Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells

Abstract

Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight ‫؍‬ 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1 ؉͞؉ ) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1 ؊͞؊ ) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.

BCL-2 in the crosshairs: tipping the balance of life and death

“…This approach yielded HA14-1 (IC 50 ¼ 9 mM for competing BAK BH3/BCL-2 interaction), 103 several micromolar affinity hits from the National Cancer Institute 3D database including Compound 6, 104 A major hurdle of small molecule library screening approaches, whether virtual or biochemical, is the chemical optimization required to achieve high-potency target-binding activity from hits that are typically in the micromolar range. An alternative strategy developed by Fesik and co-workers 115 circumvents this shortcoming of library screening by chemically linking ligands that bind to adjacent sites within a target interface, effectively converting relatively low affinity interactors into conjoined high-affinity compounds.…”

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage

“…DEVD-R110, a fluorogenic substrate for caspase-3 activity, 19 was provided by Molecular Probes, Eugene, OR. The Bcl-2 antagonist HA14-1 20 was purchased from Ryan Scientific Inc. (Isle of Palms, SC). Since this reagent slowly loses activity in the presence of water, solutions were made up in anhydrous DMSO and stored in small aliquots at −20 °C under nitrogen.…”

Small-molecule Bcl-2 inhibitors sensitise tumour cells to immune-mediated destruction

“…However, Bcl-2 antisense therapy had limited activity when tested in clinical trials, potentially owing to poor access to the intracellular compartment and inadequate reduction in Bcl-2 expression levels (Waters et al, 2000;Yuen and Sikic, 2000;Morris et al, 2002). More recently, small molecules that target the BH3-domain-binding site of Bcl-2 have been developed as Bcl-2 inhibitors (Wang et al, 2000;Oltersdorf et al, 2005). It is hypothesised that these agents displace proapoptotic BH3-only proteins (e.g., Bid and Bim) from their binding groove on Bcl-2, thus promoting interaction with Bax and Bak, and transduction of an apoptotic signal (Oltersdorf et al, 2005).…”

“…It is hypothesised that these agents displace proapoptotic BH3-only proteins (e.g., Bid and Bim) from their binding groove on Bcl-2, thus promoting interaction with Bax and Bak, and transduction of an apoptotic signal (Oltersdorf et al, 2005). One of these compounds -ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (designated HA14-1) -directly triggered apoptosis in Bcl-2-expressing leukaemia and lymphoma cell lines, and also enhanced the cytotoxicity of the antileukaemia drug cytarabine (Wang et al, 2000;Lickliter et al, 2003). A more-recently developed compound, ABT-737, bound to Bcl-2 and Bcl-x L with extremely high affinity Revised 14 December 2006; accepted 19 December 2006 (inhibitory constant K i p1 nM), and exhibited significant in vitro and in vivo activity in preclinical models of follicular lymphoma and small-cell lung cancer (Oltersdorf et al, 2005).…”