The proposal entitled Identification of Antagonists of the Molecular Chaperone Function of CBF? has been written in response to the Program Announcement PA-10-213 entitled Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). We implement a novel live cell, quenched FRET (FqRET) reporter assay for the molecular interaction of the cellular transcription factor CBF? and the HIV Vif protein in order to discover novel molecular probes for studying the mechanism whereby CBF? binds to and stabilizes Vif. Our target-biased primary assay and the development and optimization of secondary assays and counter screens are planned to identify one or more cell permeable, nontoxic molecular probes that inhibit CBF? binding to Vif. The four Specific Aims will be: (1) Develop and optimize an in-cell quenched FRET (FqRET) assay that will be used in HTS to identify compounds that inhibit the interaction of CBF? with Vif. (2) Develop and optimize orthogonal secondary screens validating that antagonists of CBF? binding to Vif are mechanistically relevant in their ability to reduce the cellular abundance of Vi as well as reduce Vif- dependent degradation of A3G. (3) Counter screen for off-target and cytotoxic hits that affect the ability of CBF? to bind to RUNX1 and function as a cellular transcription factor. And (4) validate selected hits and commercially available chemical analogs for their antiviral activities and target-specificity through functional endpoint assays and bindin studies. At this stage we will have achieved the objective of the PA of having a primary assay and complementary assay systems optimized and validated for transfer and screening at a facility within the NIH MLPCN.
The proposal entitled Identification of Antagonists of the Molecular Chaperone Function of CBF? has been written in response to the Program Announcement PA-10-213 entitled Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). The significance and ultimate goal of this proposal is to develop, optimize and validate a novel live cell assay for the molecular interaction of the cellular transcription factor CBF? and the HIV Vif protein. This assay will be appropriate to screen chemical libraries for novel molecular probes. Vif binds to the host defense factor APOBEC3G (A3G) in order to induce A3G degradation, but Vif is unstable in cells without the molecular chaperone function of CBF?. Antagonists of CBF? binding to Vif are predicted to permit the destabilization of Vif and thereby enable A3G to inhibit HIV replication. Little is know of the chaperone function of CBF?, therefore this study will satisfy an unmet need for target-biased molecular probes that bind to either CBF? or Vif and only antagonize this interaction while not interfering with the transcription cofactor function of CBF?. It goes without saying that molecular probes may not only serve a basic research function but may have value in future therapeutic development studies.
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|Salter, Jason D; Bennett, Ryan P; Smith, Harold C (2016) The APOBEC Protein Family: United by Structure, Divergent in Function. Trends Biochem Sci 41:578-594|
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|Bennett, Ryan P; Stewart, Ryan A; Hogan, Priscilla A et al. (2016) An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation. Antiviral Res 136:51-59|
|Salter, Jason D; Morales, Guillermo A; Smith, Harold C (2014) Structural insights for HIV-1 therapeutic strategies targeting Vif. Trends Biochem Sci 39:373-80|
|Smith, Harold C; Bennett, Ryan P; Kizilyer, Ayse et al. (2012) Functions and regulation of the APOBEC family of proteins. Semin Cell Dev Biol 23:258-68|