The continuous emergence of HIV-1 phenotypes resistant to the currently available drugs dictates a need to develop new therapies with alternative mechanisms of action. One such mechanism proposed in the present application is to exploit multimeric structures of a key HIV-1 enzyme integrase (IN). Our recent studies revealed a highly dynamic nature of interacting IN subunits. Interestingly, the integrase binding domain (IBD) of lens epithelium derived growth factor (LEDGF), a key cellular cofactor for HIV-1 integration, profoundly alters IN subunit-subunit interactions by """"""""locking"""""""" the free viral protein into a tetrameric state. This IN tetramer is active in the 3'-processing reaction but fails to catalyze the second biologically essential concerted integration step. These observations uncovered a potential novel mechanism of inhibiting the HIV-1 IN function and provided a compelling rationale for the following hypothesis: highly flexible multimeric forms of HIV-1 IN possess unique structural pockets that can be selectively targeted by small molecules to inhibit the concerted integration reaction. To address this premise we propose the following two principal aims.
Aim 1 will identify and characterize novel """"""""hotspots"""""""" within different multimeric forms of IN that can potentially be targeted by small molecule inhibitors. Innovative mass spectrometric protein footprinting and molecular modeling approaches will be used in these studies.
Aim 2 will develop and validate a novel fluorescence assay for high-throughput screening (HTS) of new types of HIV-1 integrase inhibitors. The assay will monitor subunit-subunit interactions between affinity tag containing and tag-free fluorescently labeled proteins. The interacting proteins will be pulled down by the affinity resin and evaluated by fluorescence measurements. The productive exchange between the two protein subunits will result in strong fluorescence, while potent inhibitors of this reaction will significantly diminish the fluorescence signal. The assay will be optimized through initial screening of small chemical libraries. These studies will define important statistical parameters and an effective protocol for future application of the novel fluorescence assay for HTS of large chemical libraries.

Public Health Relevance

Emergence of HIV-1 strains resistant to the current antiretroviral therapies is a serious clinical problem. Therefore, there is an urgent need to identify and validate new viral targets for drug discovery. One such target investigated in the present proposal is a multimeric structure of a key HIV-1 enzyme integrase.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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AIDS Discovery and Development of Therapeutics Study Section (ADDT)
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Gupta, Kailash C
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Ohio State University
Schools of Pharmacy
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Engelman, Alan; Kessl, Jacques J; Kvaratskhelia, Mamuka (2013) Allosteric inhibition of HIV-1 integrase activity. Curr Opin Chem Biol 17:339-45
Feng, Lei; Sharma, Amit; Slaughter, Alison et al. (2013) The A128T resistance mutation reveals aberrant protein multimerization as the primary mechanism of action of allosteric HIV-1 integrase inhibitors. J Biol Chem 288:15813-20
Kessl, Jacques J; Jena, Nivedita; Koh, Yasuhiro et al. (2012) Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors. J Biol Chem 287:16801-11
Wang, Hao; Jurado, Kellie A; Wu, Xiaolin et al. (2012) HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor. Nucleic Acids Res 40:11518-30
Kessl, Jacques J; Li, Min; Ignatov, Michael et al. (2011) FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75. Nucleic Acids Res 39:9009-22