Jeffrey D. Carson

Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin.

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.

Effects of oncogenic p110α subunit mutations on the lipid kinase activity of phosphoinositide 3-kinase

The PIK3CA gene, encoding the p110α catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110α, 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 PI3Kα, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.

Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor.

The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K(i) values of 380 and 320nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

Mechanism of Inhibition of Human KSP by Ispinesib

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.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Conformation-dependent ligand regulation of ATP hydrolysis by human KSP: activation of basal hydrolysis and inhibition of microtubule-stimulated hydrolysis by a single, small molecule modulator.

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.

Characterization of PI3K class IA isoforms with regulatory subunit p55α using a scintillation proximity assay

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110alpha, p110beta, and p110delta, each coexpressed with the regulatory subunit p85alpha or splice variant p55alpha. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.

Abstract 3513: Inhibition of LSD1 for the treatment of cancer

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 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

Abstract C62: Identification of GSK2126458, a highly potent inhibitor of phosphoinositide 3‐kinase (PI3K) and the mammalian target of rapamycin (mTOR)

Phosphoinositide 3‐kinase (PI3K) is a critical regulator of cell growth and transformation and its signaling pathway is one of the most commonly mutated pathways in human cancer. 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 the PI3K/AKT pathway. GSK1059615, our first PI3K clinical compound, progressed to a dose escalation study in patients with refractory malignancies. Following the discovery of GSK1059615, we sought to identify a second inhibitor with improved potency, selectivity, and pharmacokinetics. Key to our approach to achieving the desired levels of PI3K activity was to pursue structure‐based design utilizing crystallography of the more amenable PI3K as a surrogate protein. Following a chemistry lead optimization effort, the pyridylsulfonamide GSK2126458 was identified as a highly potent, orally bioavailable, pan‐PI3K and mTOR inhibitor (PI3K app Ki = 19 pM; mTORC1 app Ki = 180 pM; mTORC2 app Ki = 300 pM). Consistent with potent PI3K and mTORC2 enzyme inhibition, GSK2126458 decreased cellular levels of phosphorylated AKT (BT474 pAKT IC50 = 180 pM) and inhibited cell proliferation in a large panel of cancer cell lines (e.g. BT474 growth IC50 = 2 nM). GSK2126458 showed good exposure in four pre‐clinical animal species and exhibited in vivo activity in both pharmacodynamic and tumor growth efficacy models. GSK2126458 is being evaluated currently in human clinical trials for the treatment of cancer. The discovery, design, and optimization of GSK2126458 and related analogs will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C62.

Abstract C63: Biological characterization of GSK2126458, a novel and potent inhibitor of phosphoinositide 3‐kinase and the mammalian target of rapamycin (mTOR)

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.