Supplemental Information Structure-Guided Discovery of Phenyl-diketo Acids as Potent Inhibitors of M . tuberculosis Malate Synthase

The glyoxylate shunt plays an important role in fatty acid metabolism and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and overexpression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors guided optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics.

[1]  Bing Chen,et al.  Mycobacterium tuberculosis DeltaRD1 DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis. , 2006, Vaccine.

[2]  Elizabeth E Howell,et al.  A survey of aspartate-phenylalanine and glutamate-phenylalanine interactions in the protein data bank: searching for anion-π pairs. , 2011, Biochemistry.

[3]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[4]  Yun Tang,et al.  Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors. , 2002, Journal of medicinal chemistry.

[5]  L. Lally.  The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[6]  W. Jusko,et al.  Fluid shifts and other factors affecting plasma protein binding of prednisolone by equilibrium dialysis. , 1984, Journal of pharmaceutical sciences.

[7]  S. Remington,et al.  Structure of the Escherichia coli malate synthase G:pyruvate:acetyl‐coenzyme A abortive ternary complex at 1.95 Å resolution , 2003, Protein science : a publication of the Protein Society.

[8]  M. Yazdanian,et al.  Correlating Partitioning and Caco-2 Cell Permeability of Structurally Diverse Small Molecular Weight Compounds , 1998, Pharmaceutical Research.

[9]  Robert A. Copeland,et al.  Evaluation of enzyme inhibitors in drug discovery , 2013 .

[10]  S. Noack,et al.  13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation , 2011, PLoS pathogens.

[11]  Uwe Koch,et al.  Discovery of α,γ-Diketo Acids as Potent Selective and Reversible Inhibitors of Hepatitis C Virus NS5b RNA-Dependent RNA Polymerase , 2004 .

[12]  M. Egbertson,et al.  HIV integrase inhibitors: from diketoacids to heterocyclic templates: a history of HIV integrase medicinal chemistry at Merck West Point and Merck Rome (IRBM). , 2007, Current topics in medicinal chemistry.

[13]  P. Jeffrey,et al.  Utility of metabolic stability screening: comparison of in vitro and in vivo clearance , 2001, Xenobiotica; the fate of foreign compounds in biological systems.

[14]  Robert A Copeland,et al.  Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. , 2005, Methods of biochemical analysis.

[15]  Thomas R. Ioerger,et al.  High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism , 2011, PLoS pathogens.

[16]  K. Pethe,et al.  Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability. , 2010, Microbiology.

[17]  Sabine Ehrt,et al.  Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates. , 2010, Chemistry & biology.

[18]  R. Dayam,et al.  Novel dimeric aryldiketo containing inhibitors of HIV-1 integrase: effects of the phenyl substituent and the linker orientation. , 2008, Bioorganic & medicinal chemistry.

[19]  Roman A. Laskowski,et al.  LigPlot+: Multiple Ligand-Protein Interaction Diagrams for Drug Discovery , 2011, J. Chem. Inf. Model..

[20]  U. Sauer,et al.  A Novel Metabolic Cycle Catalyzes Glucose Oxidation and Anaplerosis in Hungry Escherichia coli* , 2003, Journal of Biological Chemistry.

[21]  H. Krebs,et al.  Synthesis of Cell Constituents from C2-Units by a Modified Tricarboxylic Acid Cycle , 1957, Nature.

[22]  Robert H. Gilman,et al.  Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by Using the Microplate Alamar Blue Assay , 1998, Journal of Clinical Microbiology.

[23]  Sujata Sharma,et al.  Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis , 2000, Nature Structural Biology.

[24]  K. Dunbar,et al.  Anion-pi interactions. , 2008, Chemical Society reviews.

[25]  L G Wayne,et al.  Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions , 1982, Infection and immunity.

[26]  W. Segal,et al.  BIOCHEMICAL DIFFERENTIATION OF MYCOBACTERIUM TUBERCULOSIS GROWN IN VIVO AND IN VITRO , 1956, Journal of bacteriology.

[27]  J. Badia,et al.  Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli. , 2001, The Biochemical journal.

[28]  E. Muñoz-Elías,et al.  Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence , 2005, Nature Medicine.

[29]  J. Knowles,et al.  Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test. , 1988, Biochemistry.

[30]  Rick Lyons,et al.  The temporal expression profile of Mycobacterium tuberculosis infection in mice. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Blanchard,et al.  Kinetic and chemical mechanism of malate synthase from Mycobacterium tuberculosis. , 2011, Biochemistry.

[32]  M. Dunn,et al.  Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. , 2009, Microbiology.

[33]  James C Sacchettini,et al.  Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis , 2006, Molecular microbiology.

[34]  N. Ford,et al.  Rational use of moxifloxacin for tuberculosis treatment. , 2011, The Lancet. Infectious diseases.

[35]  D. Behera.  Global Tuberculosis Control 2011, WHO Report 2011 , 2012 .

[36]  M. Jackson,et al.  A preference for edgewise interactions between aromatic rings and carboxylate anions: the biological relevance of anion-quadrupole interactions. , 2007, The journal of physical chemistry. B.

[37]  Orion B. Berryman,et al.  Structural criteria for the design of anion receptors: the interaction of halides with electron-deficient arenes. , 2007, Journal of the American Chemical Society.

[38]  Yang Liu,et al.  Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages , 2003, The Journal of experimental medicine.

[39]  D J Rance,et al.  The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. , 1997, The Journal of pharmacology and experimental therapeutics.

[40]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[41]  S. Remington,et al.  The product complex of M. tuberculosis malate synthase revisited , 2006, Protein science : a publication of the Protein Society.

[42]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[43]  Gilla Kaplan,et al.  Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[44]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[45]  H. Sakuraba,et al.  Structure of an archaeal alanine:glyoxylate aminotransferase. , 2008, Acta crystallographica. Section D, Biological crystallography.

[46]  S. Franzblau,et al.  Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis , 2007, Antimicrobial Agents and Chemotherapy.

[47]  James C Sacchettini,et al.  Biochemical and Structural Studies of Malate Synthase fromMycobacterium tuberculosis * , 2002, The Journal of Biological Chemistry.

[48]  M. Motyl,et al.  Basic Microbiological Techniques Used in Antibacterial Drug Discovery , 2005, Current protocols in pharmacology.

[49]  Gerald R. Fink,et al.  The glyoxylate cycle is required for fungal virulence , 2001, Nature.

[50]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[51]  P. Cardona,et al.  Fast Standardized Therapeutic-Efficacy Assay for Drug Discovery against Tuberculosis , 2010, Antimicrobial Agents and Chemotherapy.

[52]  James C. Sacchettini,et al.  Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase , 2000, Nature.