1

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”

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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

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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

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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)

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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

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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

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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

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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

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

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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

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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

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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

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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

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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

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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

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#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

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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

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#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.

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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

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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

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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

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Thank you