Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays.

Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERß loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERß and act as ERß antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.

Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK.

Constitutive overexpression and activation of NPM-ALK fusion protein [t(2:5)(p23;q35)] is a key oncogenic event that drives the survival and proliferation of anaplastic large-cell lymphomas (ALCLs). We have identified a highly potent and selective small-molecule ALK inhibitor, NVP-TAE684, which blocked the growth of ALCL-derived and ALK-dependent cell lines with IC(50) values between 2 and 10 nM. NVP-TAE684 treatment resulted in a rapid and sustained inhibition of phosphorylation of NPM-ALK and its downstream effectors and subsequent induction of apoptosis and cell cycle arrest. In vivo, NVP-TAE684 suppressed lymphomagenesis in two independent models of ALK-positive ALCL and induced regression of established Karpas-299 lymphomas. NVP-TAE684 also induced down-regulation of CD30 expression, suggesting that CD30 may be used as a biomarker of therapeutic NPM-ALK kinase activity inhibition.

An efficient rapid system for profiling the cellular activities of molecular libraries.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.

An efficient enzymatic synthesis of thiamin pyrophosphate.

Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.