Transition metals are essential, but deregulation of their metabolism causes toxicity. Here, we report that the compound NSC319726 binds copper to induce oxidative stress and arrest glioblastoma-patient-derived cells at picomolar concentrations. Pharmacogenomic analysis suggested that NSC319726 and 65 other structural analogs exhibit lethality through metal binding. Although NSC319726 has been reported to function as a zinc ionophore, we report here that this compound binds to copper to arrest cell growth. We generated and validated pharmacogenomic predictions: copper toxicity was substantially inhibited by hypoxia, through an hypoxia-inducible-factor-1α-dependent pathway; copper-bound NSC319726 induced the generation of reactive oxygen species and depletion of deoxyribosyl purines, resulting in cell-cycle arrest. These results suggest that metal-induced DNA damage may be a consequence of exposure to some xenobiotics, therapeutic agents, as well as other causes of copper dysregulation, and reveal a potent mechanism for targeting glioblastomas.
Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently there is no mechanistic or structural information on ICDH. We optimized expression and purification to a yield high enough to perform the first detailed kinetic and structural studies for Mtb ICDH-1. A large solvent kinetic isotope effect (D2OV = 3.0 ± 0.2, D2O[V/Kisocitrate] = 1.5 ± 0.3) and a smaller primary kinetic isotope effect (DV = 1.3 ± 0.1, D[V/K[2R-2H]isocitrate] = 1.5 ± 0.2) allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn2+ bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking beta sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyses the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.
Malate synthase catalyzes the Claisen-like condensation of acetyl-coenzyme A and glyoxylate in the glyoxylate shunt of the citric acid cycle. The Mycobacterium tuberculosis malate synthase G gene, glcB, was cloned, and the N-terminal His6 tagged 80 kDa protein was expressed in soluble form and purified by metal affinity chromatography. A chromogenic 4,4′-dithiodipyridine assay did not yield linear kinetics, but the generation of an active site directed mutant, C619S, gave an active enzyme and linear kinetics. The resulting mutant exhibited comparable kinetics to wild type and was used for the full kinetic analysis. Initial velocity studies were intersecting suggesting a sequential mechanism, which was confirmed by product and dead-end inhibition. The inhibition studies delineated the ordered binding of glyoxylate followed by AcCoA and the ordered release of CoA followed by malate. The pH dependence of kcat and kcat/Kgly are both bell-shaped and catalysis depends on a general base (pK 5.3) and a general acid (pK 9.2). Primary kinetic isotope effects determined using [C2H3-methyl] acetyl-CoA suggested that proton removal and carbon-carbon bond formation were partially rate-limiting. Solvent kinetic isotope effects on kcat suggested the hydrolysis of the malyl-CoA intermediate was also partially rate-limiting. Multiple kinetic isotope effects, utilizing D2O and [C2H3-methyl] acetyl-CoA, confirmed a stepwise mechanism in which the step exhibiting primary kinetic isotope effects precedes the step exhibiting the solvent isotope effects. The kinetic data and the pH dependence of the kinetic parameters were combined with existing structural and mutagenesis data to propose a chemical mechanism for malate synthase from Mycobacterium tuberculosis.
Identifying drugs for the treatment of glioblastoma (GBM), a rapidly fatal disease, has been challenging. Most screening efforts have been conducted with immortalized cell lines grown with fetal bovine serum, which have little relevance to the genomic features found in GBM patients. Patient-derived neurosphere cultures, while being more physiologically relevant, are difficult to screen and therefore are only used to test a few drug candidates after initial screening efforts. Laminin has been used to generate two-dimensional cell lines from patient tumors, preserving the genomic signature and alleviating some screening hurdles. We present here the first side-by-side comparison of inhibitor sensitivity of laminin and neurosphere-grown patient-derived GBM cell lines and show that both of these culture methods result in the same pattern of inhibitor sensitivity. We used these screening methods to evaluate the dependencies of seven patient-derived cell models: three grown on laminin and four grown as neurospheres, against 56 agents in 17-point dose-response curves in 384-well format in triplicate. This allowed us to establish differential sensitivity of chemotherapeutic agents across the seven patient-derived models. We found that MEK inhibition caused patient-sample-specific growth inhibition and that bortezomib, an FDA-approved proteasome inhibitor, was potently lethal in all patient-derived models. Furthermore, the screening results led us to test the combination of the Bcl-2 inhibitor ABT-263, and the mTOR inhibitor AZD-8055, which we found to be synergistic in a subset of patient-derived GBM models. Thus, we have identified new candidate therapeutics and developed a high-throughput screening system using patient-derived GBM samples.
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