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Transcript
Translating Structural Biology into Medical Practice; Bridging the Gap Wim Hol Department of Biochemistry University of Washington Seattle, USA Thomassen à Thuessink Lezing Universitair Medisch Centrum Groningen (UMCG) Rijksuniversiteit Groningen (RUG) Nederland 25 oktober 2007 Rembrandt’s “The anatomical lesson of Dr. Tulp” 1632 1 From Anatomy to Structural Biology In 375 years: From a scale of millimeters to a scale of Ångstroms I.e. an increase in resolution by a factor of 107 (that is about 4 % per year) Enabling to understand life at atomic resolution. What is Structural Biology? 2 Towards protein structures Gly Ala Val Ile Leu Met Phe Ser Cys Thr His ASP Lys Glu Asn Arg Gln Tyr Trp Pro Amino Acids: The Building Blocks of Proteins Towards protein structures How does the protein chain fold, flex and function? 3 Towards protein structures Protein Crystals Bram Schierbeek Towards protein structures 700 m Synchrotrons provide intense X-rays with variable wavelengths 4 Towards protein structures 100 μm A frozen crystal scooped into in a nylon loop at 100 K Towards protein structures An X-ray “Precession Photograph” of Lipoamide Dehydrogenase The intensities of the spots relate in a complex way the protein structure The “diffraction pattern” is the Fourier Transform of the entire crystal. 5 Towards protein structures An Experimental Electron Density Distribution Jan Abendroth Towards protein structures Atoms Built Into an Experimental Electron Density Distribution Jan Abendroth 6 A protein in its native state Fold of Peptide Deformylase (PDF) A possible drug target from the major malaria parasite “Rainbow-colored” Abhinav Kumar STRUCTURE-BASED DRUG DESIGN Essential region of “Target” COMPLEMENTARITY PRINCIPLE Essential region of “Target” homolog WITH SUFFICIENT SELECTIVITY 7 Jan Tinbergen UN “Tinbergen Committee” Report 1970 Rich countries should spend 1% of their GNP on aid to developing countries. The proposal was defeated. World Bank Report 2006 !903 – 1994 Only five rich countries have fulfilled the UN official development assistance target of 0.7 of GNI: Nobel Prize Economics 1969 Denmark, Luxembourg, the Netherlands, Norway, and Sweden. Jan Tinbergen Some of the major tropical diseases of today AIDS Dengue Tuberculosis Children's diarrhea Malaria Sleeping Sickness Chagas Disease Leishmaniasis Schistosomiasis Filariasis River Blindness “Neglected” and “Totally Neglected” tropical diseases 8 “It is inconceivable that of the 1233 drugs that have been approved in the last decade, only 11 were for treating tropical diseases, and of these, half were intended for livestock, not humans”. Ismail Serageldin “World Poverty and Hunger – the Challenge for Science”. Science 296: 54-58, 2002. Sleeping Sickness 9 Sleeping Sickness aka African Trypanosomiasis • Parasite: Trypanosoma brucei • Vector: Glossina spp. • Sub-Saharan Africa • ~ 500 thousand cases per year • ~ 50 thousand deaths annually • Fever, fatigue and sleeping for long periods of the day • Fatal without treatment Sleeping Sickness aka African Trypanosomiasis Blood stream form of parasite Tsetse fly Lumbar puncture for diagnosis of parasites in CNS Sleeping sickness is caused by a unicellular eukarytote: Trypanosoma brucei – a “Trypanosomatid” Other pathogenic trypanosomatids are whole set of 18 Leishmania species. These cause a spectrum of different tropical diseases, called “leishmaniasis”. Many enzymes in Trypanosoma brucei and Leishmania species are very similar in amino acid seqeunce. With thanks to Wes Van Voorhis 10 The sleeping sickness parasite Blood-stream Form Trypanosome Red Blood Cell Trypanosome Note that the sleeping sickness parasite does NOT hide in red blood cells Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Parasite and Host Human GAPDH Trypanosomal GAPDH Cofactor (co-substrate) NAD Note the difference in conformation near the ribose of the NAD cofactor in the homologous proteins of host and parasite. 11 Exploring multiple hydrophobic grooves Hydrophobic Groove “Targeted Combinatorial Chemistry” to fill the grooves optimally Hydrophobic Groove Surface of L. mexicana* GAPDH with NAD bound. •Note: Leishmania mexicana GAPDH is ~77% sequence identical to Trypanosoma brucei GAPDH and all residues in the region of interest are identical in these two pathogenic “Trypanosomatids”. So these two enzymes are used interchangeably. Designed Adenosine Derivatives 100,000 x more potent than start compound adenosine LmGAPDH + NAD LmGAPDH + NMDBA CONFORMATIONAL CHANGES in L. mexicana GAPDH UPON BINDING DIFFERENT LIGANDS Stephen Suresh, Jerri Bressi, Alex Aranov, Michael Gelb 12 Inhibition of Trypanosoma cruzi amastigote by GAPDH inhibitor β-gal reporter: Blue indicates parasite alive Trypanosoma cruzi is yet another “Trypanosomatid” Blue color of β-gal reporter means parasite growth It causes “Chagas disease” in Latin America Many enzymes in Trypanosoma brucei and Trypanosoma cruzi are very similar in amino acid seqeunce. Fred Buckner, Wes Van Voorhis Malaria 13 Malaria Some sobering facts Plasmodium falciparum and Plasmodium vivax ~500 million cases of malaria annually ~1 to 2 million deaths per year Victims mainly children and pregnant women Global status of resistance to chloroquine and sulphadoxine/ pyrimethamine, the two most widely used antimalarial drugs. Data are from the WHO. ROBERT G. RIDLEY Nature 2002; 415, 686 - 693 14 Plasmodium falciparum The major malaria parasite Host Cell Invasion Machinery Myosin Tail Interacting Protein (MTIP) and the Myosin A tail MTIP MyoA Jürgen Bosch, Stewart Turley Bill Bergman 15 P. falciparum MTIP binding the Myosin A tail MTIP N-terminal domain MTIP undergoes dramatic conformational changes upon MyoA tail binding MyoA Tail Helix MTIP C-terminal domain Jürgen Bosch, Stewart Turley Bill Bergman P. falciparum MTIP completely surrounds the Myosin A tail MTIP N-terminal Domain MTIP C-terminal Domain 16 MTIP plus the MyoA-tail (P. knowlesi complex) MyoA Oxygen Carbon Nitrogen Note the hydrophobic character of the MTIP contact surface The MTIP-MyoA Tail Interface Hydrophobic interactions Oxygen 1389 Carbon Å2 Nitrogen buried surface Leu804 Val807 Ile811 Linking Leu&Val&Ile side chains → → → New Antimalarials? Late breaking news: “Compound screening” by thermal melt fluorescence (you need only a PCR machine!) of MTIP has resulted in about 10 compounds which stops the growth of malaria parasites in cell culture at low micromolar concentrations 17 Cholera and Children’s Diarrhea Vibrio cholerae : - produces CT - ~4000 victims per year Enterotoxigenic E. coli : - produces LT and ST - ~480,000 victims per year 18 Heat-labile Enterotoxin (LT), a very close relative of Cholera toxin (CT) A subunit B pentamer Titia Sixma The secretion of Cholera toxin (CT) and Heat-labile enterotoxin (LT) by the marvelous Type 2 Secretion System (T2SS) 19 Peri-EpsD Nanobody CT Moonlander EpsH Pilin-like PDZ-EpsC Helix binding? peri-EpsM Fundamental Ferredoxin Fold N EpsI-J Pilin-like cyto-F1 Calciumbinding N M M F F peri-EpsL Fundamental Ferredoxin Fold Δ90-EpsE Secretion ATPase cyto-EpsL Actin-like N1-EpsE:cyto-EpsL Binary complex Cholera Toxin (CT) & the Type II Secretion System (T2SS) D D DD CT H C H H G G G C Periplasm G G G N J I G G K I LM M L L F E Facts and Fiction mixed J N F Cytoplasm Vibrio cholerae 20 The T2SS in Action D D DD AB5 H C H H G G G C Periplasm G G G J N I G G K I J N LM M L L F F E Cytoplasm Vibrio cholerae Facts and Fiction mixed The T2SS in Action D D DD AB5 C C H G N J H G G G G G G G J I K I LM M L L F E Facts and Fiction mixed Periplasm H N F Cytoplasm Vibrio cholerae 21 The T2SS in Action D D DD C C H G J N Periplasm H H G G G G G G G I K I J N LM M L L F F E Cytoplasm Vibrio cholerae Facts and Fiction mixed The T2SS in Action D D D DD AB5 C C H G J N H G G G G G G G I K I J LM M L L F E Facts and Fiction mixed Periplasm H N F Cytoplasm Vibrio cholerae 22 The T2SS in Action D AB5 H C D D DD H H G G G C Periplasm G G G J N I G G K I J N LM M L L F F E Cytoplasm Vibrio cholerae Facts and Fiction mixed The T2SS in Action AB5 D D D DD H C H H G G G C Periplasm G G G J N I G G K I LM M L L F E Facts and Fiction mixed J N F Cytoplasm Vibrio cholerae 23 The T2SS in Action AB5 D D D DD H C H H G G G C Periplasm G G G J N I G G K I J N LM M L L F F E Cytoplasm Vibrio cholerae Facts and Fiction mixed The T2SS in Action AB5 D D DD H C H H G G G C Periplasm G G G N J I G G K I LM M L L F E Facts and Fiction mixed J N F Cytoplasm Vibrio cholerae 24 The Interaction of Cholera toxin (CT) and Heat-labile enterotoxin (LT) with human cell surface receptors. And its inhibition CT and LT vs. human cell A B5 Ganglioside GM1 Intestinal epithelial cell CT: Cholera LT: Traveller’s & Children’s diarrhea 25 CT and LT vs. human cell A B5 Ganglioside GM1 Intestinal epithelial cell CT: Cholera LT: Traveller’s & Children’s diarrhea Cholera toxin – GM1 Receptor Interaction A subunit Toxin B pentamer Intestinal ce ll surface GM1 Receptors Ethan Merritt, Steve Sarfaty, Joseph Martial 26 GM1 Pentasaccharide bound by CT OH HO OH O HO OH O OH O O N H HO OH HOOC O O O OH HO O NH HO OH O O HO OH O HO His 13 OH IC50 = 14 nM The enemy Five receptor binding sites 27 Making ligands longer Ligand-Protein Complex 28 Pentavalent Ligand The pentavalent concept “Proper Pre-organization” 29 Gains in surface-receptor binding inhibition One-Unit Linker ??? x Single Finger Two-Unit Linker ???? x Single Finger Three-Unit Linker Four-Unit Linker ????? x Single Finger ?????? x Single Finger Erkang Fan, Zhongsheng Zhang, Jason Pickens, Jiyun Liu, et al Gains in surface-receptor binding inhibition One-Unit Linker 240 x Single Finger Two-Unit Linker 3600 x Single Finger Three-Unit Linker Four-Unit Linker 10,000 x Single Finger 104,000 x Single Finger Erkang Fan, Zhongsheng Zhang, Jason Pickens, Jiyun Liu, et al 30 Genome-wide approaches MEDICAL STRUCTURAL GENOMICS OF PATHOGENIC PROTOZOA (MSGPP) Genome Sequences Target & Ligand Selection I n f o r m a t I c s M a n Protein Production a Crystal Growth Assays g e m Medicinal Chemistry Virtual Screening Crystal Structure Determination e n t url: www.msgpp.org 31 Fragment Cocktail Crystallography A new tool in drug design Courtesy of Jürgen Bosch Fragment Cocktail Crystallography Roots Verlinde, C. et al. & Hol, W. G. J. (1997). Antitrypanosomiasis drug development based on structures of glycolytic enzymes. In Structure-Based Drug Design (Veerapandian, P., ed.), pp. 365-394. Marcel Dekker, New York. Describing the first crystallographic compound cocktail studies performed at the University of Groningen, The Netherlands, starting Oct 1990 32 Fragment Cocktail Crystallography Principle + Protein crystal with bound chemical fragment Protein crystals Cocktails of chemical fragments Probe protein pockets by soaking crystals in well-designed mixtures of 5-10 different chemicals, followed by crystal structure determinations Fragment Cocktail Crystallography Cocktail construction in MSGPP 9,500 compounds fragmentation 626 fragments isolate ring systems ACD Compound Filtering 23 frameworks (at connectivity level) 60 cocktails manual selection of compounds from each framework class 680 compounds - eliminate mutagens, known poisons - no highly functionalized compounds - retain Br containing compounds Christophe Verlinde, Erkang Fan http://faculty.washington.edu/verlinde/ 33 T. brucei Nucleoside 2-deoxyribosyltransferase plus Cocktail #4 Omni-present glycerol 1,2-DIHYDROBENZO[CD]INDOL-2-ONE Jürgen Bosch & Christophe Verlinde & Erkang Fan& & SGPP T. brucei Nucleoside 2-deoxyribosyltransferase plus Cocktail #5 Omni-present glycerol 6-AMINO-1-NAPHTHOL Jürgen Bosch & Christophe Verlinde & Erkang Fan & SGPP 34 L. major Coproporphyrinogen Oxidase plus Cocktails #61 and #68 in separate experiments 5-fluoroindole-2-carboxylic acid (FIC) from cocktail #68 Cyclopentylacetic acid from cocktail #61 Isolde LeTrong, Alberto Napuli, Liren Xiao, Ethan Merritt, Erkang Fan, Christophe Verlinde & MSGPP 2007 The two cocktail compounds bind at different sites: Ready for linking! Genome Target Selection Protein Expression Crystallization Data Collection Structure Determination Structure Analysis Structures with Ligands Bound Medicinal Chemistry & Pharmacology New Therapeutics 35 Interdisciplinary Research Essential for Progress in Medicine #1: Bring different disciplines close together spatially #2: Flexible funding of interdisciplinary projects Across all disciplines Short-to-medium term Top-light #3: Invite interdisciplinary lecturers “I-lecture” series #4: Reward interdisciplinary initiatives Award prizes for excellent interdisciplinary research #5: Create interdisciplinary buildings With flexibility – to avoid eternal occupants Bring disciplines together spatially University of Washington in Seattle The Campus on a Typical Day 36 Bring disciplines together spatially Computer Sceince Physics Chemistry Bioengineering Genome Sciences Biology Biochemistry Pharmacology Hospital Immunology Pharmacy Flexible Funding of Interdisciplinary Projects NIH “Program Projects” Buckner – Verlinde – Fan Target & Ligand Selection Hol Hol Core Core n f o r m a t I c s a Van Voorhis Protein Production n Van Voorhis Assays Hol Crystal Growth Fan Medicinal Chemistry Merritt Crystal Structure Determination a g e m Verlinde Virtual Screening e n t MSGPP Two “Cores”, Six Groups 1.5 M$ per year PLUS ~ 50% overhead 37 Translation of Research Results Into Practical Applications #1: Courses about translational applications #2: Lectures about translational success stories #3: Links with applied institutions #6: Requirements for an applied mind set E.g. Vlaams Instituut voor Biotechnology (VIB): Evaluation depends for ~ 60% on scientific impact and for ~ 40 % on application impact. Translational Medical Science Star Dr. Paul A.J. Janssen (1926-2003) Produced a total of eighty drugs; five are WHO essential drugs Galemmo et al. (2005) J. Med. Chem. 48: 1685. 38 Acknowledgements Malaria Invasion Machinery University of Washington Seattle Drexel University College of Medicine Philadelphia New York University School of Medicine New York Jürgen Bosch Stewart Turley Claudia Roach Stephen M. Bogh Thomas M. Daly Michelle L. Villasmil Na Zhou Joanne M. Morrisey Akhil B. Vaidya Lawrence W. Bergman Carlos Buscaglia Victor Nussenzweig Acknowledgements Vibrio and ETEC T2SS University of Washington Seattle University of Michigan Medical School Ann Arbor Vrije Universiteit Brussel Belgium Konstantin Korotkov Jan Abendroth Allison Kreger Stewart Turley Dan Mitchell Marissa Yanez Mark Robien Claudia Roach Brian Krumm Paul Murphy Maria Sandkvist Jan Steyaert Els Pardon Lode Wijns Michigan State University East Lansing Michael Bagdasarian 39 Acknowledgements CT and LT Multivalent Inhibitors University of Washington Seattle University of Washington Seattle University of Washington Seattle Misol Ahn Steve Sarfaty Dan Mitchell Ethan Merritt Claudia Roach Erkang Fan Zhongsheng Zhang Zheng Hou Feng Hong Ajit Ghosh Jason Pickens Guangtao Zhang Jiyun Li Wendy Minke Christophe Verlinde Xiaojang Tan University of Groningen The Netherlands Titia Sixma Kor Kalk University of Liège Belgium Joseph Martial Acknowledgements Fragment Cocktail Crystallography Origin University of Groningen, The Netherlands Christophe Verlinde Tjaard Pijning Rik Wierenga Gabby Rudenko 40 Acknowledgements Fragment Cocktail Crystallography MSGPP University of Washington, Seattle Jürgen Bosch Erkang Fan Christophe Verlinde Oleksandr Kalyuzhniy Lori Anderson Helen Neely Jenni Ross Isolde LeTrong Alberto Napoli Natascha Mueller Liren Xiao Ethan Merritt Fred Buckner Wes van Voorhis Financial Support University of Groningen, The Netherlands Dutch Organization for Scientific Research (NWO) Special WHO/UNDP/WHO program for Tropical Diseases (TDR), Geneva Hoffman La Roche, Basel, Switzerland (Klaus Müller) University of Washington, Seattle, USA Howard Hughes Medical Institute (HHMI), USA National Institute of Health (NIH), USA 41 Thank you 42
