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Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Helena Danielson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Helena Danielson)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Helena Danielson)
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2009 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 14, no 4, 395-403 p.Article in journal (Refereed) Published
Abstract [en]

A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance-based biosensor. the aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. the authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. to efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the  screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dissociation for the   complete panel of enzyme variants. hits were confirmed by visually  inspecting the complete sensorgrams. two structurally unrelated   compounds fulfilled the hit criteria, but only 1 compound was found to   (a) compete with a known NNRTI for binding to the NNRTI site, (b)   inhibit HIV-1 RT activity, and (c) inhibit HIV-1 replication in cell culture, for all 4 enzyme variants. this novel screening methodology offers high-resolution real-time kinetic data for multiple targets in parallel. it is expected to have broad applicability for the discovery of compounds with defined kinetic profiles, crucial for optimal therapeutic effects.

Place, publisher, year, edition, pages
SAGE , 2009. Vol. 14, no 4, 395-403 p.
Keyword [en]
HIV-1 Reverse transcriptase, screening, surface plasmon resonance (SPR)-biosensor, NNRTIs, microbicides
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-109362DOI: 10.1177/1087057109333977ISI: 000265948100008PubMedID: 19403922OAI: oai:DiVA.org:uu-109362DiVA: diva2:273601
Available from: 2009-10-22 Created: 2009-10-14 Last updated: 2011-07-08Bibliographically 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)
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Available from: 2011-05-19 Created: 2011-04-26 Last updated: 2011-07-01Bibliographically approved

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Elinder, MalinNordström, HelenaDanielson, U Helena

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