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Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery: SPR-biosensor Aided Interaction Studies
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Helena Danielson)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The efficacy of a drug is tightly intertwined with its interaction mechanism with the drug target. The mechanism is dependent on the physicochemical and structural characteristics of both target and drug molecule.

Drug discovery is plagued by a high attrition rate, whereas in the clinic, a major issue is drug resistance. To improve the quality of the lead compounds in the pre-clinical phase of drug discovery, and thereby reducing the attrition, a deeper understanding of interaction mechanisms is needed. We have adopted new strategies and techniques for this purpose.

A compound library was compiled for the purpose of fragment-based drug discovery. Its compatibility with the SPR platform, along with its interaction profile, was validated. The library was subsequently used in a screening campaign for novel scaffolds of human immunodeficiency virus-1 protease, not sensitive to common resistance mutations. This was achieved by the use of a target panel containing signature resistance mutations towards already approved HIV-1 protease inhibitors. 10 scaffolds were identified and deemed novel. These constitute interesting starting points for development of a new generation of HIV-1 protease inhibitors with different resistance mechanisms, which is very valuable in combination therapies.

The cause of difference in anti-viral potency in cell cultures was investigated for two iso-affinity compounds acting on the hepatitis C viral polymerase, NS5B. By SPR-aided interaction analysis with chemo- and thermodynamic characterization, filibuvir and VX-222, both same-site allosteric inhibitors in phase II clinical trials, were identified to have two different interaction mechanisms. This was ultimately suggested to cause the differences in potency.

A structure-kinetic relationship study, with a thermodynamic characterization, was performed for an approved thrombin inhibitor and five close P3-analogues. This study had the aim to better understand the basic mechanisms of the interactions. Stopped-flow spectroscopy, SPR, and calorimetry were used in parallel and their results compared before evaluation with x-ray crystallography data.

Thus, this thesis has demonstrated successful use of fragment-based drug discovery and high resolution techniques to advance projects in most stages of pre-clinical drug discovery with the aim to reduce the future drug attrition and to understand molecular interactions on a fundamental level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 923
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-171997ISBN: 978-91-554-8340-1 (print)OAI: oai:DiVA.org:uu-171997DiVA: diva2:513954
Public defence
2012-05-30, B41, BMC, Uppsala University, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2012-05-04 Created: 2012-03-31 Last updated: 2012-08-01Bibliographically approved
List of papers
1. 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
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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
2. New Scaffolds for Design of Inhibitors of Drug Resistant HIV-1 Protease Identified by Fragment Library Screening
Open this publication in new window or tab >>New Scaffolds for Design of Inhibitors of Drug Resistant HIV-1 Protease Identified by Fragment Library Screening
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(English)Article in journal (Other academic) Submitted
Keyword
HIV protease, inhibitors, fragment based drug discovery, SPR biosensor, screening, resistance
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-172215 (URN)
Available from: 2012-04-03 Created: 2012-04-02 Last updated: 2012-08-01Bibliographically approved
3. Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
Open this publication in new window or tab >>Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
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2013 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, no 3, 356-368 p.Article in journal (Other academic) Published
Abstract [en]

Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

Keyword
HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biochemistry
Identifiers
urn:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
4. Identification of Structure-Kinetic and Structure-Thermodynamic Relationships for Thrombin Inhibitors
Open this publication in new window or tab >>Identification of Structure-Kinetic and Structure-Thermodynamic Relationships for Thrombin Inhibitors
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(English)Manuscript (preprint) (Other academic)
Keyword
Thrombin, melagatran, biosensor, kinetics, thermodynamics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-172216 (URN)
Available from: 2012-04-03 Created: 2012-04-02 Last updated: 2012-08-01

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