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Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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2008 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 51, no 12, 3449-3459 p.Article in journal (Refereed) Published
Abstract [en]

Small inhibitors of matrix metalloproteinase 12 (MMP-12) have been identified with a biosensor-based screening strategy and a specifically designed fragment library. The interaction between fragments and three variants of the target and a reference protein with an active-site zinc ion was measured continuously by surface plasmon resonance. The developed experimental design overcame the inherent instability of MMP-12 and allowed the identification of fragments that interacted specifically with the active-site of MMP-12 but not with the reference protein. The interaction with MMP-12 for selected compounds were analyzed for concentration dependence and saturability. Compounds interacting distinctly with the target were further evaluated by an activity-based assay, verifying MMP-12 inhibition. Two effective inhibitors were identified, and the compound with highest affinity was confirmed to be a competitive inhibitor with an IC50 of 290 nM and a ligand efficiency of 0.7 kcal/mol heavy atom. This procedure integrates selectivity and binding site identification into the screening procedure and does not require structure determination.

Place, publisher, year, edition, pages
2008. Vol. 51, no 12, 3449-3459 p.
National Category
Natural Sciences
Research subject
URN: urn:nbn:se:uu:diva-106025DOI: 10.1021/jm8000289ISI: 000256922800012PubMedID: 18494455OAI: oai:DiVA.org:uu-106025DiVA: diva2:223359
Available from: 2009-06-11 Created: 2009-06-11 Last updated: 2014-01-23Bibliographically approved
In thesis
1. Fragment Based Drug Discovery with Surface Plasmon Resonance Technology
Open this publication in new window or tab >>Fragment Based Drug Discovery with Surface Plasmon Resonance Technology
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fragment based drug discovery (FBDD) has been applied to two protease drug targets, MMP-12 and HIV-1 protease. The primary screening and characterization of hit fragments were performed with surface plasmon resonance -technology. Further evaluation of the interaction was done by inhibition studies and in one case with X-ray crystallography. The focus of the two projects was different.

Many MMP inhibitors contain a strong zinc chelating group, hydroxamate, interacting with the catalytic zinc atom. This strategy may be the cause for the low specificity of MMP inhibitors. Using FBDD we found a fragment with an unusual strong affinity for MMP-12. An inhibition assay confirmed that it was an inhibitor but indicated a stoichiometry of 2:1. Crystallography data revealed that an adduct of the fragment was bound in the active site, with interactions both with the catalytic zinc and the S1’ pocket. This may present a new scaffold for MMP-12 inhibitors.

For HIV-1 protease the focus was on identifying inhibitors not sensitive to current resistance mutations. A fragment library for screening with SPR-technology was designed and used for screening against wild type enzyme and three variants with resistance mutations. Many of the hits were promiscuous but a number of fragments with possible allosteric inhibition mechanism were identified.

The temperature dependency of the dissociation rate and reported resistance mutations was studied with thermodynamics. A good, but not perfect correlation was found between resistance and both the dissociation data and the free energy for dissociation compared to data from wild type enzyme. However, the type of mutation also influenced the results. The flap mutation G48V displayed thermodynamic profiles not completely correlating with resistance. It was found that dissociation rate and thermodynamics may complement each other when studying resistance, but only one of them may not be enough.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1087
Fragment based drug discovery, SPR biosensor, matrix metalloproteinase-12, HIV-1 protease, resistance
National Category
Other Chemistry Topics
urn:nbn:se:uu:diva-209136 (URN)978-91-554-8775-1 (ISBN)
Public defence
2013-11-29, C2:301, BMC, Uppsala University, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2013-11-08 Created: 2013-10-14 Last updated: 2014-01-23

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Nordström, HelenaDanielson, U. Helena
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