066 protein structure as a target for ligand design

PROTEIN STRUCTURE AS A TARGET FOR

LIGAND DESIGN

Finding the target

Crystallography

Pre-clinical trials, Clinical trials

Protein structure

Drug Development Diagram

Synthesis and testing

Available compound or lead compounds, substrates

Protein structure

Lead Improvement

Biological problem

Target

Structure of target(MMP9, Cellulase, Chymotrypsin)

Proteins as a target

Matrix Metalloproteinase-9

- Zinc-dependent metalloproteinase

- Degrades Extracellular matrix components

Signal domain Fibronectin-like domain

Propeptide Hinge region

Hemopexin-like domainZn2+

Catalytic domain

92 kD

- Physiological Conditions :

- Tissue remodelling, wound healing, angiogenesis

- Pathological Conditions :

- Cancer metastasis, cardiovascular aneurysms and rupture

MMP9 IMPORTANCE

MMP9 IN METASTASIS

Peter Carmeliet, (2000) J Clin Invest 105(11) 1519-1520

MMPs in cardiovascular aneurysms and rupture

Purification of truncated form of MMP-9

Expression in E. Coli

Inclusion body

Expression using Baculovirus system

Zn2+

S P C

F

Load Wash 0.1-2.0M NaCl

Heparin-Sepharose column

55kD

Gelatin-Sepharose column

Load 2% DMSO

Characterization of truncated form of MMP-9

- TIMP-1 binds and inhibits the catalytic domain

- Fibronectin-like domain is not sufficient for heparin binding

Zn2+

C

F

~38kD

Sadatmansoori, S (2001) protein expression and purification 23(3) 447-452.

The STRUCTURE OF CELLULASES FROM THE

FUNGUS ASPERGILLUS NIGER

Problem : Cellulase producing fungi destroying cellulose-based materials.

Target : Cellulase from Fungus Aspergillus niger.

Cellulase as a Target

Ribbon Stereo Diagram of EglA

The Binding Cleft of EglA

Pd2+

Glu 116Glu 204

Met 118

The STRUCTURE OF CELLULASES FROM A

WOOD- EATING TERMITE

The problem: Termite

- $1 billion damage a year!

- 1000 lbs wood a year!

- $15 million effort a year

- Inhibition of digestive enzymes

- The best choice is Cellulase.

NtEgl Crystals

Initial crystals

Final crystal

Ribbon Representation of NtEgl

Surface potential of NtEgl

W253

F205

D57D54

W301

E412Y408

W127R361

H124

H359

Y417

Ca2+

W516

N-RE RE

The Binding Cleft of NtEgl

D210

D213 E214

D254

N252

Water Water

Ca2+

The Ca2+ Binding Site of NtEgl at pH 5.6

D213 E214

D210

D254

N252

The Ca2+ Binding Site of NtEgl at pH 2.5

H359

A55

Y417

Y408

Catalytic Acid/Base Glu 412 at pH 5.6 and 6.5

The STRUCTURE OF A SERINE PROTEASE FROM

FIRE ANT

The Problem: fire ant

Crystallization of C3

Asp 102His 57

Ser 195

PMS

- C3- Cymotrypsine- Trypsine- Elastase- Collagenase

182 195 214 228- Chymotrypsin CAG-ASGV-SSCMGDS … SWGSST-CS-TSTPGVY- Trypsin CVGFLEGGKDSCQGDS … SWGYG--CALPDNPGVY- Elastase CAGGDG-VRSGCQGDS … SFVSRLGCNVTRKPTVE- Collagenase CID-STGGKGTCDGDS … SFGAAAGCEA-GYPDAE- C3 CANDPSAQKGACKGDS … SW--SLDCALTTHPTVY

Asp 102

His 57

Ser 195PMS

Ser 217

Asp 102

His 57

Ser 195

PMS

IPS

Structure-Based Drug Design

Protein structure

Small molecule database

Docking

Protein structure

Crystallography

Molecular MechanicsDynamics calculations

Protein structure

Touching Molecules: force feedback as an aid to docking

Acknowledgement

-Dr. Edgar Meyer - Dr. Linda A. Guarino

- Dr. Hirofumi Watanabe - Dr. Arnold Wartenberg

- Dr. Sepideh Sadatmansoori - Dr. Reza Forough

- Dr. Erik Meyer

- Dr. Stanley and Rosemarie Swanson

- Robert A. Welch Foundation

- Texas Advanced Technology Program