uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Fragment Based Drug Discovery with Surface Plasmon Resonance Technology
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1087
Keyword [en]
Fragment based drug discovery, SPR biosensor, matrix metalloproteinase-12, HIV-1 protease, resistance
National Category
Other Chemistry Topics
Identifiers
URN: urn:nbn:se:uu:diva-209136ISBN: 978-91-554-8775-1 (print)OAI: oai:DiVA.org:uu-209136DiVA: diva2:656187
Public defence
2013-11-29, C2:301, BMC, Uppsala University, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-10-14 Last updated: 2014-01-23
List of papers
1. The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors: characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors
Open this publication in new window or tab >>The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors: characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors
Show others...
2013 (English)In: MedChemComm, ISSN 2040-2503, E-ISSN 2040-2511, Vol. 4, no 2, 432-442 p.Article in journal (Refereed) Published
Abstract [en]

The kinetic characteristics of hydroxamate-based inhibitors of matrix metalloproteinase (MMP)-12 were explored using an SPR biosensor-based assay and enzyme inhibition analysis. These high-affinity inhibitors were shown to dissociate very slowly from the enzyme-inhibitor complex while a carboxylate analogue had a much faster dissociation rate, verifying the importance of the hydroxamate group for the slow dissociation. Progress curve enzyme inhibition analysis confirmed that the hydroxamate compounds but not the carboxylate compound acted as time-dependent inhibitors. The slow dissociation excluded steady-state estimation of IC50-values and K-i values but also made K-i values from progress curve analysis unreliable. Although a full characterization of the inhibitors using biosensor analysis was limited by slow dissociation, it provided kinetic and mechanistic information of relevance for MMP drug discovery and avoided some pitfalls of conventional enzyme inhibition assays.

National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-196030 (URN)10.1039/c2md20268a (DOI)000314311600020 ()
Available from: 2013-03-04 Created: 2013-03-04 Last updated: 2017-12-06Bibliographically approved
2. Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
Open this publication in new window or tab >>Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library
Show others...
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.

National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-106025 (URN)10.1021/jm8000289 (DOI)000256922800012 ()18494455 (PubMedID)
Available from: 2009-06-11 Created: 2009-06-11 Last updated: 2014-01-23Bibliographically approved
3. Characterization of fragment hits interacting with MMP-12 reveals a dimerised adduct of a fragment hit in the active site
Open this publication in new window or tab >>Characterization of fragment hits interacting with MMP-12 reveals a dimerised adduct of a fragment hit in the active site
(English)Manuscript (preprint) (Other academic)
National Category
Other Chemistry Topics
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-209101 (URN)
Available from: 2013-10-14 Created: 2013-10-14 Last updated: 2014-01-23
4. Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
Open this publication in new window or tab >>Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
Show others...
2011 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 16, no 1, 15-25 p.Article in journal (Refereed) Published
Abstract [en]

A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.

Keyword
enzyme, fragment library, fragment screening, interaction analysis, SPR biosensor
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-140679 (URN)10.1177/1087057110389038 (DOI)000286063300002 ()21149860 (PubMedID)
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved
5. Identification of Fragments for Design of HIV-1 Protease Inhibitors with Allosteric Mechanisms and New Resistance Profiles
Open this publication in new window or tab >>Identification of Fragments for Design of HIV-1 Protease Inhibitors with Allosteric Mechanisms and New Resistance Profiles
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Other Chemistry Topics
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-209197 (URN)
Available from: 2013-10-15 Created: 2013-10-15 Last updated: 2014-01-23Bibliographically approved
6. From Temperature Dependence of Drug-Target Interactions to Thermodynamics and HIV Drug Resistance
Open this publication in new window or tab >>From Temperature Dependence of Drug-Target Interactions to Thermodynamics and HIV Drug Resistance
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-209130 (URN)
Available from: 2013-10-15 Created: 2013-10-14 Last updated: 2014-01-23

Open Access in DiVA

fulltext(1538 kB)1194 downloads
File information
File name FULLTEXT01.pdfFile size 1538 kBChecksum SHA-512
574699904683f75d39f141a4735278d89a80ae1d6f2f4933e762e8ffdf4706eb0dc99f7379245f69d44b68a8abd38c28021d93194c972b85ea710a574892b8be
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Nordström, Helena

Search in DiVA

By author/editor
Nordström, Helena
By organisation
Biochemistry
Other Chemistry Topics

Search outside of DiVA

GoogleGoogle Scholar
Total: 1194 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1033 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf