We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

Abstract Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in hematopoietic differentiation. LSD1 is also overexpressed in multiple tumor types. These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, irreversible, GSK LSD1 inhibitor (GSK2879552) in acute myeloid leukemia (AML) and small cell lung cancer (SCLC). GSK2879552 is a potent, selective, mechanism-based inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML and SCLC cells have a unique requirement for LSD1. While GSK2879552 treatment did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of genes whose expression increased with LSD1 inhibition. Treatment of AML cell lines with GSK2879552 increased cell surface expression of CD11b and CD86, markers associated with a differentiated immunophenotype. Six days of GSK2879552 treatment resulted in potent anti-proliferative growth effects in 19 of 25 AML cell lines representing a range of AML subtypes. Treating for longer time periods revealed sensitivity in all AML cell lines. AML blast colony forming ability was also inhibited in 4 of 5 bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and were treated upon engraftment. After 17 days of treatment, control mice had 80% GFP+ cells in the bone marrow whereas treated mice had only 2.8% GFP positive cells (p<0.012), and the treated animals survived weeks beyond control mice. Growth inhibition was also observed in a subset of SCLC cell lines. GSK2879552 treatment of mice engrafted with SCLC lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Kimberly Smitheman, Monica Cusan, Yan Liu, Michael Butticello, Melissa Pappalardi, James Foley, Kelly Federowicz, Glenn Van Aller, Jiri Kasparec, Xinrong Tian, Dominic Suarez, Jess Schneck, Jeff Carson, Patrick McDevitt, Thau Ho, Charles McHugh, William Miller, Scott Armstrong, Christine Hann, Neil Johnson, Ryan G. Kruger, Helai P. Mohammad, Shekhar Kamat. Inhibition of LSD1 for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2015-3513

Phosphoinositide 3-kinase α (PI3Kα) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3Kα and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

The phosphoinositide 3‐kinase (PI3K) pathway is among the most commonly activated pathways in human cancer. The biological role of PI3K in growth and survival of cancer cells and the prevalence of activating mutations in human cancers are well documented and a significant proportion of tumors would be predicted to benefit from inhibition of this pathway. Here we report on the characterization of the pan‐PI3K pyridylsulfonamide inhibitor GSK2126458: a very potent (PI3K app K i = 19 pM), reversible, ATP‐competitive inhibitor of wild‐type PI3K and the ‘hotspot’ activating mutants of p110 found in human cancer. GSK2126458 demonstrated good selectivity for the PI3K family of enzymes including the mTORC1 and mTORC2 complexes, when evaluated in a large panel of protein kinases. Consistent with potent PI3K enzyme inhibition, GSK2126458 decreased the cellular levels of phosphorylated AKT, p70 S6K , PRAS40 and ERK in a concentration and time dependent manner, the IC50 for pAKT in the HCC1954 breast carcinoma cell line was 2 nM. GSK2126458 induced the nuclear translocation of the FOXO3a transcription factor in a concentration dependent manner that mechanistically appeared bimodal. Growth inhibition was time and concentration dependent with a 3 day exposure resulting in a growth IC 50 (gIC 50 ) of 9 nM in HCC1954 cells with evidence of cell death. Cell death correlated with induction of caspase 3 and 7 activity suggesting apoptosis was the mechanism of cell death. GSK2126458 had a breadth of activity for potent cell growth inhibition and induction of cell death in a variety of human cancer cells. GSK2126458 demonstrated robust, dose dependent in vivo pharmacodynamic activity as measured by inhibition of phospho‐AKT in advanced BT474 breast cancer cell xenograft tumors. Inhibition of AKT phosphorylation was rapid and lasted for several hours after a single oral administration of GSK2126458. Transient increases in plasma insulin and blood glucose were observed. Evaluation of in vivo tumor growth effects of GSK2126458 in xenograft models demonstrated dose dependent tumor growth delay in several models of diverse tumor lineage and tumor regression in a breast cancer xenograft. The biological profile of GSK2126458 supports the clinical advancement of this compound and GSK2126458 has entered Phase I human clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C63.

Human kinesin spindle protein (KSP)/hsEg5, a member of the kinesin-5 family, is essential for mitotic spindle assembly in dividing human cells and is required for cell cycle progression through mitosis. Inhibition of the ATPase activity of KSP leads to cell cycle arrest during mitosis and subsequent cell death. Ispinesib (SB-715992), a potent and selective inhibitor of KSP, is currently in phase II clinical trials for the treatment of multiple tumor types. Mutations that attenuate Ispinesib binding to KSP in vitro have been identified, highlighting the need for inhibitors that target different binding sites and inhibit KSP activity by novel mechanisms. We report here a small-molecule modulator, KSPA-1, that activates KSP-catalyzed ATP hydrolysis in the absence of microtubules yet inhibits microtubule-stimulated ATP hydrolysis by KSP. KSPA-1 inhibits cell proliferation and induces monopolar-spindle formation in tumor cells. Results from kinetic analyses, microtubule (MT) binding competition assays, and hydrogen/deuterium-exchange studies show that KSPA-1 does not compete directly for microtubule binding. Rather, this compound acts by driving a conformational change in the KSP motor domain and disrupts productive ATP turnover stimulated by MT. These findings provide a novel mechanism for targeting KSP and perhaps other mitotic kinesins.

KSP, also known as HsEg5, is a kinesin that plays an essential role in the formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. Ispinesib is the first potent, highly specific small-molecule inhibitor of KSP tested for the treatment of human disease. This novel anticancer agent causes mitotic arrest and growth inhibition in several human tumor cell lines and is currently being tested in multiple phase II clinical trials. In this study we have used steady-state and pre-steady-state kinetic assays to define the mechanism of KSP inhibition by ispinesib. Our data show that ispinesib alters the ability of KSP to bind to microtubules and inhibits its movement by preventing the release of ADP without preventing the release of the KSP-ADP complex from the microtubule. This type of inhibition is consistent with the physiological effect of ispinesib on cells, which is to prevent KSP-driven mitotic spindle pole separation. A comparison of ispinesib to monastrol, another small-molecule inhibitor of KSP, reveals that both inhibitors share a common mode of inhibition.

The PIK3CA gene, encoding the p110alpha catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110alpha, H1047R, E542K and E545K, were shown to potently activate PI3K signalling in human epithelial cells. In the present study, we examine the biochemical activity of the recombinantly purified PI3K oncogenic mutants. The kinetic characterizations of the wt (wild-type) and the three 'hot spot' PI3K mutants show that the mutants all have approx. 2-fold increase in lipid kinase activities. Interestingly, the phosphorylated IRS-1 (insulin receptor substrate-1) protein shows activation of the lipid kinase activity for the wt and H1047R but not E542K and E545K PI3Kalpha, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.

3179 Centromere-associated protein E (CENP-E), a member of the kinesin-7 superfamily, is a plus end-directed microtubule motor. CENP-E is an essential component of the kinetochore since it is required for correct positioning and stabilization of attached chromosomes to spindle microtubules (MTs) at the metaphase plate. This stable arrangement of chromosomes and MTs also develops tension across aligned chromosomes and is required for checkpoint satisfaction. Dysfunction of CENP-E in cultured human tumor cells due to microinjection of antibodies or ablation of gene expression with siRNA results in a failure to satisfy the mitotic checkpoint, resulting in cell cycle arrest with bipolar mitotic spindles and misaligned chromosomes and eventual cell death. GSK923295Ais a first in class, potent, highly specific, small-molecule inhibitor of human CENP-E currently being developed as a novel anticancer agent. We have used steady-state and presteady-state kinetic assays to define the molecular basis of CENP-E inhibition by GSK923295A. GSK-923295A is a potent inhibitor of the MT-stimulated ATPase activity of the human CENP-E motor domain, but in the absence of MTs this inhibitor is ~100-fold less potent. Further steady-state analysis reveals that GSK923295A is ATP and MT uncompetitive indicating that this compound is an allosteric inhibitor of the motor domain of human CENP-E. The MT-CENP-E presteady-state kinetics of ATP binding are unaffected by GSK-923295A and are consistent with our steady-state data for GSK923295A being allosteric inhibitor. However, GSK923295A induces a dramatic decrease in the observed rates of MT-stimulated Pi release and ADP-release. Surprisingly, in the absence of MTs, GSK923295A accelerates the rate of ADP-release. Kinetic studies of ATP- and ADP-promoted dissociation of the MT-CENP-E complex revealed that GSK923295A blocks the disassociation of CENP-E from MTs, whereas CENP-E-MT association kinetic studies revealed that GSK923295A accelerates the formation of the MT-CENP-E complex in the ADP state. The MT-CENP-E association data were further supported by a pelleting assay which showed that in the absence of GSK923295A CENP-E is weakly attached to MTs in the ADP-state. However, in the presence of GSK-923295, human CENP-E is tightly bound to MTs indicating that GSK923295A stabilizes the CENPE-ADP-MT complex. Based on the presteady-state and steady-state data we conclude that the mechanism of inhibition of CENP-E by GSK923295A is distinct from allosteric inhibitors of KSP (like Monastrol and ispinesib). Ispinesib altered the ability of KSP to bind microtubules and inhibited its movement by slowing the release of ADP without trapping the microtubule-KSP-ADP intermediate. However, GSK923295A appears to “lock” CENP-E onto MTs and inhibit the movement of this motor by stabilizing the ADP-Pi state of the MT-CENP-E complex.

Kinesin spindle protein (KSP), an ATPase responsible for spindle pole separation during mitosis that is present only in proliferating cells, has become a novel and attractive anticancer target with potential for reduced side effects compared to currently available therapies. We report herein the discovery of the first known ATP-competitive inhibitors of KSP, which display a unique activity profile as compared to the known loop 5 (L5) allosteric KSP inhibitors that are currently under clinical evaluation. Optimization of this series led to the identification of biphenyl sulfamide 20, a potent KSP inhibitor with in vitro antiproliferative activity against human cells with either wild-type KSP (HCT116) or mutant KSP (HCT116 D130V). In a murine xenograft model with HCT116 D130V tumors, 20 showed significant antitumor activity following intraperitoneal dosing, providing in vivo proof-of-principle of the efficacy of an ATP-competitive KSP inhibitor versus tumors that are resistant to the other known KSP inhibitors.

Kinesin motor proteins utilize the energy from ATP hydrolysis to transport cellular cargo along microtubules. Kinesins that play essential roles in the mechanics of mitosis are attractive targets for novel antimitotic cancer therapies. Monastrol, a cell-permeable inhibitor that specifically inhibits the kinesin Eg5, the Xenopus laevis homologue of human KSP, can cause mitotic arrest and monopolar spindle formation. In this study, we show that the extent of monastrol inhibition of KSP microtubule-stimulated ATP hydrolysis is highly dependent upon ionic strength. Detailed kinetic analysis of KSP inhibition by monastrol in the presence and absence of microtubules suggests that monastrol binds to the KSP-ADP complex, forming a KSP-ADP-monastrol ternary complex, which cannot bind to microtubules productively and cannot undergo further ATP-driven conformational changes.

Tuberculosis remains a global health problem, and programs dedicated to discovery of novel compounds against Mycobacterium tuberculosis require robust assays for high-throughput screening of chemical and natural product libraries. Enzymes involved in the biosynthesis of mycolic acids, vital components of the mycobacterial cell wall, have received much attention as potential drug targets. KasA and KasB, examples of the β-ketoacyl-acyl carrier protein synthase I/II (KASI/II) class of condensing enzymes of the M. tuberculosis fatty acid synthase II system have been the focus of several studies designed to biochemically characterize these enzymes. Whilst robust methods have been developed for FabH-like proteins, fast and sensitive assays for high-throughput screening of KASI/II enzymes have not been available. Here we report the development of a direct scintillation proximity assay (SPA) for the KASI/II enzymes, KasA and KasB. The SPA was more sensitive than existing assays, as shown by its ability to measure activity using less enzyme than other assay formats, and the SPA was validated using the known KAS inhibitor thiolactomycin. In addition, the KasA and KasB SPA was adapted for use with Staphylococcus aureus FabF to show the versatility of this assay format to KAS enzymes from other pathogenic organisms.