Abstract: This application is in response to RFA-AI-19-072, ?Novel Therapeutics Directed to Intracellular HIV Targets?. The long term goal of this application is to develop stapled peptide inhibitors to disrupt intracellular protein-protein interactions (PPI) between the host and the virus to curb HIV-1 replication. PPI surfaces are hard to disrupt because of their large and flat surface of interactions. However, recent success in the development of larger biologics such as hydrocarbon stapled peptides allows targeting of PPIs. The stapled peptides are a helices from binding interfaces of PPI that are locked into their bioactive forms. Our goal is to intracellularly disrupt HIV-1 integrase (IN) interaction with the host factor INI1/hSNF5 using stapled peptides, to inhibit HIV-1 assembly, particle production and/or particle morphogenesis. It has been established that perturbing IN without affecting its enzymatic activity can inhibit late stages of HIV-1 replication such as assembly, particle production and/or particle morphogenesis. Several class II IN mutations and allosteric inhibitors of IN (ALLINI), inhibit late events and they do so by perturbing IN/IN multimerization, IN/host factor interaction or IN/RNA interactions. INI1/hSNF5 is the first IN-binding host factor to be identified. We have extensively studied its role in HIV-1 replication and found that it is required for HIV-1 late events. We found that expression of a minimal-IN-binding domain of INI1 (INI1183-292) termed S6, disrupts IN/INI1 interaction in vivo and potently inhibits HIV-1 particle production. Knocking down INI1 and use of INI1-/- cell lines also inhibit HIV-1 particle production. Interestingly, IN mutants that are defective for binding to INI1 lead to the production of morphologically defective particles. These studies together indicate that targeting IN/INI1 interaction is an effective strategy to inhibit HIV-1 particle production. However, lack of structure of INI1 and IN/INI1 interactions have precluded our ability to develop inhibitors to target this interaction. Recent developments in our laboratory in solving the NMR structure of the IN-binding Repeat 1 (Rpt1) domain of INI1, and molecular docking studies of IN/INI1 interaction have helped to overcome this knowledge gap. These structural studies have been validated by mutational, biochemical and virological studies that establish the significance of IN/INI1 interactions. During our structural studies we made an unprecedented novel discovery that INI1 Rpt1 and Trans Activating Response element (TAR) of HIV-1 genomic RNA structurally mimic each other. Nucleic acid mimicry by proteins exists in nature, but mimicry of Rpt1 to TAR is novel and has not been reported earlier. We found that both Rpt1 and TAR bind to same surface of IN C-terminal domain (CTD) and compete with each other for binding to IN with identical IC50 value of 0.005 µM. Furthermore, INI1-interaction-defective mutants of IN resulted in impairment of particle morphogenesis, indicating that these mutants do not bind to RNA in vivo. The knowledge about structural mimicry between Rpt1 and TAR have provided novel strategies to target these interactions. Based on the fact that Rpt1 domain disrupts both IN/INI1 and IN/TAR interactions, we hypothesize that peptidomimetics derived from Rpt1 have dual activity and inhibit both IN/INI1 and IN/TAR interactions. Thus, designing inhibitors using IN/INI1 interaction have the benefit of ?killing two birds in one stone?. This proposal is in collaboration with a medicinal chemist Dr. Asim Debnath (New York Blood Center). In Aim I we will design INI1-based antiviral peptides with enhanced ?-helicity, cell-penetrating properties, and resistance against proteolysis through peptide stapling. In aim II we will test the effect of Stapled peptides on IN-INI1, IN-RNA interactions and on HIV-1 replication: These studies are likely to yield novel stapled dual-active peptides that target intracellular IN/INI1 and/or IN/RNA interactions to inhibit HIV-1 late events.
|Effective start/end date||11/13/20 → 10/31/21|
- National Institute of Allergy and Infectious Diseases: $271,500.00