Harold C. Smith
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, USA.
Title: Drug Leads that Inhibit Vif and Enable APOBEC3G are Broadly Neutralizing of HIV1 Clades and Drug-Ââ€resistant Strains
Biography
Biography: Harold C. Smith
Abstract
HIV Viral infectivity factor (Vif) evokes the destruction of the host restriction factor known as APOBEC3G (A3G). Vif dimerization has been shown to be essential for Vif binding to A3G and A3G degradation. Experimental data show that in the absence of Vif, virion-Ââ€assembled A3G will hypermutate proviral DNA during reverse transcription. The open questions are: can Vif be successfully drugged and by sparing A3G, will hypermutation activity be sufficient to inhibit viral replication? High throughput screening (HTS) assays were developed using FRET for Vif dimerization. The screens were used to select compounds with dose-Ââ€dependent signals by preventing Vif FRET. A secondary assay for Vif-Ââ€dependent A3G degradation was used to identify compounds that preserved A3G. These were triaged for the ability to inhibit pseudotyped HIV replication and to have low cytoxicity. Ion torrent sequencing of integrated viral genomes revealed extensive hypermutation characteristic of A3G preferences. Following medicinal chemistry, a lead was tested in a seven day spreading infection in PBMC. The Vif dimerization FRET assay provided a robust HTS method applicable to a large library of drug-Ââ€like molecules. Quantitative HTS followed by orthogonal secondary screen and cytotox counter screening enabled selection of limited number of chemistries for pseudo typed viral testing. A lead (SMVDA) was selected with nanomolar IC50. By impairing Vif dimerization, A3G degradation was reduced and viral particle incorporation of A3G was enhanced. Proviral DNA isolated form target cells showed numerous tracts of dG to dA hypermutation that corresponded to multiple nonsense codon and sense changes. 17 different clades and 8 drug resistant strains of HIV infecting PBMC were sterilized by seven days following a single dose of SMVDA . Conclusions: Drugging Vif led to massive dG to dA hypermutation of HIV proviral DNA, such that the protein coding capacity of the virus would be severely compromised. Inhibitors of Vif are broadly neutralizing and inhibited all drug resistant strains of HIV tested. Drugging Vif therefore is achievable and the data suggest that this will serve as a firewall for viremia induced by activating reservoirs as well as a solution for rescue and salvage therapies.