2--3--dideoxyguanosine-5--triphosphate and deoxyguanosine-triphosphate

Mutations that confer resistance to nucleoside analogs do not cluster around the deoxynucleotide triphosphate (dNTP) binding site. Instead, these mutations appear to lie along the groove in the enzyme where the template-primer binds. Based on such structural data and on complementary biochemical analyses, it has been suggested that resistance to nucleoside analogs involves repositioning of the template-primer. We have prepared mutations in HIV-2 RT that are the homologs of mutations that confer resistance to nucleoside analogs in HIV-1 RT. Analysis of the behavior of HIV-2 RT mutants (Leu74Val, Glu89Gly, Ser215Tyr, Leu74Val/Ser215Tyr and Glu89Gly/Ser215Tyr) in vitro confirms the results obtained with HIV-1 RT: resistance is a function of the length of the template overhang. These analyses also suggest that the homolog in HIV-2 RT of one of the mutations that confers resistance to AZT in HIV-1 RT (Thr215Tyr) confers resistance by repositioning of the template-primer.

Topics: Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; DNA Primers; DNA, Viral; Drug Resistance, Microbial; HIV Reverse Transcriptase; HIV-1; HIV-2; Humans; Models, Molecular; Mutation; Recombinant Proteins; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Templates, Genetic; Thymine Nucleotides

Carbovir (the carbocyclic analog of 2'-3'-didehydro-2',3'-dideoxyguanosine) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. Assays were developed to assess the mechanism of inhibition by the 5'-triphosphate of carbovir of HIV-1 reverse transcriptase using either RNA or DNA templates that contain all four natural nucleotides. Carbovir-TP was a potent inhibitor of HIV-1 reverse transcriptase using either template with Ki values similar to that observed by AZT-TP, ddGTP, and ddTTP. The kinetic constants for incorporation of these nucleotide analogs into DNA by HIV-1 reverse transcriptase using either template were similar to the values seen for their respective natural nucleotides. In addition, the incorporation of either carbovir-TP or AZT-TP in the presence of dGTP or dTTP, respectively, indicated that the mechanism of inhibition by these two nucleotide analogs was due to their incorporation into the DNA resulting in chain termination. Carbovir-TP was not a potent inhibitor of DNA polymerase alpha, beta, or gamma, or DNA primase. Given the potent activity of carbovir-TP against HIV-1 reverse transcriptase and its lack of activity against human DNA polymerases, we believe that further evaluation of this compound as a potential drug for the treatment of HIV-1 infection is warranted.

Topics: Antiviral Agents; Base Sequence; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; HIV-1; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Reverse Transcriptase Inhibitors; RNA; Templates, Genetic; Thymine Nucleotides; Zidovudine

Interactions of tubulin with a number of guanine nucleotides modified at the 2' and 3' ribose hydroxyls were examined. Deoxy analogs of GTP were equal or superior to GTP in supporting tubulin polymerization, but analogs bearing either methyl or phosphate groups on the hydroxyls had significantly reduced ability to support polymerization. These substituted GTP analogs were hydrolyzed at the 5'-gamma-phosphate position, although less rapidly than GTP, at rates exceeding those of polymerization. GTP hydrolysis, however, was closely coupled to polymerization. Moreover, the partially active GTP analogs were not effective inhibitors of GTP-dependent polymerization. These data indicate that the substituted GTP analogs have reduced affinity for tubulin at the exchangeable site because of steric factors. No deoxy or substituted GDP analog was as effective as GDP itself in inhibiting GTP-supported tubulin polymerization. Furthermore, there was no apparent relationship between the ability of nucleoside 5'-triphosphates to support polymerization and that of nucleoside 5'-diphosphates to inhibit the reaction. These findings suggest that GTP and GDP may actually bind to different, mutually exclusive sites rather than to a single exchangeable site.

Topics: Animals; Binding Sites; Brain; Cattle; Deoxyguanine Nucleotides; Dideoxynucleotides; Glutamates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Substrate Specificity; Tubulin; Vinblastine