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Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
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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.

Place, publisher, year, edition, pages
2011. Vol. 16, no 1, 15-25 p.
Keyword [en]
enzyme, fragment library, fragment screening, interaction analysis, SPR biosensor
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-140679DOI: 10.1177/1087057110389038ISI: 000286063300002PubMedID: 21149860OAI: oai:DiVA.org:uu-140679DiVA: diva2:384213
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Towards a New Generation of Anti-HIV Drugs: Interaction Kinetic Analysis of Enzyme Inhibitors Using SPR-biosensors
Open this publication in new window or tab >>Towards a New Generation of Anti-HIV Drugs: Interaction Kinetic Analysis of Enzyme Inhibitors Using SPR-biosensors
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

As of today, there are 25 drugs approved for the treatment of HIV and AIDS. Nevertheless, HIV continues to infect and kill millions of people every year. Despite intensive research efforts, both a vaccine and a cure remain elusive and the long term efficacy of existing drugs is limited by the development of resistant HIV strains. New drugs and preventive strategies that are effective against resistant virus are therefore still needed. In this thesis an enzymological approach, primarily using SPR-based interaction kinetic analysis, has been used for identification and characterization of compounds of potential use in next generation anti-HIV drugs.

By screening of a targeted non-nucleoside reverse transcriptase inhibitor (NNRTI) library, one novel and highly potent NNRTI was identified. The inhibitor was selected with respect to resilience to drug resistance and for high affinity and slow dissociation – a kinetic profile assumed to be suitable for inhibitors used in topical microbicides. In order to confirm the hypothesis that such a kinetic profile would result in an effective preventive agent with long-lasting effect, the correlation between antiviral effect and kinetic profile was investigated for a panel of NNRTIs. The kinetic profiles revealed that NNRTI efficacy is dependent on slow dissociation from the target, although the induced fit interaction mechanism prevented quantification of the rate constants.

To avoid cross-resistance, the next generation anti-HIV drugs should be based on chemical entities that do not resemble drugs in clinical use, either in structure or mode-of-action. Fragment-based drug discovery was used for identification of structurally new inhibitors of HIV-enzymes. One fragment that was effective also on variants of HIV RT with resistance mutations was identified. The study revealed the possibility of identifying structurally novel NNRTIs as well as fragments interacting with other sites of the protein.

The two compounds identified in this thesis represent potential starting points for a new generation of NNRTIs. The applied methodologies also show how interaction kinetic analysis can be used as an effective and versatile tool throughout the lead discovery process, especially when integrated with functional enzymological assays.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 834
Keyword
drug discovery, fragment, screening, reverse transcriptase, microbicides, interaction analysis, NNRTIs
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-152172 (URN)978-91-554-8092-9 (ISBN)
Public defence
2011-06-09, B42, BMC, Uppsala Universitet, Husargatan 3, 751 23, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-05-19 Created: 2011-04-26 Last updated: 2011-07-01Bibliographically approved
2. Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery: SPR-biosensor Aided Interaction Studies
Open this publication in new window or tab >>Reducing Attrition via Improved Strategies for Pre-clinical Drug Discovery: SPR-biosensor Aided Interaction Studies
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:nbn:se:uu:diva-171997 (URN)978-91-554-8340-1 (ISBN)
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
3. 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1087
Keyword
Fragment based drug discovery, SPR biosensor, matrix metalloproteinase-12, HIV-1 protease, resistance
National Category
Other Chemistry Topics
Identifiers
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)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-10-14 Last updated: 2014-01-23

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