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The Career of Christopher T. Walsh

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The Career of Christopher T. Walsh The Career of Christopher T. Walsh Supergroup Meeting May 9, 2007 Teresa D. Beeson Outline ! Who is Christopher T. Walsh? ! Research ! Suicide Substrates ! Vancomycin Resistance ! Siderophores ! Biosynthesis of Natural Products 1 Biography Christopher T. Walsh Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School) Biography . 1965 A.B. in biology (Harvard Univ.) . C. Walsh, J.H. Law, and E.O. Wilson. "Purification of the Fire Ant Trail Substance." Nature 1965, 207, 320-321. 1970 Ph.D. in life sciences (Rockefeller Univ.) . Advisor Leonard B. Spector . 6 publications, all first author . "The Mechanism of Action of the Citrate Cleavage Enzyme." Ph.D. Dissertation, The Rockefeller University. 2 Biography . 1970-1972 Postdoctoral Fellow (Brandeis Univ.) . Advisor Robert H. Abeles . 8 publications . 1972-1987 Professor of Chemistry and Biology (MIT) . 1987-1995 Chair, Dept. of Biological Chemistry and Molecular Pharmacology (Harvard Medical School) . 1991-present Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School) . 1992-1995 President of Dana Farber Cancer Institute Biography . Authored over 650 publications since 1965 . Average of over 15 publicatons per year for 42 years . Former students and post docs include: . Michael Marletta (Berkeley) . Robert Pascal (Princeton) . Peter Schultz (Scripps) . Greg Verdine (Harvard) . Yian Shi (Colorado State) . Thomas Wandless (Stanford) 3 Affiliations . National Academy of Sciences . Institute of Medicine . American Academy of Arts and Sciences. American Academy of Microbiology . Pharmaceutical consultant (Merck, Roche, and Abbot) . Scientific advisor (Microbia, Genzyme, Immunogen, Kosan Biosciences, and Millennium) . Board of Directors (Critical Therapeutics, Kosan, Immunogen, Leukosite. Microbia, Transform, and Vicuron) Awards . Eli Lilly Award in Biochemistry . Arthur C. Cope Scholar Award in Organic Chemistry . Repligen Award in Biological Chemistry . Alfred Bader Award in Bioorganic and Bioinorganic Chemistry . Promega Biotechnology Research Award from the American Society for Microbiology (ASM). 4 Suicide Substrates Suicide Substrates ! What is a suicide substrate? ! A competitive inhibitor that is converted to an irreversible inhibitor at the active site of the enzyme O O O O HO HN Me N Me N Me HN Me NADPH, O2 – H2O P450 P450 OH OH O OH Acetaminophen Imino quinone Alkylated Enzyme Tylenol ! The enzyme causes it's own destruction by unmasking a latent functional group Walsh, C. T. Tetrahedron, 1982, 38, 871. 5 Suicide Substrates ! Suicide substrates in medicine ! Chemotherapy Me O O O Me H F HN HO NH2 H H NH2 O O N F F H OH 5-Fluorouracil DFMO Formestane ! Antibiotics O OH NH2 O N O N O S H O O Sulbactam D-cycloserine ! Suicide substrates for research ! Can be used study the role of proteins in vitro or in vivo Walsh, C. T. Tetrahedron, 1982, 38, 871. Suicide Substrates ! Enzyme Target: E. coli B Alanine Racemase ! Alanine racemase epimerizes L-alanine O O O Me alanine racemase Me Me OH OH OH NH2 NH2 NH2 L-alanine D-alanine ! D-Alanine incorporated into bacterial cell walls ! D-Ala-D-Ala binding site for Vancomycin ! Racemases not found in mammalian cells ! Natural Antibiotics for Alanine Racemase NH2 O O O H2N O OH N O H NH2 D-cycloserine O-carbamoyl-D-serine Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. 6 Suicide Substrates ! Haloalanines also shown to be bacteriocidal ! Time-dependant inhibition of alanine racemase O O Cl OH F OH NH2 NH2 D-!-chloroalanine D-!-fluoroalanine ! Deuterated analogues more potent antibiotics D D O D D O Cl OH F OH NH2 NH2 ! What is the mode of enzyme deactivation? Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. Suicide Substrates ! Observations from the Walsh Lab ! Time-dependant inactivation of alanine racemase by fluoroalanine ! Enzyme activity was never regained after filtration or competitive binding ! 14C-Labeled fluoroalanine caused 14C-labeling of enzyme ! Haloalanines deemed to be irreversible inhibitors Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. 7 Suicide Substrates ! Observations from the Walsh Lab ! Alanine racemase did not epimerize haloalanines O O O Me alanine racemase Me Me OH OH OH NH2 NH2 NH2 L-alanine D-alanine O O alanine racemase Me – Cl OH OH NH3 Cl NH2 O Pyruvate ! D-haloalanines much faster than L-haloalanines but... ! Turnovers before inactivation ~800 for all haloalanines ! Is a common inactivator or "suicide substrate" formed? Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. Suicide Substrates ! Explaining the observations O O – Cl O x O– NH3 NH3 Alkylated Enzyme ! Simple nucleophilic attack by enzyme we would expect less turnovers before inactivation for Cl than F ! Haloalanines must be suicide substrates Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. 8 Suicide Substrates ! Explaining the observations O H Cl O– H O O H N – O O O H P OH –O O Cl O– P OH HO O NH3 HO Me N Me N H H Pyridoxal-P Aldimine complex O O – Cl O– O H N H N O H –H+ O H – – –O – Cl O P OH P OH O O HO HO Me N Me N H H Enamino acid complex Electrophilic species Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. Suicide Substrates ! Explaining the observations O O– H N O O H – O O H2O H2O Me P OH O– O– O HO NH3 O Me N Pyruvate H Electrophilic species Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. 9 Suicide Substrates ! Explaining the observations O O– H N O O H – O O H2O H2O Me P OH O– O– O HO NH3 O Me N Pyruvate H Electrophilic species O O– H N H O – O O H2O P OH O O– HO O Me N H Alkylated Enzyme Enzyme nucleophilic attack Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. Suicide Substrates ! Natural D-serine antibiotics determined to be suicide substrates NH2 O O O O O H2N O OH Me O OH N O H NH2 NH2 D-cycloserine O-carbamoyl-D-serine O-acetyl-D-serine ! Turnovers per inactivation also ~800 ! D-serine does not inactivate alanine racemase O HO OH NH2 D-serine Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313. 10 Suicide Substrates ! Propargyl suicide substrates operate in a similar manner O O O O– • O– O– H N H N NH O H O H O –O –O –O P OH P OH P OH O O O HO HO HO N Me N Me N Me H H H O H+ O O– O O– Me N O H Me N O– –O O H P OH –O Me NH O P OH 3 HO O HO N Me N Me H H Johnston, M.; Jankowski, D.; Marcotte, P.; Tanaka, H.; Esaki, N.; Soda, K.; Walsh, C. T. J. Am. Chem. Soc., 1979, 21, 4690. Suicide Substrates ! Allyl sulfoxide suicide substrates undergo 2,3-rearrangement O O S O R O O – O S O S O– R Cl N O– H !-Elim R N H N OH H – OH HO3PO – HO3PO OH – HO3PO N Me N Me N Me H H H sulfenate ester OH O SR O– O Johnston, M.; Raines, R.; Walsh, C.; Firestone, R. A. J. Am. Chem. Soc., 1980, 4241. 11 Vancomycin Resistance Vancomycin Resistance ! Background: Vancomycin's mode of Action Disaccharide pentapeptide units are cross-linked to build rigid bacterial cell walls Vancomycin-PG-D-Ala-D-Ala complex sterically prevents further cell wall assemply Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. 12 Vancomycin Resistance ! The Key Missing H-Bond ! Key H-bond lost due to replacement of D-Alanine for D-Lactate ! 1000-fold reduction in Kd Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. Vancomycin Resistance ! 5 Genes Necessary for Vancomycin Resistance P vanR vanS vanH vanA vanX vanR PvanH ! All 5 encoded proteins isolated and characterized by Walsh Lab Protein Activity Function VanR Transmembrane histidine kinase Initiates signal transduction VanS Response regulator Activates vanH,A,X transcription VanH D-specific !-keto acid reductase Generates D-lactate VanA Depsipeptide ligase for D-Ala-D-lactate Generates D-Ala-D-lactate VanX D-Ala-D-Ala dipeptidase Removes D-Ala-D-Ala Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. 13 Vancomycin Resistance ! VanR and VanS: Enteroccocal Bacteria's "Sensor" In the absence of Vancomycin, VanH,A,X genes are not transcribed VanS located in the bacterial cell wall, phosphorylated upon external signal Phosphate transferred to VanR, which causes conformational change VanR-PO3 is DNA-binder to promoter region of vanH,A,X, induces transcription Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. Vancomycin Resistance ! Key Change from Alanine to Lactate O OH O VanH O Me Me O O Pyruvate D-Lactate OH Me O Me O H O VanA O N Me H2N O H2N O O O Me O Me D-Lactate D-Ala-D-Lactate D-Ala-D-Ala Me O O H N VanX 2 H2N H2N O O O Me Me D-Ala-D-Ala 2 D-Ala Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. 14 Vancomycin Resistance ! The Key Missing H-Bond ! Key H-bond lost due to replacement of D-Alanine for D-Lactate ! 1000-fold reduction in Kd Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21. Siderophores 15 Siderophores ! “Bacterial-Host Iron Wars” ! Iron required for bacterial growth ! Concentration of Fe(III) in water 10–18 M ! Concentration of Fe(III) in human serum ~10–24 M ! Bacterial growth requires cytoplasmic Fe(III) concentration of ~10–6 M Fischbach, M.
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