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Towards a New Generation of Anti-HIV Drugs: Interaction Kinetic Analysis of Enzyme Inhibitors Using SPR-biosensors
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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 [en]
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: urn:nbn:se:uu:diva-152172ISBN: 978-91-554-8092-9OAI: oai:DiVA.org:uu-152172DiVA: diva2:412938
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
List of papers
1. Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
Open this publication in new window or tab >>Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
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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
Keyword
HIV-1 Reverse transcriptase, screening, surface plasmon resonance (SPR)-biosensor, NNRTIs, microbicides
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-109362 (URN)10.1177/1087057109333977 (DOI)000265948100008 ()19403922 (PubMedID)
Available from: 2009-10-22 Created: 2009-10-14 Last updated: 2011-07-08Bibliographically approved
2. Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy
Open this publication in new window or tab >>Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy
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2010 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1873-2968, Vol. 80, no 8, 1133-1140 p.Article in journal (Refereed) Published
Abstract [en]

The importance of slow dissociation of non-nucleoside reverse transcriptase inhibitors (NNRTIs) for antiviral effect has been investigated. The kinetic characteristics of a series of NNRTIs interacting with wild type and drug resistant variants of HIV-1 RT (EC 2.7.7.49) were analyzed by SPR biosensor technology. The antiviral effect was determined in MT-4 and peripheral blood mononuclear cells. Due to extremely slow dissociation rates and a complex interaction mechanism, rate constants could not be quantified. Instead, interaction characteristics were qualitatively analyzed using simulated sensorgrams. The simplest model describing these interactions adequately was an induced fit mechanism, i.e. a mechanism involving the formation of an initial enzyme-inhibitor complex subsequently transformed into a more stable complex. Differences in rates of dissociation from the initial complex and rates of relaxation from the induced complex explained (1) the differences in the amounts of formed complex, (2) the stability of the complex and (3) the antiviral efficacies of the compounds. The effect of NNRTI binding site mutations also correlated with these kinetic characteristics. MIV-170 was the most effective inhibitor of wild type and mutant HIV-1 in cell culture, a property that was associated with the formation of the largest amount of complex and the slowest relaxation and dissociation rates. This study supports the hypothesis that the efficacy of anti-HIV drugs is dependent on slow dissociation from the target, thereby maximizing the duration of the inhibitory effect. It also illustrates the strength of simulating interaction data for qualitative analysis of tight-binding drugs and the importance of resolving the kinetic mechanism of drug-target interactions.

Keyword
HIV reverse transcriptase, NNRTI, MIV, kinetics, biosensor, SPR
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-129428 (URN)10.1016/j.bcp.2010.06.035 (DOI)000281936800003 ()20599774 (PubMedID)
Available from: 2010-08-15 Created: 2010-08-15 Last updated: 2011-07-08Bibliographically approved
3. 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: 2014-01-23Bibliographically approved
4. Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
Open this publication in new window or tab >>Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
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2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 3, 699-708 p.Article in journal (Refereed) Published
Abstract [en]

A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).

National Category
Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-140680 (URN)10.1021/jm1010513 (DOI)000286798100002 ()21207961 (PubMedID)
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2016-08-17Bibliographically approved

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