7 research outputs found
Mechanistic studies of the Class I ribonucleotide reductase from Escherichia coli
Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Vita.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. The class I RNRs are found in many bacteria, DNA viruses, and all eukaryotes including humans, and are composed of two homodimeric subunits: R1 and R2. RNR from Escherichia coli (E. coli ) serves as the prototype of this class. R1 has the active site where nucleotide reduction occurs, and R2 contains the diferric-tyrosyl radical (Y Ā· ) cofactor essential for radical initiation on R1. The rate-determining step in E. coli RNR has recently been shown to be a physical step prior to generation of the putative thiyl radical (S Ā· ) on C439. Thus, the chemistry of nucleotide reduction is kinetically invisible, which has precluded detection of intermediates in the reduction process with the normal substrate. Perturbation of the system using mechanism-based inhibitors and site-directed mutants of R1 and R2 has provided the bulk of the insight into the reduction mechanism by inference.(cont.) The work described in this thesis makes use of two mechanism-based inhibitors, 20 - azido - 20 - deoxyuridine - 50 - diphosphate (N3UDP) and 20 - deoxy - 20,20 - difluorocytidine - 50 - diphosphate (dFdCDP), and one active site mutant, E441Q R1, to further our understanding of the catalytic capabilities of RNR. The results provide strong support for a 30 - ketodeoxynucleotide intermediate postulated to lie on the normal reduction pathway, as well as for the elimination of nitrogenous base in the active site of R1 during inhibition. The studies further show that under physiologically relevant reducing conditions, inhibition of RNR by the clinically important nucleotide analog dFdCDP is a result of covalent modification. An essential part of these studies was the development of a robust, high-yielding enzymatic method for the selective 50 - phosphorylation of cytidine, 20 - deoxycytidine, 20 - deoxyuridine and their analogs that are not amenable to standard chemical phosphorylation methods.by Erin Jelena Artin.Sc.D
A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model
Structure of the Nitrogen-Centered Radical Formed during Inactivation of E. coli Ribonucleotide Reductase by 2ā-Azido-2ā-deoxyuridine-5ā-diphosphate: Trapping of the 3ā-Ketonucleotide
Differential Aspartate Usage Identifies a Subset of Cancer Cells Particularly Dependent on OGDH
Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention
Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-Ī±2Ī²2 of <i>E. coli</i> Ribonucleotide Reductase
The <i>Escherichia coli</i> ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: Ī± and Ī². Incubation of the E441Q-Ī± mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4ā²-radical of a 3ā²-keto-2ā²-deoxycytidine 5ā²-diphosphate. To identify the structure of the nucleotide radical [1ā²-<sup>2</sup>H], [2ā²-<sup>2</sup>H], [4ā²-<sup>2</sup>H], [5ā²-<sup>2</sup>H], [Uā<sup>13</sup>C, <sup>15</sup>N], [Uā<sup>15</sup>N], and [5,6 -<sup>2</sup>H] CDP and [Ī²-<sup>2</sup>H] cysteine-Ī± were synthesized and incubated with E441Q-Ī±2Ī²2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of <sup>2</sup>H at C4ā² and C1ā² altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5ā²-diphosphate. The relationship of this radical to the disulfide radical anion is discussed
A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model
D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II
Discovery of AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1 Mutant Cancers
Somatic point mutations
at a key arginine residue (R132) within
the active site of the metabolic enzyme isocitrate dehydrogenase 1
(IDH1) confer a novel gain of function in cancer cells, resulting
in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite.
Elevated 2-HG levels are implicated in epigenetic alterations and
impaired cellular differentiation. IDH1 mutations have been described
in an array of hematologic malignancies and solid tumors. Here, we
report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1
mutant enzyme that exhibits profound 2-HG lowering in tumor models
and the ability to effect differentiation of primary patient AML samples
ex vivo. Preliminary data from phase 1 clinical trials enrolling patients
with cancers harboring an IDH1 mutation indicate that AG-120 has an
acceptable safety profile and clinical activity