Topological Specificity in Inhibitor Recognition by

Proteolytic Enzymes

Jeni Lauer-Fields

Matrix Metalloproteinases

Seiki, Current Opinion Cell Biology ( 2002) 14, 624-632.

Bode, Cellular Molecular Life Sci. ( 1999) 55, 639-652.

MMP Structures Superimposed

Tissue Inhibitors of Metalloproteinases

Regulate activity of MMPsForm tight non-covalent 1:1 complex with MMPsRegulate ECM turnover and other cellular processesTwo domains, with N-terminal domain retaining most of the inhibitory activityFour human TIMPs (1-4), constitutive and regulated expression patterns

Abraham et al. Current Vascular Pharmacology ( 2005) 3, 369-379.

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

Comparison of Sarafotoxin and TIMP Structures

TIMP-1 SRT-6b

Determinants of MMP Binding

TIMP-1

~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

Brew et al. Biochim. Biophys. Acta, 1477 (2000) 267-283.

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

Inhibition of MMPs by Sarafotoxin Variant STX-S4-CT

0.0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100Inhibitor (µM)

Relative Activity (Vi/Vo)

MMP-1MMP-2MMP-3MMP-9MT1-MMP

Apparent Ki Values Sarafotoxin Model

PeptidesInhibitor MMP-1

(µM) MMP-2 (µM)

MMP-3 (µM)

MMP-9 (µM)

STX NI NI > 100 NI STX-V1 6 NI NI > 100 NI STX-A4 21.5 ± 0 .6 41.8 ± 3 .5 > 100 24.5 ± 0 .9 STX-S4 22.0 ± 2 .2 35.0 ± 4 .4 > 100 29.3 ± 0 .0 STX-CT > 100 > 100 > 100 25.3 ± 3 .5 STX-S4 -CT 4.5 ± 0.0 21.6 ± 2 .2 >100 1.0 ± 0.1 Ac-STX-S4 -CT > 100 NI NI 66.0 ± 4 .0 Abu -STX-S4 -CT > 100 NI > 100 > 100 Ac-Abu-STX-S4 -CT NI NI NI NI

NI: No inhibition detected

Circular Dichroism Spectra

CD Spectra Sarafotoxin Model Peptides

-15

-10

-5

0

5

10

15

195 205 215 225 235 245Wavelength (nm)

Mean Residue Ellipticity

STXSTX-V16STX-A4STX-S4STX-CTSTX-S4-CTAc-STX-S4-CTAbu-STX-S4-CT

Molecular Docking with MMP-1

PDB file generated by PatchDock

Apparent Ki Values and CD Spectra

Inhibitor MMP-1 (µM)

MMP-2 (µM)

MMP-3 (µM)

MMP-9 (µM)

STX-S4 -CT 4.5 ± 0.0 21.6 ± 2 .2 > 100 1.0 ± 0.1 STX-S4 -CT-A16 > 50 > 100 > 100 > 100 STX-S4 -CT-S5 4.5 ± 0.1 11.9 ± 0 .3 > 100 6.7 ± 0.1 STX-S4 -CT-S1 8 12.3 ± 0 .4 34.2 ± 0 .7 > 100 6.9 ± 0.0

-10

-8

-6

-4

-2

0

2

4

6

8

10

195 205 215 225 235 245

Wavelength (nm)

Mean Residue Ellipticity

STX-S4-CTSTX-S4-CT-A16STX-S4-CT-S5STX-S4-CT-S18

Current Work

2D NMR Spectra for STX-S4 and STX-S4-CTMake a new model based on NMR dataRepeat docking and analysis with NMR-based model

Biomolecular Proton NMR

http://www.embl.de/nmr/sattler/lab/

Biomolecular Proton NMR

http://www.embl.de/nmr/sattler/lab/

SummaryEndothelin-fold is a good template for peptide-based inhibitors of MMPsEnhance selectivity between MMPs and other metalloproteinases (ADAM/ADAMTS)Inhibition is related to endothelin-fold as well as N-terminal chargeMechanism may be similar to TIMPs

AcknowledgementsChemistry and Biochemistry

Gregg FieldsFrank MariMare Cudic

Biomedical Sciences

Keith BrewVijaya Iragavarapu

Shuo WeiTyrone FernsGina Spruill

Imperial College

Hideaki NagaseRob Visse