BiochemistryConformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin ( Communicated by Joseph S. Fruton, June 21, 1982 ABSTRACT Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl Pepstatin is a naturally occurring inhibitor of aspartyl proteinases (1); it has the amino acid composition isovaleryl (Iva)-ValVal-Sta-Ala-StaOH, in which Sta is the residue of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid] (2). The interaction ofpepstatin with aspartyl proteases is characterized by small dissociation constants [i.e., 4.6 x 10-11 M with porcine pepsin (3) and 1.5 x 10-10 M with penicillopepsin (unpublished results)]. It Structure I Penicillopepsin is an aspartyl proteinase isolated from the mold Penicillium janthinellum. Its crystal structure (6, 7) has been refined at 1.8-A resolution (8) to a conventional R-factor § of 0.136 for those reflections with I, the intensity of Bragg reflections, equal to or greater than the estimated standard deviation of the intensity measurement derived from counting statistics [I 2 o-(I)]. The aspartyl proteinases are characterized by a long binding cleft that can accommodate 7 or 8 amino acid residues of an oligopeptide substrate in an extended conformation. The two catalytically important aspartyl residues, Asp&jand Asp-213, are centrally located in this binding cleft (Asp32and Asp-215 in the porcine pepsin sequence numbering).Fluorescence measurements on the aspartyl proteinases with specific substrates have indicated that conformational mobility of groups in the active site may play an important role in the mechanism of these enzymes (9). MATERIALS AND METHODSThe enzyme, penicillopepsin, was prepared, purified, and crystallized as described (6,7,10). Crystals of the native enzyme The pepstatin derivative Iva-Val-Val-StaOEt was synthesized by the following procedure. t-Butoxycarbonyl (Boc)-StaOEt (12) was deprotected with 4 M HCl in dioxane and sequentially coupled with Boc-L-valine, Boc-L-valine, and isovaleryl anhydride by using procedures previously described for-the synthesis of pepstatin analogues (13) 6137The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

IDH mutations and tumorgenicity Kate Yen1, Fang Wang1, Sung Choe1, Stefanie Schalm1, Erica Hansen1, Kimberly Straley1, Janeta Popovici-Muller1, Jeremy Travins1, Hua Yang1, Lee Silverman1, William Kaelin2, Stefan Gross1, Lenny Dang1, Frank Salituro1, Jeff Saunders1, David Schenkein1, Michael Su1, Scott Biller1 Mutations in the isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) genes are present in ∼16% of acute myeloid leukemia, and these mutations cause neomorphic enzyme activity that results in the production of 2-hydroxyglutarate (2HG). Mutational and epigenetic profiling of a large patient cohort of acute myeloid leukemia (AML) revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. To further investigate the intrinsic effect of 2HG on hematopoietic differentiation, we studied erythroleukemia cell lines transfected with IDH1 and IDH2 mutant alleles which overexpress the mutant enzyme, have high levels of 2HG, and exhibit GM-CSF independent growth. Further investigation showed that GATA1, a transcriptional factor known to direct myeloid differentiation, is also down-regulated by IDH2m in these cells along with its downstream direct target SLC4A1. These results demonstrate that the IDH2m can perturb the expression of transcription factors that could lead to alterations in myeloid differentiation. These data suggest that an inhibitor of IDH1/2 mutations could correct for the altered gene expression patterns seen in IDH1/2 mutant AML tumors leading to a profound effect on hematopoietic differentiation, proliferation and tumor growth. We are currently studying the global effects of IDH1/2 mutant overexpression on the methylation of histones and DNA to have a broader understanding of the biological consequence of the IDH1/2 gain of function mutations. Furthermore, treatment of these cell lines with compounds specific for either IDH1 or IDH2 mutant enzymes has a profound effect on their biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-164. doi:1538-7445.AM2012-LB-164

3509 Mutations in the isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) genes are present in ∼16% of acute myeloid leukemia, and cause a neomorphic enzyme activity that results in the production of 2-hydroxyglutarate (2HG). Mutational and epigenetic profiling of a large patient cohort of acute myeloid leukemia (AML) has revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and an impaired hematopoietic differentiation. To further investigate the intrinsic effect of 2HG on hematopoietic proliferation and differentiation, we transfected an erythroleukemia cell line (TF-1) with either IDH1 or IDH2 mutant alleles. These cells overexpress the mutant enzyme, have high levels of 2HG, and exhibit GM-CSF independent growth. Consistent with clinical observations, overexpression of the IDH mutant proteins led to hypermethylation of both histones and DNA. These results suggest that mutations in IDH1/2 could lead to epigenetic rewiring of cells that could facilitate the gain of function phenotype. To gain a broader understanding of the biological consequence of the IDH1/2 gain of function mutations we have generated small molecules that are capable of selectively inhibiting IDHm enzymes. Upon compound treatment in vitro, we are able to reverse hypermethylation of both histones and DNA in Idhm expressing cells. These compounds are also suitable for use in vivo and upon compound treatment are able to lower 2HG by >90% in tumor xenograft models. These data suggest that an inhibitor of IDH1/2 mutations could correct the altered gene expression patterns seen in IDH1/2 mutant AML tumors and potentially lead to a profound effect on hematopoietic differentiation, proliferation and tumor growth. Disclosures: Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership. Schalm:Agios Pharmaceuticals: Employment, Equity Ownership. Hansen:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment. Kernytsky:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:Agios Pharmaceuticals: Employment, Equity Ownership. Liu:Agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:Agios Pharmaceuticals: Employment, Equity Ownership. Travins:Agios pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals: Employment, Equity Ownership. Gross:Agios Pharmaceuticals: Employment, Equity Ownership. Dang:Agios Pharmaceuticals: Employment, Equity Ownership. Salituro:Agios Pharmaceuticals: Consultancy, Equity Ownership. Saunders:Agios Pharmaceuticals: Consultancy, Equity Ownership. Dorsch:Agios Pharmaceuticals: Employment, Equity Ownership. Agresta:Agios Pharmaceuticals: Employment. Schenkein:Agios Pharmaceuticals: Employment, Equity Ownership. Su:Agios Pharmaceuticals: Employment, Equity Ownership. Biller:Agios Pharmaceuticals: Employment, Equity Ownership.

Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. Here, we describe the mechanism of action and cellular effects of AG-348, an allosteric activator of the red cell isoform of pyruvate kinase (PKR). Hundreds of mutant alleles of PKR have been identified and are known to have deleterious effects on catalytic activity, protein stability, or protein expression. We demonstrate that AG-348 can potently activate a spectrum of recombinantly expressed PKR mutant proteins, including mutations that span distinct subdomains of the enzyme. The R532W mutation is quite sensitive to AG-348 modulation, with over 4-fold activation of the enzyme activity, even as the mutation renders PKR insensitive to stimulation by its endogenous allosteric regulator fructose 1,6-bisphosphate (FBP) (Figure A). Crystallographic analysis reveals that very few mutations associated with PKD occur within the AG-348 binding pocket, accounting for its broad activity. The binding of AG-348 attenuates the thermostability defect of several mutant alleles of PKR, including the commonly observed R510Q mutant that has a half-life of ∼2% of the half-life of wild-type PKR when incubated at 53°C. Pre-incubation of the R510Q protein with AG-348 restores the half-life to ∼70% that of the wild-type enzyme (Figure B). PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the PKR substrate phosphenolpyruvate (PEP) and deficiency in the PKR product adenosine triphosphate (ATP). PKD red cells from several patients with distinct compound heterozygous PKR mutations exposed to AG-348 had increased PKR enzyme activity (up to 4-fold over control) and showed consistent dose and time-dependent metabolic responses (Figure C), including sharp reductions in PEP (up to 70% compared to control) and increases in ATP levels (up to 100% over control). These responses were observed in patient samples harboring PKR mutations that we had studied biochemically (including R486W and R510Q), but also in an instance where the mutation had not previously been biochemically characterized (A495V). In these ex-vivo settings, ATP levels in AG-348 treated cells can reach levels that are typical of normal, non-PKD red cells. These data support the hypothesis that drug intervention with AG-348 may restore glycolytic pathway activity and normalize red cell metabolism in vivo. This therapeutic approach may be an effective way to correct the underlying pathology of PKD and, importantly, provide clinical benefit to patients. Disclosures: Kung: Agios Pharmaceuticals: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals: Employment, Equity Ownership. Histen:Agios Pharmaceuticals: Employment, Equity Ownership. Hill:Agios Pharmaceuticals: Employment, Equity Ownership. Si:Agios Pharmaceuticals: Employment, Equity Ownership. Kernytsky:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:Agios Pharmaceuticals: Employment, Equity Ownership. DeLaBarre:Agios Pharmaceuticals: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals: Employment, Equity Ownership. Ho:Agios Pharmaceuticals: Employment, Equity Ownership. Salituro:Agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:Agios Pharmaceuticals: Employment, Equity Ownership. Agresta:Agios Pharmaceuticals: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals: Employment, Equity Ownership. Dang:Agios Pharmaceuticals: Employment, Equity Ownership.