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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