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
Protease Activity, Inhibition and Ligand Interaction Analysis: Developments and Applications for Drug Discovery
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present study has focused on characterising protease-ligand interactions in the context of drug discovery. The proteases that have been studied are human matrix metallopeptidase 12 (MMP-12), HIV-protease and Hepatitis C virus (HCV) NS3/NS4A protease. These studies have involved kinetic characterisation of protease-inhibitor interactions using biosensor technology, as well as determination of inhibition and activity regulation by using activity assays.

The regulation of MMP-12 activity by calcium was proposed, based on the study of the calcium dependence of MMP-12 activity. Furthermore, it was shown that the high affinity of hydroxamate-based inhibitors of MMP-12 were due to slow dissociation of the enzyme-inhibitor complex by using a new biosensor assay for the study of interactions between MMP-12 and ligands.

A study of the pH-dependency of protease-inhibitor interactions revealed that the interaction kinetics of HIV-protease inhibitors differed with pH in a way that could be related to the inhibitor structures. This suggested that the forces of interaction are different in the association and dissociation phases of an interaction. Furthermore, it demonstrated the usefulness of pH as a variable in characterising protein-ligand interactions.

Results applicable in the discovery of drugs against Hepatitis C were obtained, with the analysis of structure-activity relationships of novel inhibitors. Furthermore, the mode of binding imposed by key functional groups of the inhibitors was explored by investigating the effect of pH on the interactions with NS3.

The results show the importance of using appropriate model systems for drug discovery by selecting relevant targets and assay conditions. Furthermore, the usefulness of kinetic rate information in drug discovery is demonstrated. Thus, by contributing to the knowledge of protease-ligand interactions, applicable to both protease inhibitor interactions and protease activity regulation, this thesis is expected to have an impact on the field of protease inhibitor development and drug discovery in general.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2007. , p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 294
Keywords [en]
Biochemistry, proteases, kinetics, biosensors, inhibition, calcium, drug discovery
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-7822ISBN: 978-91-554-6866-8 (print)OAI: oai:DiVA.org:uu-7822DiVA, id: diva2:170100
Public defence
2007-05-11, B7:113a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2017-05-04Bibliographically approved
List of papers
1. Analysis of the pH-dependencies of the association and dissociation kinetics of HIV-1 protease inhibitors
Open this publication in new window or tab >>Analysis of the pH-dependencies of the association and dissociation kinetics of HIV-1 protease inhibitors
2003 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 16, no 4, p. 203-212Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-95760 (URN)10.1002/jmr.623 (DOI)
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2017-12-14Bibliographically approved
2. Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS3
Open this publication in new window or tab >>Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS3
Show others...
2006 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 14, no 2, p. 544-559Article in journal (Refereed) Published
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-75612 (URN)10.1016/j.bmc.2005.08.045 (DOI)16213143 (PubMedID)
Available from: 2006-03-04 Created: 2006-03-04 Last updated: 2018-01-14Bibliographically approved
3. Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors
Open this publication in new window or tab >>Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors
Show others...
2007 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 15, no 12, p. 4057-4068Article in journal (Refereed) Published
Abstract [en]

There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P1 C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.

Keywords
Bioisostere, HCV, Hepatitis C, NS3, Protease inhibitor
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-17091 (URN)10.1016/j.bmc.2007.03.089 (DOI)000246870400010 ()17449253 (PubMedID)
Available from: 2008-06-16 Created: 2008-06-16 Last updated: 2018-01-12Bibliographically approved
4. Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation
Open this publication in new window or tab >>Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation
2006 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 398, no 3, p. 393-398Article in journal (Refereed) Published
Abstract [en]

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the K-D was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and > 100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pK(a) values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate-and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

Keywords
active conformation, affinity, calcium binding, human macrophage elastase, matrix metallopeptidase-12 (MMP-12), pH-dependency
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-95763 (URN)10.1042/BJ20051933 (DOI)000240793800008 ()16737445 (PubMedID)
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2017-12-14Bibliographically approved
5. The high affinity of hydroxamate-based MMP-12 inhibitors is due to an inherently slow dissociation rate of the enzyme-inhibitor complex
Open this publication in new window or tab >>The high affinity of hydroxamate-based MMP-12 inhibitors is due to an inherently slow dissociation rate of the enzyme-inhibitor complex
(English)Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-95764 (URN)
Available from: 2007-04-19 Created: 2007-04-19 Last updated: 2009-11-27Bibliographically approved

Open Access in DiVA

fulltext(3117 kB)1389 downloads
File information
File name FULLTEXT01.pdfFile size 3117 kBChecksum SHA-1
b195c6d68fdc41c84e6597a2933eeac3088f315060777156e189f7e3fe3545197d58f10d
Type fulltextMimetype application/pdf
cover(421 kB)88 downloads
File information
File name COVER01.pdfFile size 421 kBChecksum SHA-1
fc484cd7ee03fa0514221fdf56293f99d268ffa3498912c8b196ffc757827ae768f4fdfe
Type coverMimetype application/pdf
Buy this publication >>

By organisation
Department of Biochemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1389 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: 1295 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