Nicholas D. Adams

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.

Reductive carboxylation supports redox homeostasis during anchorage-independent growth

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Discovery of GSK1070916, a Potent and Selective Inhibitor of Aurora B/C Kinase

The Aurora kinases play critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. Selective inhibitors may provide a new therapy for the treatment of tumors with Aurora kinase amplification. Herein we describe our lead optimization efforts within a 7-azaindole-based series culminating in the identification of GSK1070916 (17k). Key to the advancement of the series was the introduction of a 2-aryl group containing a basic amine onto the azaindole leading to significantly improved cellular activity. Compound 17k is a potent and selective ATP-competitive inhibitor of Aurora B and C with K(i)* values of 0.38 +/- 0.29 and 1.5 +/- 0.4 nM, respectively, and is >250-fold selective over Aurora A. Biochemical characterization revealed that compound 17k has an extremely slow dissociation half-life from Aurora B (>480 min), distinguishing it from clinical compounds 1 and 2. In vitro treatment of A549 human lung cancer cells with compound 17k results in a potent antiproliferative effect (EC(50) = 7 nM). Intraperitoneal administration of 17k in mice bearing human tumor xenografts leads to inhibition of histone H3 phosphorylation at serine 10 in human colon cancer (Colo205) and tumor regression in human leukemia (HL-60). Compound 17k is being progressed to human clinical trials.

Mutant IDH1 Enhances the Production of 2-Hydroxyglutarate Due to Its Kinetic Mechanism.

The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT)

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

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.

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 A35: Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growth

Epithelial cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)1. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, an essential hallmark of cells capable of metastasis2. Previous study showed that detachment from the ECM is associated with enhanced reactive oxygen species (ROS) levels due to suppression of glucose metabolism in the pentose phosphate pathway3. Here we used metabolic flux analysis to identify an unconventional metabolic pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumor spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidative metabolism of both glucose and glutamine was suppressed in the spheroids, whereas reductive formation of citrate from glutamine was enhanced. Enhanced reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1) rather than mitochondrial IDH2, because this activity was eliminated in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. Reductive carboxylation occurred in the absence of hypoxia, a well-known inducer of reductive glutamine metabolism4, 5. IDH1dependent reductive carboxylation mitigated mitochondrial ROS during spheroid growth, and IDH1 deletion or inhibition blunted spheroid growth in a ROS-dependent manner. Ablating expression of IDH2 or the mitochondrial citrate transporter did not substantially alter reductive labeling, but suppressed spheroid growth. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism. During anchorage-independent culture, reductive carboxylation produces cytosolic citrate, some of which is then imported into the mitochondria and metabolized in the TCA cycle. This results in the net transfer of NADPH from the cytosol to the mitochondria, mitigating mitochondrial ROS and maximizing cell growth. References: 1. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature 2004; 432:332-7. 2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144:646-74. 3. Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 2009; 461:109-13. 4. Wise DR, Ward PS, Shay JE, Cross JR, Gruber JJ, Sachdeva UM, Platt JM, DeMatteo RG, Simon MC, Thompson CB. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability. Proceedings of the National Academy of Sciences of the United States of America 2011; 108:19611-6. 5. Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K, Jewell CM, Johnson ZR, Irvine DJ, Guarente L, et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 2012; 481:380-4. Citation Format: Lei Jiang, Alexander Shestov, Lance S. Terada, Nicholas D. Adams, Michael T. McCabe, Beth Pietrak, Stan J. Schimidt, Benjamin Schwartz, Ralph J. DeBerardinis. Cytosolic reductive carboxylation is required for mitochondrial redox homeostasis during anchorage-independent cell growth. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A35.

Abstract C38: Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients9 cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients9 cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients9 cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.