Furthermore, care must be taken in selecting the cell lines used to assess on-target activity

Furthermore, care must be taken in selecting the cell lines used to assess on-target activity. as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer. Anti-apoptotic proteins such as BCL-2, BCL-XL and MCL-1 maintain cell survival by binding and sequestering their pro-apoptotic counterparts, such as BAK, BAX, or the BCL-2 homology 3 (BH3)-only proteins BAD and BIM.1, ALK inhibitor 1 2, 3 Because cancer cells must survive amidst a variety of environmental stresses, they often express high basal levels of these BCL-2 family complexes and have been described to be primed for death’.4 For the past two decades, teams of basic and translational scientists have worked to generate small-molecule inhibitors of these proteinCprotein interactions, with the aim of driving cancer cells to initiate apoptosis. Although drugging these interactions has proven particularly challenging, intensive structure-based efforts have enabled the design of potent and cell-active BCL-2 family inhibitors. ABT-737 was among the first molecules described,5 followed soon thereafter by an orally bioavailable molecule, ABT-263 (navitoclax).6 Both molecules mimic BAD, with high affinity for BCL-2, BCL-XL and BCL-W, and both molecules have demonstrated impressive anti-tumor activity preclinically.7, 8 Although navitoclax also demonstrated promising signs of clinical activity, its development has been complicated by dose-limiting thrombocytopenia, the result of BCL-XL inhibition.9, 10 This prompted the DKK2 development of ABT-199/GDC-0199 (venetoclax), a BCL-2-selective inhibitor that maintains anti-tumor efficacy while sparing platelets.11 Selective BCL-XL inhibitors have also been generated12, 13, 14, 15 and the most potent molecules A-1155463 and A-1331852 ALK inhibitor 1 demonstrate significant anti-tumor effects alone or in combination with chemotherapeutics (manuscript submitted). None of the BCL-2 family inhibitors described above can inhibit MCL-1, and hence, not surprisingly, this protein has emerged as a potential resistance factor for these agents.16, 17, 18, 19 MCL-1 has also been implicated in mediating resistance to a variety of commonly used chemotherapeutic agents,20, 21, 22 and so generating small molecules capable of inhibiting MCL-1 represents an attractive approach for circumventing drug resistance. MCL-1 is a compelling ALK inhibitor 1 cancer target in its own right, having been implicated in mediating the survival of multiple tumor types.23 The gene locus is amplified in a variety of tumor types, including breast cancer and non-small cell lung cancer (NSCLC),24 and the MCL-1 protein has been shown to mediate survival in models of multiple myeloma,25, 26 acute myeloid leukemia27 and NSCLC28, 29 and MYC-driven lymphomas.30 A variety of approaches for inhibiting MCL-1 have been described, including the use of BH3 peptides31, 32, 33, 34 and small molecules35, 36, 37, 38, 39 that bind MCL-1 directly or inhibit its expression indirectly.18, 40, 41, 42 Of the direct small-molecule inhibitors reported, none possess MCL-1 affinity within a range that would be expected to confer on-target cellular effects. Indirect MCL-1 inhibitors include cyclin-dependent kinase inhibitors such as roscovitine, flavopiridol, seliciclib, dinaciclib, and SNS-032, which inhibit the phosphorylation of the RNA polymerase 2 C-terminal domain and the elongation of transcripts, including mRNA from HCC-1806 cells treated for 8?h with similar concentrations of A-1210477. (c) HCC-1806 cells were incubated with increasing concentrations (0.1, 0.5, 1, 5, 10, and 20?was released from the mitochondria of permeabilized H929 cells treated with the BIM2A peptide, which has selective affinity for MCL-1,49 but not the BAD peptide, which focuses on BCL-2, BCL-XL and BCL-W (Number 4a). When intact H929 cells were treated with the A-1210477 analog A-1208746, cytochrome was observed in cytosolic fractions within 4?h at concentrations as low as 3?released into the ALK inhibitor 1 cytosol. (b) H929 and RS4;11 cells were incubated with increasing concentrations of MCL-1 inhibitors or the BCL-2-selective inhibitor ABT-199 (venetoclax) for 48?h.