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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

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Outline • Introduction

• FRET TripleHelicalPeptides substrate assay (fTHP)

• Primary HTS assay

• Secondary HTS assay

• Compound analysis

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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.

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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.

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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.

Adopted from : Biomol.Chem.2010;8,1237-1258

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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.

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FRET THP substrate assay(fTHP)

Mca(7-methoxycoumarin-4-yl)acetyl

Dnp 2,4-dinitrophenyl

Cleave by MMP-family

Signal detected

Adopted from Eurogentec

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Primary HTS campaign to identify inhibitors of MMP-13

 S/B : 2.4 ± 0.04 (n = 52 plates)

Average Z′-factor :0.82 ± 0.06

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 Z-factors of the primary HTS campaign

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MMP-13 primary HTS campaign scattergram

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 compoundsFigure 2. MMP-13 primary HTS campaign scattergram. Graphed are the percent

inhibition values for every well tested in the primary HTS campaign.

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 Primary HTS assay frequency distribution analysis

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.

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Primary HTS assay frequency distribution analysis

 Fig3. (B) without the fluorescent artifacts

Mean inhibition value of 0.835% ± 0.026 (R2 = 0.993; n = 59,470)

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

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 Secondary screen of MMP-13 inhibitorsTable 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.

 25 in

hibited

MMP-13 a

ctivi

ty

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Structure similarity

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.

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

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Relative percent activity of MMPs in the presence of 100 μ M inhibitorInhibitor MMP-1 MMP-2 MMP-3 MMP-8 MMP-9 MMP-12 MMP-13C 25 7 63 2 14 2 4 Broad spectrumE 47 49 96 50 27 21 35 ExceptionH 96 100 100 100 100 100 36 Effective against MMP- 8&13M 85 19 96 34 27 33 35 Effective against MMP-13Q 94 100 96 80 99 100 62 Effective against MMP-2&13R 27 17 87 8 3 1 14T 68 100 75 32 100 75 45 Based onV 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 42A′ 96 73 95 36 75 64 35C′ 88 80 90 100 81 94 44

12 subset of MMP-13 inhibitor for counter screening

18MVC

E R5,5-disubstitutedpyrimidine-2,4,6-triones

General class MMP inhibitors.

Bind to MMP active site Zn2+

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Similar

X ThiazolopyrimidineMMP-13 selective inhibitor

Q WWarner-Lambert pyrimidinediones

similar+

Effective against MMP-13 triple-helical peptidase activity compared with MMP-13 single-stranded peptidase activity 

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H C’

Virtually identical structurally

T A’

Autofluorescent compund

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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