The Effect of Alpha-HGA on HIV-1 Replication

Different classes of anti HIV-1 drugs are now available for treatment of HIV-1 infection. Although improved, these drugs show adverse effects and their long-term efficiency is severely hampered by the emergence of resistant viruses. Therefore, less toxic and more effective anti HIV-1 therapeutics agents are still needed. This thesis aimed to evaluate the anti HIV-1 activity of the small molecule, alpha-hydroxy glycineamide (αHGA), and to investigate its mode of action against HIV-1. Here we showed that αHGA inhibits HIV-1 replication and that HIV-1 particles with aberrant core structures are formed in the presence of αHGA. We also showed that αHGA inhibits the replication of clinical HIV-1 isolates with acquired resistance to reverse transcriptase and protease inhibitors but has no effect on the replication of any of ten different RNA and DNA viruses. Alpha-HGA affected the ability of the HIV-1 capsid protein to assemble into tubular or core structures in vitro and in vivo. In silica molecular modeling studies indicated a possible interaction of αHGA with the aspartate 51 (D51) of p24. This amino acid residue, located in the N-terminal domain of the capsid protein, has been shown to play a key role in virus assembly and maturation by forming a β-hairpin structure upon proteolytic cleavage of the Gag precursor protein. We introduced three different D51 substitution mutations into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro CA assembly and virus infectivity. The results showed that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could not rescue the structural integrity of the capsid nor viral infectivity. Surprisingly, in direct p24 binding studies αHGA was found to bind to the hinge region between the Nand C-terminal domains of the HIV-1 capsid protein and not to the p24 interaction surfaces. Importantly, αHGA only bound to dimerized p24 and not to monomeric protein. Binding to the flexible hinge region of p24 would indicate an allosteric effect of αHGA on the protein affecting its ability to assemble into capsids. Since drug transport is an important aspect of drug function, we investigated the mechanism of [C]αHGA uptake by human T cell line. Uptake of [C]αHGA into H9 cells was timeand dosedependent. The uptake properties showed low temperature dependency (Q10<2) and the cellular uptake of [C] labeled αHGA was not inhibited by increasing concentrations of cold competitors. The metabolic inhibitors, NaN3 and NaF, had no effect on the cellular uptake of [C] labeled αHGA. Kinetic analysis of compound uptake, studies with metabolic inhibitors, saturation studies, and temperature coefficient value of αHGA uptake indicated that this compound enters H9 cells by a mechanism of passive diffusion.