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Wim HolDepartment of Biochemistry
University of WashingtonSeattle, USA
Thomassen à Thuessink LezingUniversitair Medisch Centrum Groningen (UMCG)
Rijksuniversiteit Groningen (RUG)Nederland
25 oktober 2007
Translating Structural Biology
into Medical Practice;Bridging the Gap
1632
Rembrandt’s “The anatomical lesson of Dr. Tulp”
2
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.
From Anatomy to Structural Biology
What is Structural Biology?
3
Gly
Ser
ASPHis
Ala
Val
Ile
Leu
Met
Phe
Tyr
Pro
Gln
Cys
Thr
AsnGlu
Lys
Arg
Trp
Towards protein structures
Amino Acids: The Building Blocks of Proteins
How does the protein chain fold, flex and function?
Towards protein structures
4
Protein Crystals
Bram Schierbeek
Towards protein structures
700 m
Synchrotrons provide intense X-rays with variable wavelengths
Towards protein structures
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100 μm
Towards protein structures
A frozen crystal scooped into in a nylon loop at 100 K
An X-ray “Precession Photograph” of Lipoamide Dehydrogenase
The intensities of the spots relate in a complex way the protein structureThe “diffraction pattern” is the Fourier Transform of the entire crystal.
Towards protein structures
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An Experimental Electron Density Distribution
Towards protein structures
Jan Abendroth
Towards protein structures
Atoms Built Into an Experimental Electron Density DistributionJan Abendroth
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Fold of Peptide Deformylase (PDF)A possible drug target from the major malaria parasite
“Rainbow-colored”
Abhinav Kumar
A protein in its native state
COMPLEMENTARITY PRINCIPLE
WITH SUFFICIENT SELECTIVITY
Essential region of “Target”homolog
Essential region of “Target”
STRUCTURE-BASED DRUG DESIGN
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Jan Tinbergen
Jan Tinbergen
!903 – 1994
Nobel Prize Economics 1969
World Bank Report 2006
Only five rich countries have fulfilled the UN official development assistance target of 0.7
of GNI:
Denmark, Luxembourg, the Netherlands, Norway, and Sweden.
UN “Tinbergen Committee” Report 1970
Rich countries should spend 1% of their GNP on aid to developing countries.
The proposal was defeated.
AIDSDengue
TuberculosisChildren's diarrhea
MalariaSleeping SicknessChagas DiseaseLeishmaniasis
SchistosomiasisFilariasis
River Blindness
“Neglected” and “Totally Neglected” tropical diseases
Some of the major tropical diseases of today
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“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
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• 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 Sicknessaka African Trypanosomiasis
With thanks to Wes Van Voorhis
Blood stream form of parasite
Tsetse fly
Lumbar puncturefor 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.
Sleeping Sicknessaka African Trypanosomiasis
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Note that the sleeping sickness parasite does NOT hide in red blood cells
The sleeping sickness parasiteBlood-stream Form
Red Blood Cell
Trypanosome
Trypanosome
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)from Parasite and Host
Note the difference in conformation near the ribose of the NAD cofactorin the homologous proteins of host and parasite.
Cofactor (co-substrate) NADHuman GAPDHTrypanosomal GAPDH
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•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.
“Targeted Combinatorial Chemistry” to fill the grooves optimally
Hydrophobic Groove
Hydrophobic Groove
Exploring multiple hydrophobic grooves
Surface of L. mexicana* GAPDH with NAD bound.
Designed Adenosine Derivatives100,000 x more potent than start compound adenosine
CONFORMATIONAL CHANGES in L. mexicana GAPDH UPON BINDING DIFFERENT LIGANDS
LmGAPDH + NAD LmGAPDH + NMDBA
Stephen Suresh, Jerri Bressi, Alex Aranov, Michael Gelb
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Inhibition of Trypanosoma cruzi amastigote by GAPDH inhibitor
Fred Buckner, Wes Van Voorhis
Blue color of β-gal reporter means parasite growthTrypanosoma cruzi is yet another “Trypanosomatid”
It causes “Chagas disease” in Latin America
Many enzymes in Trypanosoma brucei and Trypanosoma cruzi are very similar in amino acid seqeunce.
β-gal reporter: Blue indicates parasite alive
Malaria
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Plasmodium falciparum and Plasmodium vivax ~500 million cases of malaria annually~1 to 2 million deaths per yearVictims mainly children and pregnant women
MalariaSome sobering facts
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
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Plasmodium falciparumThe major malaria parasite
Host Cell Invasion MachineryMyosin Tail Interacting Protein (MTIP) and the Myosin A tail
Jürgen Bosch, Stewart Turley Bill Bergman
MTIPMyoA
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P. falciparum MTIP binding the Myosin A tail
Jürgen Bosch, Stewart Turley Bill Bergman
MTIP undergoes dramatic
conformational changes upon
MyoA tail binding
MTIP MTIP NN--terminal terminal
domaindomain
MyoA Tail Helix
MTIP MTIP CC--terminal terminal
domaindomain
P. falciparum MTIP completely surrounds the Myosin A tail
MTIP C-terminal Domain
MTIP N-terminal Domain
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MTIP plus the MyoA-tail (P. knowlesi complex)
MyoA
Note the hydrophobic character of the MTIP contact surface
Oxygen NitrogenCarbon
The MTIP-MyoA Tail InterfaceHydrophobic interactions
Linking Leu&Val&Ile side chains →→→ New Antimalarials?
1389 Å2 buried surfaceOxygen Nitrogen
Leu804 Val807 Ile811Carbon
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
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Choleraand
Children’s Diarrhea
Vibrio cholerae :- produces CT - ~4000 victims per year
Enterotoxigenic E. coli :- produces LT and ST - ~480,000 victims per year
19
A subunit
B pentamer
Titia Sixma
Heat-labile Enterotoxin (LT),a very close relative of Cholera toxin (CT)
The secretion of
Cholera toxin (CT) and
Heat-labile enterotoxin (LT)by the marvelous
Type 2 Secretion System (T2SS)
20
M M
Peri-EpsDNanobody
EpsHPilin-like
Δ90-EpsESecretion ATPase
cyto-EpsLActin-like
peri-EpsMFundamental Ferredoxin
Fold
CTMoonlander PDZ-EpsC
Helix binding?
N1-EpsE:cyto-EpsLBinary complex
N N
EpsI-JPilin-like
peri-EpsLFundamental Ferredoxin
Foldcyto-F1Calcium-binding
FF
L
DDD
D
FLF
E
C
M M L
CCT
I I
GH
GGGGG
G G
H H
J K J
Cholera Toxin (CT) & the Type II Secretion System (T2SS)
Cytoplasm
Vibrio cholerae
Periplasm
Facts and Fiction mixed
N N
21
L
DDD
D
FLF
E
C
M M L
CAB5
I I
GH
GGGGG
G G
H H
J K J
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
L
DDD
D
FLF
E
C
M M LI IJ K J
GH
GGG
GGG G
H H
CCAB5
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
22
L
DDD
D
FLF
E
C
M M L
C
I IJ K J
GH
GGG
GGG G
H H
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
C C
L
DDD
FLF
E
M M LI IJ K J
AB5
GH
GGG
GGG G
H H
D D
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
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C C
L
DDD
FLF
E
M M L
AB5
D D
I I
GH
GGGGG
G G
H H
J K J
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
C C
L
DDD
FLF
E
M M L
AB5
D D
I I
GH
GGGGG
G G
H H
J K J
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
24
C C
L
DDD
FLF
E
M M L
AB5
D D
I I
GH
GGGGG
G G
H H
J K J
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
L
DDD
D
FLF
E
C
M M L
C
I I
GH
GGGGG
G G
H H
J K J
AB5
Facts and Fiction mixed
The T2SS in Action
N N
Cytoplasm
Vibrio cholerae
Periplasm
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The Interaction of
Cholera toxin (CT) and
Heat-labile enterotoxin (LT)with
human cell surface receptors.And its inhibition
Intestinal epithelial cell
CT and LT vs. human cell
Ganglioside GM1
CT: Cholera LT: Traveller’s & Children’s diarrhea
A
B5
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Intestinal epithelial cell
Ganglioside GM1
A
B5
CT and LT vs. human cell
CT: Cholera LT: Traveller’s & Children’s diarrhea
Toxin
GM1 Receptors
Intestinal cell surface
A subunit
B pentamer
Cholera toxin – GM1 Receptor Interaction
Ethan Merritt, Steve Sarfaty, Joseph Martial
27
GM1 Pentasaccharide bound by CT
IC50 = 14 nM
OOH
OHHO
HO
OOH
OHO
NH
O
OOH
O
HO
OOH
OH
HO
NH
O
HOHOOC
O
OOH
OHO
HOOH
His 13
The enemy
Five receptor binding sites
28
Making ligands longer
Ligand-Protein Complex
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Pentavalent Ligand
The pentavalent concept
“Proper Pre-organization”
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??? xSingleFinger
???? xSingle Finger
????? xSingle Finger
?????? xSingle Finger
Gains in surface-receptor binding inhibitionOne-Unit Two-Unit Three-Unit Four-Unit
Linker Linker Linker Linker
Erkang Fan, Zhongsheng Zhang, Jason Pickens, Jiyun Liu, et al
240 xSingleFinger
3600 xSingle Finger
10,000 xSingle Finger
104,000 xSingle Finger
Gains in surface-receptor binding inhibitionOne-Unit Two-Unit Three-Unit Four-Unit
Linker Linker Linker Linker
Erkang Fan, Zhongsheng Zhang, Jason Pickens, Jiyun Liu, et al
31
Genome-wide approaches
MEDICAL STRUCTURAL GENOMICS OF PATHOGENIC PROTOZOA (MSGPP)
Target & Ligand Selection
Protein Production
Assays Crystal Growth
Medicinal Chemistry Virtual
Screening
Crystal Structure
Determination
InformatIcs
M
a
n
a
g
e
m
e
n
t
Genome Sequences
url: www.msgpp.org
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Fragment Cocktail Crystallography
A new tool in drug designCourtesy of Jürgen Bosch
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
Fragment Cocktail CrystallographyRoots
33
Probe protein pockets by soaking crystals in well-designed mixtures of 5-10 different chemicals,
followed by crystal structure determinations
Fragment Cocktail CrystallographyPrinciple
Protein crystals
+
Cocktails of chemical fragments
Protein crystal with bound chemical fragment
Christophe Verlinde, Erkang Fan http://faculty.washington.edu/verlinde/
Fragment Cocktail CrystallographyCocktail construction in MSGPP
ACD Compound Filtering
9,500 compoundsfragmentation
23 frameworks (at connectivity level)
626 fragmentsisolate ring systems
680 compounds
manual selection of compounds
from each framework class- eliminate mutagens, known poisons- no highly functionalized compounds- retain Br containing compounds
60 cocktails
34
T. brucei Nucleoside 2-deoxyribosyltransferaseplus Cocktail #4
Jürgen Bosch & Christophe Verlinde & Erkang Fan& & SGPP
1,2-DIHYDROBENZO[CD]INDOL-2-ONE
Omni-present glycerol
Jürgen Bosch & Christophe Verlinde & Erkang Fan & SGPP
6-AMINO-1-NAPHTHOL
Omni-present glycerol
T. brucei Nucleoside 2-deoxyribosyltransferaseplus Cocktail #5
35
Isolde LeTrong, Alberto Napuli,Liren Xiao, Ethan Merritt,
Erkang Fan, Christophe Verlinde & MSGPP 2007
5-fluoroindole-2-carboxylic acid (FIC) from cocktail #68 Cyclopentylacetic acid from cocktail #61
The two cocktail compoundsbind at different sites:
Ready for linking!
L. major Coproporphyrinogen Oxidaseplus Cocktails #61 and #68 in separate experiments
Target Selection
Protein Expression
Crystallization
Data Collection
Structure Determination
Structure Analysis
Medicinal Chemistry & Pharmacology
Structures with Ligands Bound
Genome
New Therapeutics
36
#1: Bring different disciplines close together spatially
#2: Flexible funding of interdisciplinary projectsAcross all disciplinesShort-to-medium termTop-light
#3: Invite interdisciplinary lecturers“I-lecture” series
#4: Reward interdisciplinary initiativesAward prizes for excellent interdisciplinary research
#5: Create interdisciplinary buildingsWith flexibility – to avoid eternal occupants
Interdisciplinary ResearchEssential for Progress in Medicine
The Campus on a Typical DayUniversity of Washington in Seattle
Bring disciplines together spatially
37
Computer Sceince
Bioengineering
Genome Sciences
Biochemistry
Pharmacology
Immunology
Chemistry
Physics
Hospital
Pharmacy
Biology
Bring disciplines together spatially
Flexible Funding of Interdisciplinary ProjectsNIH “Program Projects”
Buckner – Verlinde – FanTarget & Ligand Selection
Van VoorhisProtein Production
Van VoorhisAssays
HolCrystal Growth
FanMedicinal Chemistry
VerlindeVirtual
Screening
MerrittCrystal
Structure Determination
HolCore
nformatIcs
HolCore
a
n
a
g
e
m
e
n
t
MSGPPTwo “Cores”, Six Groups
1.5 M$ per year PLUS ~ 50% overhead
38
#1: Courses about translational applications
#2: Lectures about translational success stories
#3: Links with applied institutions
#6: Requirements for an applied mind setE.g. Vlaams Instituut voor Biotechnology (VIB):
Evaluation depends for ~ 60% on scientific impact and for ~ 40 % on application impact.
Translation of Research ResultsInto Practical Applications
Dr. Paul A.J. Janssen (1926-2003)
Translational Medical Science Star
Produced a total of eighty drugs; five are WHO essential drugs
Galemmo et al. (2005) J. Med. Chem. 48: 1685.
39
University of Washington
Seattle
Jürgen BoschStewart TurleyClaudia Roach
Acknowledgements Malaria Invasion Machinery
Drexel University College of Medicine
Philadelphia
Stephen M. BoghThomas M. Daly
Michelle L. VillasmilNa Zhou
Joanne M. MorriseyAkhil B. Vaidya
Lawrence W. Bergman
New York University School of Medicine
New York
Carlos BuscagliaVictor Nussenzweig
University of Washington
Seattle
Konstantin KorotkovJan AbendrothAllison KregerStewart TurleyDan Mitchell
Marissa YanezMark Robien
Claudia RoachBrian KrummPaul Murphy
Acknowledgements Vibrio and ETEC T2SS
Vrije UniversiteitBrusselBelgium
Jan SteyaertEls PardonLode Wijns
University of MichiganMedical School
Ann Arbor
Maria Sandkvist
Michigan State University
East Lansing
Michael Bagdasarian
40
University of Washington
Seattle
Misol AhnSteve SarfatyDan Mitchell
Ethan MerrittClaudia Roach
Acknowledgements CT and LT Multivalent Inhibitors
University of Washington
Seattle
Wendy MinkeChristophe Verlinde
Xiaojang Tan
University of Washington
Seattle
Erkang FanZhongsheng Zhang
Zheng HouFeng HongAjit Ghosh
Jason PickensGuangtao Zhang
Jiyun Li University of LiègeBelgium
Joseph Martial
University of GroningenThe Netherlands
Titia SixmaKor Kalk
Christophe VerlindeTjaard PijningRik Wierenga
Gabby Rudenko
Acknowledgements Fragment Cocktail Crystallography
Origin
University of Groningen, The Netherlands
41
Jürgen BoschErkang Fan
Christophe VerlindeOleksandr Kalyuzhniy
Lori Anderson Helen NeelyJenni Ross
Isolde LeTrongAlberto Napoli
Natascha MuellerLiren Xiao
Ethan MerrittFred Buckner
Wes van Voorhis
Acknowledgements Fragment Cocktail Crystallography
MSGPPUniversity of Washington, Seattle
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
Financial Support
42
Thank you
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