Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases alpha and beta.

4'-Azidothymidine (ADRT) is a novel nucleoside analogue that exhibits potent inhibitory activity against the replication of human immunodeficiency virus (HIV) in lymphocytes. The mechanisms by which ADRT inhibits HIV reverse transcriptase (HIV-RT) as ADRT 5'-triphosphate (ADRT-TP), the active intracellular metabolite of ADRT, and as the ADRT-MP molecule incorporated into DNA were examined and compared to their effects on human DNA polymerases alpha and beta. Inhibition of HIV-RT by ADRT-TP is competitive against TTP and is more potent against RNA to DNA synthesis (Ki = 0.009 microM versus Km = 3.3 microM for TTP) than it is against DNA to DNA synthesis (Ki = 0.95 microM versus Km = 16.3 microM for TTP). ADRT-TP is also a more potent inhibitor for primer elongation on RNA template than on DNA template. ADRT-TP is a poor inhibitor of human DNA polymerases alpha (Ki = 62.5 microM) and beta (Ki = 150 microM) (Chen et al., 1992). The consequences of ADRT incorporation into DNA are strikingly different for the HIV-RT and for human DNA polymerases alpha and beta. DNA polymerases alpha and beta incorporate a single ADRT-MP molecule into nascent DNA at a very slow rate and continue to elongate. They are unable to incorporate a second consecutive ADRT-MP. However, HIV-RT is able to efficiently incorporate two consecutive ADRT molecules. Incorporation of two consecutive ADRT-MP molecules by HIV-RT prevents further DNA chain elongation. Incorporation of two ADRT-MP molecules separated by one deoxyribonucleoside monophosphate (dAMP, dCMP, or dGMP) also abolishes DNA chain elongation by HIV-RT.

Synthesis and anti-HIV activity of 4'-azido- and 4'-methoxynucleosides.

A series of nucleosides were synthesized in which the 4'-hydrogen was substituted with either an azido or a methoxy group. The key steps in the syntheses of the 4'-azido analogues were the stereo- and regioselective addition of iodine azide to a 4'-unsaturated nucleoside precursor followed by an oxidatively assisted displacement of the 5'-iodo group. The 4'-methoxynucleosides were made via epoxidation of 4'-unsaturated nucleosides with in suit epoxide opening by methanol. Reaction-mechanism considerations, empirical conformation rules, NMR-based conformational calculations, and NOE experiments suggest that the 4'-azidonucleosides prefer a 3'-endo (N-type) conformation of the furanose moiety. When evaluated for their inhibitory effect on HIV in A3.01 cell culture, all the 4'-azido-2'-deoxy-beta-D-nucleosides exhibited potent activity. IC50's ranged from 0.80 microM for 4'-azido-2'-deoxyuridine (6c) to 0.003 microM for 4'-azido-2'-deoxyguanosine (6e). Cytotoxicity was detected at 50-1500 times the IC50's in this series. The 4'-methoxy-2'-deoxy-beta-D-nucleosides were 2-3 orders of magnitude less active and less toxic than their azido counterparts. Modifications at the 2'- or 3'-position of the 4'-substituted-2'-deoxynucleosides tended to diminish activity. Further evaluation of 4'-azidothymidine (6a) in H9, PBL, and MT-2 cells infected with HIV demonstrated a similar inhibitory profile to that of AZT. However, 4'-azidothymidine (6a) retained its activity against HIV mutants which were resistant to AZT.

Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses.

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.

Quantitative estimation of leishmanial antibody titers by enzyme-linked immunosorbent assay.

The response to immunization against Leishmania braziliensis in rabbits was followed by measurement of antibody by enzyme-linked immunosorbent assay (ELISA), passive hemagglutination, complement fixation, and countercurrent immunoelectrophoresis. Passive hemagglutination and complement-fixation titers were interpreted according to standard methods, whereas ELISA exact titers were derived by the equation Y = a-b log X, which describes the straight line that results when absorbances are plotted against test serum dilutions. Peak titer was measured at 32 +/- 1 days after initial injection, irrespective of the assay method. After seven days, the reciprocal antibody titer was 1,130 by ELISA, 0 by passive hemagglutination, and 8 by complement fixation. Precipitin bands were shown by countercurrent immunoelectrophoresis only at peak titer. With sera from hamsters experimentally infected with Leishmania, 94% showed leishmanial antibody by ELISA, compared with 92% by complement fixation and 65% by passive hemagglutination. When 31 samples of sera from human patients with cutaneous leishmaniasis were tested only by ELISA, 23 were positive for leishmanial antibodies.

Partial purification of amastigotes from cutaneous lesions of American leishmaniasis.

Amastigotes of Leishmania mexicana, L. mexicana amazonensis, L. brasiliensis, and L. enriettii were isolated from lesions in infected animals. Numbers of amastigotes recovered ranged from 1 X 10(7) to 7 X 10(8), depending on the strain of leishmania. Trypsinization disassociated the lesions and released the parasites. After 18 to 24 hr incubation at 37 C in tissue culture media with antibiotics, many of the intact host cells attached to the flask. Amastigotes were collected from the media in relatively pure preparations. Electron microscopy revealed no morphological alterations of the amastigotes and minimal contamination by membranes and cell fragments.