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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
ƒ Plasmodium falciparum and Plasmodium vivax
ƒ ~500 million cases of malaria annually
ƒ ~1 to 2 million deaths per year
ƒ 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