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. 2011 Apr;55(4):1366-76.
doi: 10.1128/AAC.01183-10. Epub 2011 Jan 18.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile

Christian Callebaut  1 , Kirsten Stray, Luong Tsai, Matt Williams, Zheng-Yu Yang, Carina Cannizzaro, Stephanie A Leavitt, Xiaohong Liu, Kelly Wang, Bernard P Murray, Andrew Mulato, Marcos Hatada, Tina Priskich, Neil Parkin, Swami Swaminathan, William Lee, Gong-Xin He, Lianhong Xu, Tomas Cihlar
Affiliations

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile

Christian Callebaut et al. Antimicrob Agents Chemother. 2011 Apr.

Abstract

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

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Figures

FIG. 1.
FIG. 1.
Structure of GS-8374.
FIG. 2.
FIG. 2.
Binding of GS-8374 and TMC-126 in the active site of HIV-1 protease. (A) Overlap of GS-8374 and TMC-126; (B) view of solvent-accessible surface of HIV-1 protease along with TMC-126; (C) view of solvent-accessible surface of HIV-1 protease along with the diethylphosphonate moiety of GS-8374; (D and E) top view (D) and front view (E) of diethylphosphonate group of GS-8374 bound in the active site of HIV-1 protease.
FIG. 3.
FIG. 3.
Resistance profile of GS-8374. Twenty-four patient-derived multi-PI-resistant viruses were evaluated for their sensitivity to GS-8374 and clinically approved PIs using the Monogram PhenoSense assay. Genotypes of PI-resistant viruses, values for individual EC50s, and fold resistance for each tested PI are presented in Tables S10, S11, and S12 in the supplemental material. ATV, atazanavir; APV, amprenavir; IDV, indinavir; LPV, lopinavir; NFV, nelfinavir; RTV, ritonavir; SQV, saquinavir; TPV, tipranavir; DRV, darunavir.
FIG. 4.
FIG. 4.
Effect of ritonavir on in vitro hepatic metabolism of GS-8374 and clinically approved PIs. (A) PIs (3 μM) were incubated with pooled human hepatic microsomal fraction in the absence or presence of 1 μM RTV as described in Materials and Methods; (B) inhibition of GS-8374 microsomal metabolism by various concentrations of ritonavir. Data shown represent the means and standard deviations from two independent experiments performed in duplicate.
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