High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator's laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen.