Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker

Abstract

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.

Keywords: Apoptosis; Biomarkers; Cell-based activity; Mcl-1; Oncology.

Figure 1

(a) Structure of compound 1, a 7-hydroxyquinoline identified by high-throughput screening as possessing Mcl-1-selective inhibitory activity relative to the related Bcl-2 family protein Bcl-xL; (b) Structure of the piperazine-substituted hydroxyquinoline 9, which displays selective inhibition of Mcl-1 relative to Bcl-xL as indicated by fluorescence polarization assay.

Figure 2

Calculated binding mode of compound 9 in Mcl-1. Modeling suggests a hydrogen bond between the phenol of the hydroxyquinoline and the carbonyl of Asn260 may orient the ethylpiperazine group into one of the four major lipophilic pockets found in the Mcl-1/BH3-only peptide binding groove.

Figure 3

(a) EC₅₀ values from cell proliferation assay experiments (viability determined by PrestoBlue staining) for the Mcl-1 inhibitor 9; Results indicated are the mean of two independent cell growth inhibition experiments conducted in triplicate (n=6) (b) Results of BH3 profiling assay on the lymphoid cell lines SU-DHL6 and SU-DHL10, myeloma cell line NCI-H929, and mouse leukemia cell lines Bcl2-1863 and Mcl1-1780. These cell lines display a range of mitochondrial depolarization upon exposure to various BH3-only peptides (Bim, Puma, Noxa, Bad). For example, SU-DHL6 is highly “primed” for all BH3 peptides tested but SU-DHL10 has low levels of mitochondrial priming. Priming values are derived from two independent assays conducted in triplicate (n=6); (c) Correlation between antiproliferative activity of 9 (EC₅₀ represented in M) and BH3 profiling response (% mitochondrial depolarization, Bim peptide assayed at 0.3 M). An excellent correlation is observed between priming state and antiproliferative response observed in these compound 9 –treated cell lines (R²=0.81).

Figure 4

(a) Compound 9 promotes cytochrome c release in a fashion that may be correlated with the extent of mitochondrial priming in a series of human-derived lymphoid cell lines (SU-DHL6, SU-DHL8, SU-DHL10) and two mouse leukemia cell lines (Bcl2-1863 and Mcl1-1780); (b) Cell viability of mouse leukemia cell lines Bcl2-1863 and Mcl1-1780 following treatment with ABT-263 or compound 9 as measured by DAPI assay; (c) Percentage of cells that demonstrate apoptosis, as detected by Annexin V –positive staining, following treatment with compound 9 at the indicated concentrations. Results depicted are representative of two independent experiments and are consistent for dose-response trends.