Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

A-1155463, a highly potent and selective BCL-X L inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-X L -dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanismbased and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-X L biology as well as a productive lead structure for further optimization.

G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC 50 : 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.KEYWORDS: G9a, methyltransferase, A-366, methylation, epigenetics H istone methyltransferases (HMTs), a class of enzymatic "writers" of epigenetic marks, have recently emerged as targets of potential therapeutic value.1,2 They catalyze the methylation of histone lysines and arginines utilizing Sadenosyl-methionine (SAM) as the cofactor/methyl-source. This process can result in either the activation or repression of transcription.3,4 Dysregulation of methylation at specific histone sites (alterations in the "histone code") has been implicated in many cancers.5−7 Hence, targeting HMT activity has been the subject of much investigation in the field of oncology, even recently reaching human clinical trials. 8 Euchromatic histone methyltransferase 2 (EHMT2), also known as G9a, is an HMT that is primarily responsible for the dimethylation of lysine 9 on histone H3 (H3K9). Several reports have highlighted its link to a variety of cancers. It is upregulated in hepatocellular carcinoma, 9 B cell acute lymphoblastic leukemia, 10 and lung cancers. 11 In addition, elevated expression of G9a in aggressive lung cancer correlates with poor prognosis, while its knockdown in highly invasive lung cancer cells suppressed metastasis in an in vivo mouse model. 12 In prostate cancer cells (PC3), G9a knockdown caused significant morphological changes and inhibition of cell growth. 13While small molecule inhibitors of G9a (Figure 1) have been reported as early as 2005, 14 BIX01294 (1) was the first reported potent and selective inhibitor. 15 It was found to reduce H3K9 dimethyl levels in cells and notably was not a SAM-competitive inhibitor. Recently, a second G9a inhibitor, UNC0638 (2), was disclosed. Quinazoline 2, which incorporated a lysine-mimic via an n-propyl-pyrrolidine exhibited a >10-fold enhancement in potency over 1 along with selectivity over a panel of 17 other epigenetic targets. 16−19 Our efforts toward the identification of chemically distinct G9a inhibitors commenced with a chemical diversity subset screen of our compound collection. The assay format employed was a peptide-based AlphaLISA, measuring the levels of H3K9 dimethylation. Several clusters of related chemical matter were identified, but a singleton stood out: spiro[cyclobutane-1,3′-indol]-2′-amine (3) as having robust potency (IC 50 : 153 nM). The low molecular weight of this compound translated to a high binding efficiency index (BEI) value of 27. 20 Such a feature rendered 3 as an attractive starting point for chemistry efforts aimed at further potency optimization.The importance of the various subunits of 3 was initially interrogated by evaluation of a ...