Isolation and characterization of a small antiretroviral molecule affecting HIV-1 capsid morphology

Abstract

Background: Formation of an HIV-1 particle with a conical core structure is a prerequisite for the subsequent infectivity of the virus particle. We have previously described that glycineamide (G-NH2) when added to the culture medium of infected cells induces non-infectious HIV-1 particles with aberrant core structures.

Results: Here we demonstrate that it is not G-NH2 itself but a metabolite thereof that displays antiviral activity. We show that conversion of G-NH2 to its antiviral metabolite is catalyzed by an enzyme present in bovine and porcine but surprisingly not in human serum. Structure determination by NMR suggested that the active G-NH2 metabolite was alpha-hydroxy-glycineamide (alpha-HGA). Chemically synthesized alpha-HGA inhibited HIV-1 replication to the same degree as G-NH2, unlike a number of other synthesized analogues of G-NH2 which had no effect on HIV-1 replication. Comparisons by capillary electrophoresis and HPLC of the metabolite with the chemically synthesized alpha-HGA further confirmed that the antiviral G-NH2-metabolite indeed was alpha-HGA.

Conclusion: alpha-HGA has an unusually simple structure and a novel mechanism of antiviral action. Thus, alpha-HGA could be a lead for new antiviral substances belonging to a new class of anti-HIV drugs, i.e. capsid assembly inhibitors.

Figure 1

Antiviral activity of G-NH₂ and characterization of G-NH₂ metabolite obtained after dialysis against FCS or PS. (A) H9 cells (10⁵) were infected with the SF-2 strain of HIV-1 and cultured in medium containing 100 μM G-NH₂ and either 10% fetal calf serum (FCS) or human serum (HS). Ten days post-infection, the level of p24-antigen in the culture supernatants was assayed with a p24-ELISA. (B) H9 cells infected with the SF-2 strain of HIV-1 were cultured in medium containing 10% human serum (HS) and either 100 μM G-NH₂ or a dialysis solution (DS) of 1/10 dilution of 1 mM G-NH₂ dialyzed against FCS. Untreated cultures without any addition of G-NH₂ or DS served as controls. (C) Infected H9 cells were cultured in the presence of 10% boiled porcine serum (BPS) or non-boiled porcine serum (PS). The infected cultures were cultured with the addition of 100 μM G-NH₂, DS or were left untreated. Virus production was assessed using an RT assay. Error bars indicate standard deviations from quadruple cultures.

Figure 2

HPLC analysis of G-NH₂ and the G-NH₂-derived metabolite Met-X. HPLC chromatograms of dialysis solution (DS) after dialyzing 1 mM G-NH₂ against porcine serum (PS) at 37°C (A) and at 4°C (B) or human serum at 37°C. The dialysis solutions were analyzed with a cationic ion-exchange column (Theoquest Hypersil SCX, Thermo), and the absorbance was measured at 206 nm.

Figure 3

Conversion of G-NH2 to Met-X in different sera. ¹⁴C-labeled G-NH₂ was incubated with sera from different species at different time points as indicated in the figure. Conversion to Met-X was analyzed by HPLC. Percent conversion to Met-X for respective sera is depicted.

Figure 4

Chemical structure and production of G-NH₂-derived metabolite after dialysis against porcine serum. The chemical structures of doubly labeled glycine with two ¹³C- and one ¹⁵N-isotopes (A) which was transformed into labeled glycineamide (B). The latter was dialyzed against porcine serum at 37°C, and the ¹³C₂ ¹⁵N-labeled product (C) here is referred to as Met-X. This compound was purified by HPLC and concentrated before being analyzed with NMR.

Figure 5

Comparison of Met-X with α-HGA. HPLC analysis of synthetically produced α-HGA and Met-X, the latter produced enzymatically after dialyzing 1 mM G-NH₂ against PS at 37°C, is depicted in panel A, and capillary electrophoresis analysis of α-HGA and Met-X in panel B.

Figure 6

Biological and antiviral comparison of α-HGA and some structurally related compounds. Chemical structures of glycine, glycineamide (G-NH₂), α-HGA, oxamic acid, oxamide, α-methoxy glycineamide and Boc-α-methoxy glycineamide (A). Antiviral activity of 100 μM of respective compound added to HIV-1 SF-2 infected H9 cells cultured in the presence of 10% fetal bovine serum (B). Dose response of the antiviral activity of synthetically produced α-HGA (C).