A set of mutations [Ala-62 -> Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddl). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The Ki value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.

From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 M when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively. NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion.We prepared ϳ9,000 different extracts from solitary colonies obtained by incubation of bacterial samples from various sources, including soil specimens collected in Japan. The methanol extract from each bacterial colony was tested for antihuman immunodeficiency virus type 1 (HIV-1) activity by using a colorimetric assay employing a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide [MTT]) and MT4 cells (9). A crude extract of strain SKH-2344 (FERMP-14239), a Streptomyces species strain isolated from soil collected in Yamaguchi, was found to be active against HIV-1 (data not shown). The strain was then grown in culture medium and subjected to fermentation. On day 5 of fermentation, 20 liters of culture broth medium was centrifuged, and the supernatant was concentrated in vacuo to yield a paste, which was resuspended in 1 liter of methanol for 12 h. The methanol extract was then filtered and concentrated to yield a pale yellow oily specimen (fraction A). The pellet (the mycelium) was homogenized in 10 liters of methanol with agitation for 12 h, and the filtered solution was concentrated in vacuo to yield a yellow oily specimen (fraction B). Fractions A and B were combined, dissolved in water-saturated butanol, and concentrated in vacuo. The extract was column chromatographed on silica gel with ethyl acetate-methanol-water (10:5:0.25) and purified (Ͼ99%) by high-performance liquid chromatography with CH 3 CN-H 2 O-CF 3 COOH (38:62:0.1) to yield a white powder (100 mg), designated NP-06.Amino acid analysis of the NP-06 hydrolysate revealed the presence of one residue each of Ser, Ile, Leu, and Tyr; two residues each of Phe, Ala, Val, and Asp; and four residues each of Cys and Gly, although whether the two Asp residues represented Asn was to be confirmed. Thin-layer chromatography analysis of the hydrazine hydrolysate of NP-06 revealed that the C terminus was Trp. NP-06 could not be sequenced by the Edman degradation method, although the physical properties of NP-06 showed that it was a peptide. Therefore, nuclear magnetic resonance (NMR) spectroscopy was used to determine the amino acid sequence of NP-06. Inspection of the relayed signals arising from the amide resonances in a homonuclear Hartmann-Hahn spectroscopy experiment revealed the presence of 21-amino-acid spin systems. One Leu, one Ile, one Ser, two Val, two Ala,...

We determined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activities of various dideoxynucleoside analogs by using phytohemagglutinin-activated peripheral blood mononuclear cells (PHA-PBMs) and resting PBMs (R-PBMs) as target cells. The comparative order of anti-HIV-1 activity in PHA-PBMs was azidothymidine (AZT) > dideoxycytidine (ddC) > dideoxythymidinene (d4T) > dideoxyinosine (ddI) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) > 2-␤-fluoro-dideoxyadenosine (F-ara-ddA), while that in R-PBMs was ddC > ddI, PMEA, and F-ara-ddA, >> AZT and d4T. A pronucleotide, bis-(S-acetylthioethanol)phosphotriester-ddAMP, which bypasses the anabolic monophosphorylation step for the intracellular delivery of ddAMP, was highly active both in PHA-PBMs and R-PBMs. These data may have basic and clinical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy with dideoxynucleosides, and in the development of active pronucleotides.

The changes in viremia levels and the development of drug-related mutations were examined in 26 patients with symptomatic human immunodeficiency virus type 1 (HIV-1) infection participating in a randomized trial comparing alternating (A) and simultaneous (S) regimens of zidovudine and didanosine therapy. Patients on both arms had significant reduction in serum RNA copies from baseline throughout the 2 years of study. Significant differences between the two arms were demonstrated over the first 2-3 months of therapy. Analyses with nested polymerase chain reaction revealed that the emergence of the didanosine-related position 74 Leu-->Val mutation was significantly blocked in both regimens, while the zidovudine-related mutation at codon 215 was not affected. Determination of the overall durability of the antiviremic effect of the A and S regimens of zidovudine and didanosine and clinical implications of the results require further research.

The syntheses and biological activities of fluorobutynol 11 and (E)- and (Z)-fluorobutenols 8a,d and 9a,d are described. Alkylation of adenine with bromofluorobutyne 13a afforded intermediate 14 which was converted to fluorobutynol 11. Aldehyde 16a and (carbethoxyfluoromethyl)-triphenylphosphonium bromide furnished (E)- and (Z)-fluorobutenoates 19a and 20a accompanied by regioisomer 21a. A similar reaction of compound 16d afforded Z- and E-esters 19d and 20d. Reduction of the mixture of 19a and 20a with DIBALH gave (E)- and (Z)-fluoroalkenols 8a and 9a. Similarly, the Z-ester 19d gave (Z)-fluoroalkenol 9d. Both 19d and 20d were reduced with NaBH4 to give (Z)- and (E)-fluoroalkenols 9d and 8d. Hydrogenation of 19a and 20a afforded fluoro ester 23. A similar reduction of 8a and 9a led to fluoro alcohol 24 and the defluorinated product 25 which were separated by chromatography on a Bio-Rad AG 1-X2 (OH-) column. (Z)-Fluorobutenol 9a is a substrate for adenosine deaminase, whereas the E-isomer 8a is inert toward the enzyme. By contrast, analogue 8a inhibited the replication and cytopathic effect of HIV-1 in ATH8 cells with an IC50 of approximately 100 microM, but the Z-isomer 9a was inactive. This effect was accompanied by 36% cytotoxicity at 100 microM. Compounds 11 and 8d inhibited the growth of murine leukemia L1210 culture with IC50 = 89 and 60 microM, respectively.

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