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

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Presentation on theme: "Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes Jeni Lauer-Fields."— Presentation transcript:

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

2 Matrix Metalloproteinases Seiki, Current Opinion Cell Biology ( 2002) 14,

3 Bode, Cellular Molecular Life Sci. ( 1999) 55, MMP Structures Superimposed

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-1 SRT-6b

7 Determinants of MMP Binding TIMP-1 ~75% of contacts are from region including residues 1-4 and residues  -amino and carbonyl groups of Cys1 coordinate Zn ++ -OH of residue 2 displaces MMP-bound H 2 O necessary for hydrolysis 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- NH 2 H-CSCSDMTDKECLYFCMSEMS- NH 2 Ac-CSCSDMTDKECLYFCMSEMS- NH 2 H-XSXSDMTDKEXLYFXMSEMS-NH 2 Ac-XSXSDMTDKEXLYFXMSEMS- NH 2

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

10 Apparent K i 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 K i 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

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


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