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
Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Show others and affiliations
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
Description
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).

Place, publisher, year, edition, pages
2011. Vol. 54, no 3, 699-708 p.
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:uu:diva-140680DOI: 10.1021/jm1010513ISI: 000286798100002PubMedID: 21207961OAI: oai:DiVA.org:uu-140680DiVA: diva2:384215
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. Revealing Secrets of Synaptic Protein Interactions: A Biosensor based Strategy
Open this publication in new window or tab >>Revealing Secrets of Synaptic Protein Interactions: A Biosensor based Strategy
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels.

The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin.

Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery.

AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction.

The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 73 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1133
Keyword
Surface plasmon resonance, biosensor, AMPA receptor, GABAA receptor, ligand-gated ion channel, A-kinase anchoring protein, caldendrin, calmodulin, HIV, fragment based drug discovery
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Biochemistry; Neuroscience
Identifiers
urn:nbn:se:uu:diva-220879 (URN)978-91-554-8916-8 (ISBN)
Public defence
2014-05-16, B42, Husargatan 3, 751 23 Uppsala, 13:15 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme
Available from: 2014-04-25 Created: 2014-03-22 Last updated: 2014-04-29

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Authority records BETA

Elinder, MalinSeeger, ChristianBrandt, PeterDanielson, U Helena

Search in DiVA

By author/editor
Elinder, MalinSeeger, ChristianBrandt, PeterDanielson, U Helena
By organisation
Department of Biochemistry and Organic ChemistryBiochemistry
In the same journal
Journal of Medicinal Chemistry
Other Basic Medicine

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 909 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