature of the instrumentation available. Higher-throughput kinetic studies of signaling pathways are greatly facilitated by new confocal, liquid handling-enabled, high content screening (HCS) platforms. In the present work, we have implemented one such instrument, the BD(TM) Pathway HT bioimager (BD Biosciences, Rockville, MD), for studying regulation of neuronal signaling pathways.