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Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3( S )-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors. Squalene epoxidase (SQLE) is a key enzyme in cholesterol biosynthesis and is a target for hypercholesteremia and cancer drug development. Here the authors present the crystal structures of the human SQLE catalytic domain alone and bound with small molecule inhibitors, which will facilitate the development of next-generation SQLE inhibitors.

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (m1DH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the m1DH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of m1DH1 impaired the growth of IDH1-mutant—but not IDH1-wild-type—glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that m1DH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

Somatic mutations in the isocitrate dehydrogenase 2 gene ( IDH2 ) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG) 1 – 8 . Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants 9 , 10 . In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2 -mutant AML 11 . Here we describe two patients with IDH2 -mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans , such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis , which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH -mutant malignancies. A new mechanism of acquired clinical resistance in two patients with acute myeloid leukaemia driven by mutant IDH2 is described, in which a second-site mutation on the wild-type allele induces therapeutic resistance to IDH2 inhibitors.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase 2 , 3 , yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17–IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI–IκBζ regulatory axis could be an important new strategy for the treatment of IL-17–IκBζ-mediated autoimmune diseases. The immunoregulatory metabolite itaconate and its dimethyl derivative induce electrophilic stress and react with glutathione to induce both Nrf2-dependent and Nrf2-independent responses, resulting in AF3-mediated inhibition of the inflammation-related protein IκBζ.

Dapagliflozin, a Selective SGLT2 Inhibitor, Improves Glucose Homeostasis in Normal and Diabetic Rats Songping Han 1 , Deborah L. Hagan 1 , Joseph R. Taylor 1 , Li Xin 1 , Wei Meng 2 , Scott A. Biller 2 3 , John R. Wetterau 1 4 , William N. Washburn 2 and Jean M. Whaley 1 1 Metabolic Diseases Biology, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 2 Metabolic Diseases Chemistry, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 3 Novartis Institute for Biomedical Research, Cambridge, Massachusetts 4 Cerenis Therapeutics, Ann Arbor, Michigan Corresponding author: Jean M. Whaley, Bristol-Myers Squibb Research and Development, 311 Pennington Rocky Hill Rd., Mail Code 21-2.01, Pennington, NJ 08534. E-mail: jean.whaley{at}bms.com Abstract OBJECTIVE— The inhibition of gut and renal sodium-glucose cotransporters (SGLTs) has been proposed as a novel therapeutic approach to the treatment of diabetes. We have identified dapagliflozin as a potent and selective inhibitor of the renal sodium-glucose cotransporter SGLT2 in vitro and characterized its in vitro and in vivo pharmacology. RESEARCH DESIGN AND METHODS— Cell-based assays measuring glucose analog uptake were used to assess dapagliflozin's ability to inhibit sodium-dependent and facilitative glucose transport activity. Acute and multi-dose studies in normal and diabetic rats were performed to assess the ability of dapagliflozin to improve fed and fasting plasma glucose levels. A hyperinsulinemic-euglycemic clamp study was performed to assess the ability of dapagliflozin to improve glucose utilization after multi-dose treatment. RESULTS— Dapagliflozin potently and selectively inhibited human SGLT2 versus human SGLT1, the major cotransporter of glucose in the gut, and did not significantly inhibit facilitative glucose transport in human adipocytes. In vivo, dapagliflozin acutely induced renal glucose excretion in normal and diabetic rats, improved glucose tolerance in normal rats, and reduced hyperglycemia in Zucker diabetic fatty (ZDF) rats after single oral doses ranging from 0.1 to 1.0 mg/kg. Once-daily dapagliflozin treatment over 2 weeks significantly lowered fasting and fed glucose levels at doses ranging from 0.1 to 1.0 mg/kg and resulted in a significant increase in glucose utilization rate accompanied by a significant reduction in glucose production. CONCLUSIONS— These data suggest that dapagliflozin has the potential to be an efficacious treatment for type 2 diabetes. EC50, half-maximal response FPG, fasting plasma glucose SGLT, sodium-glucose cotransporter Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 20 March 2008. DOI: 10.2337/db07-1472. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-1472 . All of the work described herein was conducted in its entirety at Bristol-Myers Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted March 18, 2008. Received October 15, 2007. DIABETES

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers. Cancer cells are metabolically adaptable and the identification of specific vulnerabilities is challenging. Here the authors identify a subset of neuroendocrine cell lines exquisitely sensitive to inhibition of SQLE, an enzyme in the cholesterol biosynthetic pathway, due to the toxic accumulation of pathway intermediate squalene.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele–alcohol interaction may be an even greater human public health hazard than previously appreciated. About 40% of East Asians and over 500 million people worldwide carry a specific polymorphism, ALDH2*2, and exhibit “Asian flush” after alcohol drinking. We generated a mouse strain with this engineered polymorphism and demonstrated its resemblance to human carriers in terms of defective alcohol metabolism. With this model, we show that murine ALDH2*2 increases ALDH2 protein turnover and promotes chemical-induced liver tumor development. Importantly, ALDH2 is unstable in ALDH2*2 human liver samples and is significantly down-regulated in human liver tumors. Data from our mouse and clinical studies suggest that ALDH2 is a liver tumor suppressor and the ALDH2*2 polymorphism is a risk factor for liver cancer.

Muraglitazar, a Novel Dual (α/γ) Peroxisome Proliferator–Activated Receptor Activator, Improves Diabetes and Other Metabolic Abnormalities and Preserves β-Cell Function in db/db Mice Thomas Harrity 1 , Dennis Farrelly 1 , Aaron Tieman 1 , Cuixia Chu 1 , Lori Kunselman 1 , Liqun Gu 1 , Randolph Ponticiello 1 , Michael Cap 1 , Fucheng Qu 2 , Chunning Shao 2 , Wei Wang 2 , Hao Zhang 2 , William Fenderson 3 , Sean Chen 2 , Pratik Devasthale 2 , Yoon Jeon 2 , Ramakrishna Seethala 1 , Wen-Pin Yang 3 , Jimmy Ren 1 , Min Zhou 1 , Denis Ryono 2 , Scott Biller 2 , Kasim A. Mookhtiar 1 , John Wetterau 1 , Richard Gregg 1 , Peter T. Cheng 2 and Narayanan Hariharan 1 1 Department of Metabolic Diseases Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 2 Department of Metabolic Diseases Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 3 Department of Applied Genomics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey Address correspondence and reprint requests to Narayanan Hariharan, PhD, Metabolic Diseases, HWP-21-2.02, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543. E-mail: narayanan.hariharan{at}bms.com Abstract Muraglitazar, a novel dual (α/γ) peroxisome proliferator–activated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03–50 mg · kg −1 · day −1 for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg · kg −1 · day −1 for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg · kg −1 · day −1 for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases high–molecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (α/γ) PPAR activator muraglitazar 1 ) exerts potent and efficacious antidiabetic effects, 2 ) preserves pancreatic insulin content, and 3 ) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels. ACO, acyl coenzyme-A oxidase FFA, free fatty acid HMW, high molecular weight LMW, low molecular weight MMW, medium molecular weight PPAR, peroxisome proliferator–activated receptor WAT, white adipose tissue Footnotes S.B. is currently affiliated with Novartis Institute Bio-Med Research, Cambridge, Massachusetts. K.A.M. is currently affiliated with Advinus Therapeutics, Pune, India. J.W. is currently affiliated with the Department of Cardiovascular Pharmaceuticals, Pfizer, Ann Arbor, Michigan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted September 26, 2005. Received May 23, 2005. DIABETES

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.