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The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.

Somatic gain-of-function mutations in IDH1 have been identified in multiple tumor types including AML, glioma, cholangiocarcinoma and chondrosarcoma. The neo-enzymatic activity of mutant IDH1 results in accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), leading to a hyper-methylation phenotype, a block in cell differentiation, and tumor growth. Using a structure-based drug design approach, we have developed a highly potent covalent inhibitor of mutant IDH1. LY3410738 modifies a single cysteine (Cys269) in the allosteric binding pocket and rapidly inactivates the enzyme with a KI/Kinact = 84,257 M-1sec-1. The compound selectively inhibits the 2-HG in IDH1 mutant tumor cells without depleting the levels of a-ketoglutarate. Using patient-derived primary AML cells, we demonstrated that LY3410738 was more potent than AG120 in reversing the block in differentiation associated with IDH1 mutant activity. In vivo, LY3410738 displayed prolonged pharmacodynamic activity, depleting 2-HG levels in tumors at low circulating exposures and for an extended time after clearance of compound. Importantly, LY3410738 has the ability to cross the blood-brain barrier and can achieve concentrations in the brain that exceed those needed to engage the target. Consistent with this, LY3410738 effectively inhibits 2HG in orthotopic glioma models. Using patient-derived IDH1 mutant orthotopic AML models, we demonstrated that LY3410738 effectively inhibited 2-HG, induced differentiation, and cleared AML from mice. Collectively, LY3410738 represents the first covalent brain-penetrant mutant IDH1 inhibitor with potential for Best-in-Class activity. Citation Format: Nathan Brooks, Robin DeWalt, Serge Boulet, ZhaoHai Lu, Lisa Kays, Rachel cavitt, Sandra Gomez, John Strelow, Paul Milligan, Kenneth Roth, Renato Bauer, Stephen Antonysamy, Patric Hahn, Zoran Rankovic, Denis McCann, Gary Mo, Ramon Tiu, Timothy Burkholder, Sandaruwan Geeganage, Raymond Gilmour. Identification and characterization of LY3410738, a novel covalent inhibitor of cancer-associated mutant Isocitrate Dehydrogenase 1 (IDH1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-274.

Encoded by Kaposi's sarcoma-associated herpesvirus, viral macrophage-inflammatory protein-II (VMIP-II) is unique among CC chemokines in that it has been shown to bind to the CXC chemokine receptor CXCR4 as well as to a variety of CC chemokine receptors. This unique binding ability allows vMIP-II to block infection by a wide range of human immunodeficiency virus type I (HIV-1) strains, but the structural and dynamic basis for this broad range of binding is not known. 15N T1, T2 and 15N[-HN] nuclear Overhauser effect (NOE) values of vMIP-II, determined through a series of heteronuclear multidimensional nuclear magnetic resonance (NMR) experiments, were used to obtain information about the backbone dynamics of the protein. Whereas almost all chemokine structures reveal a dimer or multimer, vMIP-II has a rotational correlation time (tauc) of 4.7 +/- 0.3 ns, which is consistent with a monomeric chemokine. The rotational diffusion anisotropy, D parallel/D perpendicular, is approximately 1.5 +/- 0.1. The conformation of vMIP-II is quite similar to other known chemokines, containing an unstructured N-terminus followed by an ordered turn, three beta-strands arranged in an antiparallel fashion, and one C-terminal alpha-helix that lies across the beta-strands. Most of the protein is well-ordered on a picosecond time scale, with an average order parameter S2 (excluding the N-terminal 13 amino acids) of 0.83 +/- 0. 09, and with even greater order in regions of secondary structure. The NMR data reveal that the N-terminus, which in other chemokines has been implicated in receptor binding, extends like a flexible tail in solution and possesses no secondary structure. The region of the ordered turn, including residues 25-28, experiences conformational exchange dynamics. The implications of these NMR data to the broad receptor binding capability of vMIP-II are discussed.