This document summarizes the development and results of high-throughput screening assays to identify inhibitors of matrix metalloproteinase 13 (MMP-13), an enzyme implicated in osteoarthritis. A primary screen using a fluorescent resonance energy transfer peptide substrate identified several autofluorescent compounds as false positives. A secondary screen using chromatographic and fluorescence techniques on 30 compounds found some that selectively inhibited MMP-13 over other MMPs. The best inhibitors were further analyzed for structural similarity and inhibitory profiles against different MMPs.

1. Student:黃德偉 5-Year Impact factor : 3.231
Instructor:楊智惠教授 PubMed ID: 18358729
DOI: 10.1016/j.bmc.2008.03.004 ISSN: 0968-0896
Impact factor:2.951 1

2. Outline
• Introduction
• FRET TripleHelicalPeptides substrate
assay (fTHP)
• Primary HTS assay
• Secondary HTS assay
• Compound analysis
2

3. Introduction
• Osteoarthritis (OA) is an age-related debilitating
disease affecting more than 80% of people over the
age of 75.
• Cartilage destruction associated with OA has been
shown to be due to increased catabolism rather
than decreased synthesis.
• However, Matrix MetalloProtease (MMP) family
has several members that can cleave collagen.
3

4. Introduction
• Proteases with potential roles in OA include MMP-
1,MMP-13, and members of the a disintegrin and
metalloproteinase with thrombospondin motifs.
• MMP-13 is believed to be the more prominent
collagenase than MMP-1 in OA.
• Goal of OA researchers is the design of selective
MMP-13 inhibitors.
4

5. Introduction
• Established using FRET substrates with Mca
as fluorophore and Dnp as quencher
• Triple Helical Peptides(THP) substrates identify
unique MMP-13 inhibitors due to distinct
conformational features that interact with
secondary binding sites found within MMPs.
5
Adopted from :
Biomol.Chem.2010;8,1237-1258

6. Introduction
• Can be utilized for the identification of non-active site-
binding inhibitors.
• Exosites have been shown to represent unique
opportunities for the design of selective inhibitors
• Initial clinical trials with MMP inhibitors were
disappointing, with one of the problematic features being a
lack of selectivity.
• Selective exosite-binding inhibitors for MMP-13 could
represent a potential next generation in metalloproteinase
therapeutics for OA. 6

7. FRET THP substrate assay(fTHP)
7
Mca
(7-methoxycoumarin-4-yl)acetyl
Dnp
2,4-dinitrophenyl
Cleave by MMP-family
Signal detected
Adopted from Eurogentec

8. Primary HTS campaign to identify inhibitors of MMP-13
8
S/B : 2.4 ± 0.04 (n = 52 plates)
Average Z′-factor :0.82 ± 0.06

9. Z-factors of the primary HTS campaign
9

10. MMP-13 primary HTS campaign scattergram
10
The negative inhibition
scatter is attributed to
the substrate fluorophore
(7-methoxycoumarin)
excitation wavelength,
making the primary HTS
campaign assay prone to
the interference from
fluorescent compounds
Figure 2. MMP-13 primary HTS campaign scattergram. Graphed are the percent inhibition
values for every well tested in the primary HTS campaign.

11. Primary HTS assay frequency distribution analysis
11
Fig3. (A) with fluorescent
artifacts
Mean inhibition value of
0.833% ± 0.031
(R2 = 0.990; n = 64,928)
Fluorescent
compounds
within the
screening
file.

12. Primary HTS assay frequency distribution analysis
12
Fig3. (B) without the fluorescent
artifacts
Mean inhibition value of
0.835% ± 0.026
(R2 = 0.993; n = 59,470)

13. Identify inhibitors of MMP-13 within the
large subset of autofluorescent compounds
found in the primary HTS campaign
13

14. Secondary screen of MMP-13 inhibitors
14
Table 3.Compounds examined in the secondary screen
• An RP-HPLC based secondary screen
was performed on 30 of these
compounds.
• one single-stranded (Knight fSSP)
• one triple-helical (fTHP-15)
Result
• Compounds C, R, V, W, A′, C′ best
inhibitors
• Compounds J and L were better
inhibitors against the SSP substrate
• Compound Q was a better inhibitor
against the THP substrate.

15. Structure similarity
15
Figure 5 Bioactivity clustering (structure–activity analysis) of 42 compounds tested
in dose–response experiment for non-fluorescent compounds. Results from a
cheminformatics tool contained within the PubChem website are shown.
Red arrow indicates columns
containing MMP-13 primary HTS and
dose–response experiment results.

16. Figure 4 Inhibition of MMP-13 by 30 different compounds, as monitored by RP-HPLC
and fluorescence spectroscopy. The change in RP-HPLC peak areas or relative
fluorescence units for 10 nM MMP-13 hydrolysis of 10 μM fTHP-15 or 5 μM Knight
fSSP was monitored

17. 17
Relative percent activity of MMPs in the presence of 100 μ M inhibitor
Inhibitor MMP-1 MMP-2 MMP-3 MMP-8 MMP-9 MMP-12 MMP-13
C 25 7 63 2 14 2 4 Broad spectrum
E 47 49 96 50 27 21 35 Exception
H 96 100 100 100 100 100 36 Effective against MMP- 8&13
M 85 19 96 34 27 33 35 Effective against MMP-13
Q 94 100 96 80 99 100 62 Effective against MMP-2&13
R 27 17 87 8 3 1 14
T 68 100 75 32 100 75 45 Based on
V 91 0 43 10 0 0 15 1.Relative level of inhibition
W 98 47 97 80 66 72 25 2.Uniqueness of substrate profile
X 93 100 92 94 100 92 42
A′ 96 73 95 36 75 64 35
C′ 88 80 90 100 81 94 44
12 subset of MMP-13 inhibitor for
counter screening

18. 18MVC
E R
5,5-disubstitutedpyrimidine-2,4,6-triones
General class MMP
inhibitors.
Bind to MMP
active site Zn2+

19. 19
Similar
X Thiazolopyrimidine
MMP-13
selective
inhibitor
Q W
Warner-Lambert
pyrimidinediones
similar
+
Effective against
MMP-13 triple-
helical peptidase
activity compared
with MMP-13 single-
stranded peptidase
activity

20. 20
H C’
Virtually
identical
structurally
T A’
Autofluorescent
compund

21. References
• Pictures sources : Wikipedia, Eurogentec, Biomol.Chem
• J. Liquid Chromatogr. Relat. Technol., 26 (2003), pp.
2225–2254
• Crit. Rev. Oral Biol. Med., 4 (1993), pp. 197–250
• Osteoarthr. Cartilage, 11 (2003), pp. 801–809
• Arthritis Res., 4 (2002), pp. 157–164
• Expert Opin. Ther. Patents, 15 (2005), pp. 253–269
• Biochemistry, 40 (2001), pp. 5795–5803
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