The antiviral activity of G-NH₂ was tested in HIV-1 infected H9 cells cultured in medium containing human (HS), porcine (PS) or fetal calf serum (FCS). When FCS was used, 100 μM G-NH₂ repeatedly abolished HIV infectivity (Fig. 1A, FCS). Similar results were also obtained when infected cells were cultured in PS (data not shown). However, no antiviral activity was observed when the infected cells were cultured in HS (Fig. 1A, HS). An explanation for this could be that G-NH₂ had to be converted by an enzyme present in FCS and PS, but not in HS, to acquire its antiviral activity. To test this hypothesis, 1 mM G-NH₂ was dialyzed against FCS (pre-dialyzed against PBS five times to clear it from low molecular weight material) at 37°C over night. The dialysis solution (DS) obtained which contained the presumed G-NH₂ metabolite, was then added to infected H9 cells cultured in medium containing HS (Fig. 1B, DS). Infected cell cultures to which 100 μM G-NH₂ or no drug had been added served as controls. The results of a typical experiment are shown in Fig. 1B. G-NH₂ showed no antiviral activity, however, infected cells cultured in human serum with DS at a 1/10 dilution, corresponding to ~100 μM of possible G-NH₂-FCS product, showed virus replication that was completely inhibited (Fig. 1B, DS).

To further test if G-NH₂ was converted to the active antiviral substance by an enzyme present in porcine or calf serum, HIV-1-infected H9 cells were cultured in medium containing normal PS or boiled PS (BPS). The cells were then treated with 100 μM G-NH₂, with the DS at a 1/10 dilution or were left untreated. A typical experiment is depicted in Fig. 1C. Infected cells without any test compound and cultured in medium containing PS or BPS served as controls (Fig. 1C, Untreated). In contrast to what was observed in cells cultured with BPS and treated with DS, G-NH₂ showed no antiviral activity in cells cultured with medium containing BPS (Fig. 1C). DS, however, repeatedly inhibited HIV replication regardless of the infected cells being cultured in the presence of PS or BPS (Fig. 1C, DS).

DS derived from 1 mM G-NH₂ dialyzed against pre-washed HS or PS was analyzed by HPLC using a cationic ion-exchange column. With G-NH₂ dialyzed against PS at 37°C (Fig. 2A) but not at 4°C (Fig. 2B), a peak designated Met-X (retention time at 2.9 min) was always observed in addition to the G-NH₂ peak (at 6.2 min). Dialysis of G-NH₂ in HS gave no such change in the HPLC pattern (Fig. 2C). The unknown peak fraction obtained by dialysis of G-NH₂ at 37°C was also isolated and tested for its antiviral activity (see below).

Furthermore, we tested a number of different animal sera for their ability to convert ¹⁴C-G-NH₂ to the antiviral metabolite X (Met-X). The conversion of G-NH₂ to Met-X was detected by the migration pattern in HPLC. As shown in Figure 3, sera from human, rat, mouse, and bird did not convert G-NH₂ but sera from rabbit, monkey, cat, dog, pig, horse and cow were successful in conversion.

¹³C₂/¹⁵N-labeled glycine (Fig. 4A) was transformed to ¹³C₂/¹⁵N-labeled G-NH₂ (Fig. 4B) by Fmoc peptide synthesis. In order to produce ¹³C₂/¹⁵N-labeled Met-X (Fig. 4C), ¹³C₂/¹⁵N-labeled G-NH₂ (Fig. 4B) was dialyzed against PS or FCS as described above. The ¹³C¹⁵N-labeled Met-X was then purified by HPLC and the peak fraction containing labeled Met-X was concentrated by lyophilization before being analyzed by NMR.

Based on the NMR analysis (¹H NMR, coupled ¹H-¹³C NMR, and 2D ¹H-¹⁵N HSQC NMR) one of the possible structures of the unknown compound Met-X was determined as α-hydroxy-glycineamide (α-HGA).

α-HGA was chemically synthesized and compared to Met-X. The HPLC chromatogram of α-HGA was identical to that of Met-X (Fig. 5A). Furthermore, the antiviral activity of the HPLC peak fraction identified as Met-X and α-HGA were tested in H9 cells infected with the HIV-1 SF-2 virus and in chronically infected ACH-2 cells. Both substances had similar antiviral activity (data not shown). The HPLC peak fractions identified as Met-X and α-HGA were also analyzed by capillary electrophoresis using bare silica capillaries under the same experimental conditions. The analysis of Met-X revealed some impurities which were well separated from the major peak containing Met-X (Fig. 5B). The pure α-HGA sample gave a symmetrical single peak, which had strikingly similar migration times to Met-X. The reproducibility was high (RSD = 1.14%; n = 4). Comparison of the UV spectra of the substances in separate experiments further revealed identical absorbance properties. Furthermore, the structural information gained by proton and carbon NMR analyses resulted in identical chemical shift values (data not shown).

Both α-HGA and Met-X treatment at concentrations corresponding to 10 μM resulted in similarly significant changes in virion core morphology (data not shown). Pleomorphic virus particles with distorted, irregular packing of aberrant core structures, partly devoid of dense core material, were seen. Virions having double core structures and occasionally viral cores bulging off from viral envelope were also observed.

α-HGA and some other structurally related compounds (Fig. 6A) at drug concentrations of 100 μM were tested for a possible inhibitory effect on HIV-1 replication in infected H9 cells in the presence of FCS. As shown in Fig. 6B, both α-HGA and G-NH₂ abolished HIV-1 replication. By contrast, oxamic acid, oxamide, α-methoxy glycineamide, and Boc-α-methoxy glycineamide did not show any significant effect on HIV-1 replication. The 50% inhibitory concentration (IC₅₀) in HIV-1 SF-2 infected H9 cells ranged from 4 to 6 μM for both α-HGA and Met-X. A typical dose response curve for α-HGA is depicted in Fig. 6C.

Peripheral blood mononuclear cells (PBMC), H9 and ACH-2 cells were cultured in complete RPMI-1640, and HeLa-tat cells was cultured in complete DMEM medium supplemented with 10% serum and antibiotics. Porcine and human sera (PS and HS) (Biomeda), fetal calf serum (FCS; Invitrogen) oxamic acid and oxamide (Sigma) were used. Glycineamide (G-NH₂) and α-hydroxy glycineamide (α-HGA; manufactured to order by Pharmatory Oy, Oulu, Finland) were kindly provided by Tripep AB, Stockholm, Sweden. ¹³C₂/¹⁵N-labeled Fmoc-glycine (Isotech) was transformed to ¹³C₂/¹⁵N-labeled G-NH₂ by Fmoc peptide synthesis. ¹³C₂/¹⁵N-labeled G-NH₂ was dialyzed against PS or FCS to produce ¹³C₂/¹⁵N-labeled metabolite of G-NH₂ which will be referred to as Met-X (Fig. 2A, B and 2C).

HIV-1 stock of SF-2 from H9 cells was prepared as described previously 31, and 50% tissue culture infectious dose (TCID₅₀) was determined. H9 cells were infected with SF-2 at 100 TCID₅₀ by incubating for 2 hours at 37°C. The cells were then pelleted, washed and resuspended in complete RPMI medium containing HS or PS, and the test compound was added. Cells were cultured for 11 days, and the growth medium was changed seven days post-infection. The HIV-1 p24 antigen contents were assayed at day 7 and 11 post infection essentially as described elsewhere 32 (see below). For RT-assay, the manufacturer's procedure was followed (Cavidi Tech AB, Uppsala, Sweden).

¹³C₂/¹⁵N-labeled or unlabeled G-NH₂ was enzymatically transformed to Met-X by dialysis against FCS or PS at 37°C. Dialysis was performed with 10 ml of serum in a dialysis tubing (5 kD MWCO) that was prewashed by dialyzing 5× against PBS under constant stirring. After 24 hours, the dialysis solution (DS) containing Met-X was analyzed by injecting it onto a 250 × 10 mm, 5 μm cationic ion-exchange column, Theoquest Hypersil SCX, (Thermo), with 90% 0.1 M KH₂PO₄pH 4.5/10% acetonitrile as mobile phase at isocratic flow. The absorbance was measured at 206 nm. Lyophilized ¹³C₂/¹⁵N-labeled Met-X was also analyzed as above except that a mobile phase of 90% 2.5 mM formic acid pH 3/10% acetonitrile was used. All HPLC chromatograms were compared using retention time as an indicator. Once the structure of Met-X was indicated by NMR (see below) to be α-HGA, the HPLC properties of Met-X and chemically synthesized α-HGA were analyzed under the same conditions.

The HPLC peak fraction containing ¹³C₂/¹⁵N-labeled Met-X was isolated, lyophilized, and analyzed with NMR. Due to the low natural abundance of ¹³C- and ¹⁵N-nuclei, a commercially available labeled glycine with two 99% ¹³C- and one 99% ¹⁵N-isotopes (Fig. 4A) was used as starting material. The labeled glycine was transformed into G-NH₂ (Fig. 4B) which was dialyzed against PS or FCS to obtain labeled Met-X. The ¹³C/¹⁵N-labeled Met-X was purified by HPLC and concentrated by lyophilization before being analyzed with NMR. The samples were analyzed on a Bruker DPX 300 MHz, a Jeol Eclipse⁺500 MHz and Bruker DMX 600 MHz spectrometers.

Capillary electrophoresis experiments were carried out at 20°C with a BioFocus 3000 system (Bio-Rad) which was equipped with a fast scanning UV-Vis detector. Fused silica tubing (50 and 365 μm inner and outer diameter, respectively) was purchased from MicroQuartz and cut to a length of 23 cm (with 18.5 cm effective length). Sodium phosphate buffer (0.05 M) at pH 7.4 was used as a background electrolyte. The polarity was set from positive to negative (with the detection point closer to the cathode). The capillary was flushed with the buffer for 1 minute before each run. The Met-X solution obtained from the dialysis procedure was diluted two fold in the buffer solution and filtered through a syringe disc filter (Ultra free-MC 5 000 NMWL, Millipore) prior to injection by pressure (3 psi·s). α-HGA was dissolved in the buffer at 10 mM concentration and injected by pressure (3 psi·s). The applied voltage was 10 kV in all experiments resulting in 50 μA current.

p24-ELISA of infected cell culture supernatants was performed essentially as described elsewhere 32. Briefly, rabbit anti-p24 coated micro-well plates (MWP) were blocked with 3% BSA in PBS at 37°C for 30 minutes. Supernatants from infected cells were added to the plates, followed by incubated at 37°C for 1 hour. The MWPs were washed three times, and biotinylated anti-p24 antibody (1:1 500) was added. One hour after incubation, the MWPs were washed and incubated with HRP-conjugated streptavidine (1:2 000) for 30 minutes. Finally, the MWPs were washed and detected by adding the substrate o-Phenylenediamine Dihydrochloride (Sigma). Recombinant p24 at fixed concentrations was used as a standard. The plates were read in a Labsystems multiscan MS spectrometer. For RT-ELISA, the manufacturer's procedure was followed (Cavidi Tech).

The antiviral activity of α-HGA and some other structurally related compounds was tested in infected H9 cells in the presence of FCS at drug concentrations of 100 μM. H9 cells were infected as described above and cultured in medium containing oxamic acid, oxamide, α-methoxy glycineamide and Boc-α-methoxy glycineamide.

The sera from different animal species were diluted 10-fold in 50 mM potassium phosphate buffer pH 8.0. To 100 μl (10% serum) were added 0.1 μCi [¹⁴C]G-NH₂ (radiospecificity: 56 mCi/mmol), and the samples were incubated for 1, 6 or 24 hours at 37°C. At these time points, 200 μl cold methanol was added, and the samples were left on ice for another 15 minutes. After centrifugation at 15,000 rpm, the supernatants were subjected to HPLC analysis using a SCX-partisphere column (Whatman). The following gradient was used to separate G-NH₂ and Met-X (α-HGA): 5 mM buffer B (5 mM NH₄H₂PO₄ pH 3.5) (10 minutes); linear gradient to 83% buffer C (0.3 M NH₄H₂PO₄ pH 3.5) (6 minutes); equilibration 83% buffer C (2 minutes); linear gradient to 100% buffer B (6 minutes); equilibration 100% buffer B (6 minutes). The retention times of G-NH₂ and α-HGA were 12 and 2 minutes, respectively.

H9 cells were infected with HIV-1 SF-2 at 100 TCID₅₀ by incubating for 2 hours at 37°C. After seven days of incubation, medium containing Met-X or α-HGA was added. Cells were cultured for an additional four days, and progeny virus was analyzed by transmission electron microscopy (TEM). The HIV-1-infected H9 cells were fixed freshly upon embedding in epon, essentially as described before 24. Sections were made approximately 60 nm thick to allow accommodation of the volume of the core structure parallel to the section plane. Duplicate samples were used and minimal beam dose technique was employed throughout. Evaluation of morphology was done with series of electron micrographs to depict different categories of virus morphology. Similar results were also obtained with chronically infected ACH-2 cells induced to replicate HIV-1.