The extensive use of antiviral drugs has resulted in the emergence of mutant viral strains that are resistant to the currently available nucleoside therapeutics. Using a combination of drugs that target different viral enzymes (termed highly active anti-retroviral therapy or HAART) has helped, however undesirable side effects and toxicity are often prevalent and reports of multidrug resistance are increasing. As a result, the need for new viral enzymatic targets is critical. In that regard, the nucleocapsid protein of HIV offers forth an attractive target. NCp7 is a small zinc-binding protein that has been shown to be critical in both the early and late stages of the HIV-1 replication process. NCp7 (and the NCp7 domain of the Gag precursor polyprotein) has several key roles in HIV replication, including direct participation in RNA genome recognition and packaging, promoting annealing of the tRNA primer to initiate reverse transcription, and facilitating strand transfer during reverse transcription. NCp7 contains two CCHC-type zinc finger domains that play critical RNA-binding roles during replication, and single-atom Cys-to-Ser mutations that disrupt zinc coordination are sufficient to completely block viral replication. Several approaches to designing inhibitors have been tried, most commonly, use of zinc-ejectors, however to date these have exhibited significant toxicity and poor specificity. A problem that has hindered the identification of viable inhibitors is the inherent flexibility of the protein itself. As such, flexible nucleosides, known s fleximers designed and developed in the Seley-Radtke laboratory may provide a solution. In that regard, a series of flexible nucleobase (flexbase) inhibitors of NCp7 have been identified by means of a virtual screening process. The flexbase analogues are in excellent alignment with the central guanosine residue known to be critical to the biologically relevant conformations of NCp7, thus form the basis for this application. Notably, the flexbases should not result in zinc ejection thereby overcoming the problems associated with other approaches.
The rapidly increasing reports of resistance to currently used antiviral therapeutics is driving the search for new enzyme targets. This application focuses on the synthesis, biological screening, structural studies of protein-inhibitor complexes, as well as construction of a viable computational model for the HIV nucleocapsid protein NCp7. The heterocyclic 'flexbase' analogues that form the basis for this application should provide a strategic approach to inhibiting NCp7. NCp7 is anticipated to be much less susceptible to the development of resistance due to its multifunctional role in several HIV replication processes. The impact of this project goes beyond just global health research, but will also provide valuable hands on, cross-disciplinary training for students.
