1. Matrix metalloproteinase
Andressa Obice &Tony Voong
November 29, 2010
Biochemistry

2. Overview Family of highly-homologous, zinc dependent endopeptidases
Groups: collagenases, gelatinases,
stromelysins, matrilysins, membrane-type
MMPs (MT-MMPs), and Other MMPs
Function:
-degrade matrix/ non-matrix proteins
Biological processes:
-embryogenesis, tissue repair/remodeling,
wound healing, angiogenesis
Diseases:
-atheroma, arthritis, cancer, tissue
ulceration, nephritis, fibrosis, aneurysms
Regulated by tissue inhibitors of metalloproteinases (TIMPS)

3. Structure Most MMPs: -propetide domain -catalytic domain -hinge region
-hemopexin domain
MMP-7, MMP-26, MMP-23,
- no hinge region
- no hemopexin domain
MMP-23
-cysteine-rich domain,
-immunoglobulin-like domain g

4. Catalytic domain Hinge region Hemopoxin Domain About 170 amino acids
Spherical shape
Catalytic zinc ion
(zinc-binding motif)
“Met-turn”
Fibronectin type II repeats
Hinge region
Variable lengths
No determinable structure
Hemopoxin Domain
About 200 amino acids
Propeptide domain
About 80 amino acids
Must be removed before
enzyme is active
“Cysteine switch”
Furin cleavage site

5. Tertiary (3D) Structure of MMPs
Catalytic Domain:
5-stranded-β-pleated sheet
3 α-helices
Connective loops
2 zinc ions
(1 catalytic/1 structural)
Up to 3 calcium ions
3 histidines
“S1’ pocket”
Fibronectin Type II domains
-2 antiparrallel β sheets, connected with a short
α-helix, 2 disulfide bonds

6. Propetide Domain Hemopoxin Domain 3 α chains connecting loops
4-bladed β-propeller
1 disulfide bond
(first/fourth blades)
1 calcium ion
1 chloride ion

7. Propeptide Domain
Catalytic Domain
Fibronectin Domain
Hemopexin Domain
ProMMP-2-TIMP-2 Complex

8. Inhibition of
MMP by TIMP

9. Function

10. Degradation Over expression of MMP Alterations of TIMP ECM
 Proteolytic activity
ECM molecules from tissue  cell surface molecules alters cell–matrix and cell–cell interactions and the release of growth factors that are bound to the ECM makes them available for cell receptors. In addition, a number of non-ECM molecules are also potential substrates of MMPs alters cellular behavior and phenotypes
- cell migration, differentiation, growth, inflammatory, processes, neovacularization, apoptosis, etc.
Figure  shows example Role of matrix metalloproteinases in the pathogenesis of abdominal aortic aneurysms (MMP-2 and MMP-9)

11. Therapeutics Pro TNF- TNF -  TNF -  converting enzyme (TACE) MMP
Rheumatoid arthritis
Crohn’s disease
Cancer
Stroke

12. TNF- Pro TNF- sTNF- Stages of Inhibition 1. pro-TNF- processing
2. pro-TNF- synthesis
Pro-inflammatory cytokine
TNF-a is one of the primary cytotoxic proteins of the human immune system and its potent cell-killing effects are mediated through interaction with two cell-surface receptors
- Receptor- Mediated Apoptosis

13. TACE Catalytic Domain
Transmembrane glycoprotein
Pro-domain
Catalytic domain
Disintegrin
Cysteine-rich region
Transmembrane segment / Cytoplasmic tail
Green – Disulfide bridges
Yellow – Helices (hC active site-helix)
Red – Beta sheets (sI through sIV)
Pink – catalytic zinc sphere
White – His-His-His-Met-Pro (inhibitor)

14. TACE and MMP Inhibition of TACE and MMP-1 TACE / MMP
- same catalytic site / active site
- are zinc endopeptidases
- MMP inhibitors also inhibit TACE
- Marimastat / princomastat = failed clinical studies musculoskeletal side effects.
Differences
- try to exploit differences in primary and secondary structure ( active site of s1 pocket) (narrow channel in s1 & s3) = ability to build selectivity over MMP
- use of non-hydroxamate zinc-binding group (ZBG) = thiol

15. Channel

16. TACE Inhibitor Standard nomenclature for substrate inhibitor
Second picture – TAPI-2  effectiveness of hydroxamate ZBG (zinc-binding group
- s1=hydrophobic with isobutyl
- s2=shallow subtle cleft with tert-butyl
- s3=large cleft with methyl group

17. `
While great deal is known regarding those elements of structure-based design important for the development of eith MMP or TACE specific inhibitors, a comparable understanding for TACE / ADAM 10 has yet to be achieved.
- poor oral bioavailability
- decreased potency in vivo
- low selectivity = low success rate for specificity of TACE inhibitors devoid MMP (especially MMP-1 [1,313] collegenase)
Unwanted side effects
Lack of selectivity
Poor oral bioavailability
Decreased potency in vivo

18. TMI-1 & TAPI-2 Vs. TACE & ADAM 10
Poor oral bioavailable small size TNF- antagnoist
Hard because MMP’s have distinct but often overlapping substrate specificities
Designing more potent and selective novel TACE inhibitors
Better understanding of TACE selectivity through structure 2E and TMI-1 of selectivity over 1,9, and 13 of MMP. Table 1 shows the phylogenetic profile of TMI-1 shows more selective for TACE rather than ADAM-10 ( comparing Ki values)
- study of TAPI-2 and TMI-1 binding to ADAM 10
Pharmacology - Docycycline – inhibit MMP activity
Future…

19. Questions?

20. References
Chen, Taosheng. A Practical Guide to Assay Development and High-throughput Screening in Drug Discovery. Boca Raton: CRC, Print. 26
"Crystal Structure of the Catalytic Domain of Human Tumor Necrosis Factor-α-converting Enzyme — PNAS." Proceedings of the National Academy of Sciences. Web. 5 Nov
DasGupta, Sirshendu, et al., Current perspective of TACE inhibitors: A review, Bioorganic & Medical Chemistry. (2008), doi: /j.bmc
E.F. Healy, et al., Acetylenic inhibitors of ADAM10 and ADAM17: In silico analysis of potency and selectivity, J. Mol. Graph. Model. (2010), doi: /j.jmgm
Levin, J. I., et al. Acetylenic TACE inhibitors. Part 2: SAR of six-membered cyclic sulfonamide hyroxamates, Bioorganic & Medical Chemistry. (2005), doi: /j.bmcl
M. J. Eck, S. R. Sprang. The Structure Of Tumor Necrosis Factor-Alpha At 2. 6 A Resolution. Implications For Receptor Binding. J. Biol. Chem. V
Rao, B. Govinda, et al., Novel thiol-based TACE inhibitors:Rational design, synthesis, and SAR of thiol containing aryl sulfonamides, Bioorganic & Medical Chemistry. (2007), doi: j.bmcl