Background:  GS-8374 is a novel bis-tetrahydrofuran-based peptidomimetic HIV protease inhibitor (PI) with a unique diethyl-phosphonate motif incorporated into its molecule. Here we compare its in vitro activity, resistance, and toxicity profile with that of clinically used PI.   

Methods:  Protease inhibition was evaluated using synthetic fluorescent substrates. Standard p24 ELISA or XTT-based assays were used to determine antiretroviral activity and cytotoxicity. Effect on lipid accumulation and insulin-stimulated glucose uptake was assessed in human and mouse adipocytes, respectively. Resistance was characterized using the PhenoSense assay against a panel of 24 PI-resistant mutant viruses (average of 10 protease mutations). Drug resistance selection experiments were conducted in MT-2 cells infected with HIV-1/IIIb.

Results:  GS-8374 is a potent inhibitor of Pr (K_i = 8 pM) with no effects on host aspartic proteases. GS-8374 inhibits HIV-1 in acutely and chronically infected T-cell lines, primary CD4⁺ lymphocytes and macrophages with a potency comparable to that of darunavir, atazanavir, and lopinavir. Cytotoxicity of GS-8374 in various human cell types is minimal (CC₅₀ >50 µM). In contrast to ritonavir, lopinavir, and several other PI, GS-8374 shows minimal effects on the lipid accumulation and insulin-stimulated glucose uptake in adipocytes. In the PhenoSense assay with patient-derived protease mutants, GS-8374 exhibited a mean EC₅₀ fold change of 6.2 (range 0.6 to 26) relative to the wild type control virus, whereas darunavir and brecanavir had a mean fold change of 29.8 (1.0 to 157) and 23.6 (1.2 to 121), respectively. Although tipranavir exhibited similar fold change as GS-8374 (5.9, range 0.5 to 27), its mean EC₅₀ against the panel of mutants was 90-fold higher than that of GS-8374. All the other marketed PI showed a mean fold change ranging from 60 to >200. The exposure of wild type HIV-1 to increasing concentrations of lopinavir, atazanavir, and darunavir for 6 months resulted in the selection of specific mutations in protease, whereas parallel selection with GS-8374 did not induce any genotypic changes in protease.

Conclusions:  GS-8374, a phosphonate-containing PI, exhibits a favorable in vitro pharmacology profile with potent antiretroviral activity and low toxicity that has been consistently observed in multiple assay systems. The in vitro resistance profile of GS-8374 is superior to all tested PI, including darunavir. Collectively, these results support further evaluation of this novel PI.