1. Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes
Jeni Lauer-Fields

2. Matrix Metalloproteinases
Zinc metalloenzymes
Primary role; degradation of extracellular matrix molecules
Signalling pathways
Digestion of perlecan by MMP-3 and -13 releases FGF-2
Physiological processes including wound healing, cytokine and growth factor regulation
Pathological processes including periodontal disease, atherosclerosis, tumor cell metastasis
Seiki, Current Opinion Cell Biology ( 2002) 14,

3. MMP Structures Superimposed
Zinc metalloenzymes
Primary role; degradation of extracellular matrix molecules
Signalling pathways
Digestion of perlecan by MMP-3 and -13 releases FGF-2
Physiological processes including wound healing, cytokine and growth factor regulation
Pathological processes including periodontal disease, atherosclerosis, tumor cell metastasis
Bode, Cellular Molecular Life Sci. ( 1999) 55,

4. Tissue Inhibitors of Metalloproteinases
Regulate activity of MMPs
Form tight non-covalent 1:1 complex with MMPs
Regulate ECM turnover and other cellular processes
Two domains, with N-terminal domain retaining most of the inhibitory activity
Four human TIMPs (1-4), constitutive and regulated expression patterns
Abraham et al. Current Vascular Pharmacology ( 2005) 3,

5. Endothelin-Fold as a Scaffold for TIMP-Based Inhibitors
Endothelins are 21 residue proteins with vasoactive properties
Family members; endothelins and sarafotoxins
Contain 2 disulfide bonds and moderate -helical content
Comparison with TIMP three-dimensional structure, obvious similarities
PDB file: 1SRB

6. Comparison of Sarafotoxin and TIMP Structures
TIMP SRT-6b

7. Determinants of MMP Binding
~75% of contacts are from region including residues 1-4 and residues 66-69
-amino and carbonyl groups of Cys1 coordinate Zn++
-OH of residue 2 displaces MMP-bound H2O necessary for hydrolysis
TIMP-1
Brew et al. Biochim. Biophys. Acta, 1477 (2000)

8. Sarafotoxin Variants H-CSCKDMTDKECLYFCHQDVIW-OH
H-CSCKDMTDKECLYFCHQD-OH
H-CSCKDMTDKECLYFCVQD-OH
H-CSCADMTDKECLYFCHQD-OH
H-CSCSDMTDKECLYFCHQD-OH
H-CSCKDMTDKECLYFCMSEMS-NH2
H-CSCSDMTDKECLYFCMSEMS-NH2
Ac-CSCSDMTDKECLYFCMSEMS-NH2
H-XSXSDMTDKEXLYFXMSEMS-NH2
Ac-XSXSDMTDKEXLYFXMSEMS-NH2

9. Inhibition of MMPs by Sarafotoxin Variant STX-S4-CT

10. Apparent Ki Values Sarafotoxin Model Peptides
NI: No inhibition detected

11. Circular Dichroism Spectra

12. CD Spectra Sarafotoxin Model Peptides

13. Molecular Docking with MMP-1
PDB file generated by PatchDock

14. Apparent Ki Values and CD Spectra

15. Current Work 2D NMR Spectra for STX-S4 and STX-S4-CT
Make a new model based on NMR data
Repeat docking and analysis with NMR-based model

16. Biomolecular Proton NMR

17. Biomolecular Proton NMR

20. Summary
Endothelin-fold is a good template for peptide-based inhibitors of MMPs
Enhance selectivity between MMPs and other metalloproteinases (ADAM/ADAMTS)
Inhibition is related to endothelin-fold as well as N-terminal charge
Mechanism may be similar to TIMPs

21. Acknowledgements Keith Brew Vijaya Iragavarapu Shuo Wei Tyrone Ferns
Biomedical Sciences
Keith Brew
Vijaya Iragavarapu Shuo Wei
Tyrone Ferns
Gina Spruill
Chemistry and Biochemistry
Gregg Fields
Frank Mari
Mare Cudic
Imperial College
Hideaki Nagase
Rob Visse