Inhibition of human immunodeficiency virus type 1 replication in human cells by Debio-025, a novel cyclophilin binding agent

Abstract

Debio-025 is a synthetic cyclosporine with no immunosuppressive capacity but a high inhibitory potency against cyclophilin A (CypA)-associated cis-trans prolyl isomerase (PPIase) activity. A lack of immunosuppressive effects compared to that of cyclosporine was demonstrated both in vitro and in vivo. For three cyclosporines, the inhibitory potential against PPIase activity was quantitatively correlated with that against human immunodeficiency virus type 1 (HIV-1) replication. Debio-025 selectively inhibited the replication of HIV-1 in a CD4+ cell line and in peripheral blood mononuclear cells: potent activity was demonstrated against clinical isolates of various HIV-1 subtypes, including isolates with multidrug resistance to reverse transcriptase and protease inhibitors. Simian immunodeficiency virus and HIV-2 strains were generally resistant to inhibition by Debio-025; however, some notable exceptions of sensitive HIV-2 clinical isolates were detected. In two-drug combination studies, additive inhibitory effects were found between Debio-025 and 19 clinically used drugs of different classes. Clinical HIV-1 isolates that are naturally resistant to Debio-025 and that do not depend on CypA for infection were identified. Comparison of the amino acid sequences of the CypA binding domain of the capsid (CA) protein from Debio-025-sensitive and -resistant HIV-1 isolates indicated that resistance was mostly associated with an H87Q/P exchange. Mechanistically, cyclosporines competitively inhibit the binding of CypA to the HIV-1 CA protein, which is an essential interaction required for early steps in HIV-1 replication. By real-time PCR we demonstrated that early reverse transcription is reduced in the presence of Debio-025 and that late reverse transcription is almost completely blocked. Thus, Debio-025 seems to interfere with the function of CypA during the progression/completion of HIV-1 reverse transcription.

FIG. 1.

Structural formulas of Cs and Debio-025. The top parts of the structures represent the cyclophilin binding domain, and the lower parts represents the calcineurin binding domains.

FIG. 2.

Comparison of the inhibitory effects of Debio-025 against sensitive and resistant HIV-1 isolates. (A) Inhibitory effects against 10 Debio-025-sensitive isolates: 92RW016 (⋆), 92UG037 (•), NL4-3 (○), JR-CSF (+), SF162 (□), SLKA (▵), ROJO (⋄), WEJO (▿), 92BR025 (×), and 92UG046 (▪). (B) Inhibitory effects against eight Debio-025-resistant isolates: Ba-L (+), 92BR014 (□), TEKI (⋄), CMU02 (⋆), 93BR020 (▿), Jv1083 (▵), BCF01 (×), and 52-52 (○). The activity of Debio-025 against HIV-1 isolates in human PBMCs was determined; curve fitting was performed with the XLfit4 (version 4.2.2) software add-in (IDBS, Guildford, Surrey, United Kingdom) for Microsoft Excel; regular lines indicate the results from one representative PBMC assay for each virus isolate, and each data point represents the mean of three replicates per concentration of Debio-025 tested in the assay; boldface lines indicate the global curve fits for the 10 sensitive combined and the 8 resistant isolates combined; IC₅₀ values represent the means ± standard deviations of the mean IC₅₀ values determined for the 10 sensitive and 8 resistant virus isolates whose data are included here (see Table 4 for data virus isolate 52-52 and Table 3 for data for all other virus isolates).

FIG. 3.

Debio-025 blocks HIV-1 infection during early reverse transcription. CD4⁺ HeLa cells (TZM) were infected with DNase-treated NL4-3 HIV-1 together with Debio-025 or without Debio-025 (0.5 or 2.5 μM). Target cell DNA was isolated at the indicated times and was used to detect early (top) and late (bottom) reverse transcripts. Data are the means ± standard deviations from duplicate experiments.