alpha-P-borano ddNTP analogs: Substrate/inhibitory properties for kinases and polymerases and inhibition of wild type and drug resistant HIV replication.

摘要

Ever since human immunodeficiency virus (HIV) was identified as the causative agent of acquired immunodeficiency syndrome (AIDS), many efforts have been made towards finding a cure. Presented herein is the kinetic and biochemical evaluation of a new class of anti-HIV nucleotide analogs that contain alpha- P-borano modifications.;Antiviral alpha-P-borano substituted dideoxy nucleoside triphosphates (ddNTPalphaBs) are chain terminating nucleotide analogs that show promise for HIV therapy by targeting HIV reverse transcriptase (RT). In order for nucleoside analogs to become active substrates for HIV-RT, they must be phosphorylated by intracellular kinases to their corresponding triphosphates. Here, the effects of nucleobase, ribose, and alpha-phosphate substitutions on substrate specificities, along with the stereospecificity of inhibition and phosphorylation by creatine (CK) and pyruvate kinases (PK), were investigated. CK and PK showed opposite stereospecificities with respect to alpha- Pborano modification and a preference of binding of ddNTPs was observed for CK. Also, the alpha-P-borano analog of the active triphosphate of D4T, a drug currently used for treatment of HIV, Rp-D4T-TPalphaB, was enzymatically synthesized utilizing the stereospecificity of nucleoside diphosphate kinase (NDPK).;Inhibition of the RNA-directed DNA synthesis by the Rp-D4T-TPalphaB isomer was shown to be 30-fold and 4-fold greater than D4T-TP in drug resistant and wild type HIV-RT lysates, respectively. In addition, a 4'-C-methyl-TTPalphaB analog was found to be a chain terminator and was incorporated into DNA by wild type HIV-RT with only a 2-3 fold lower efficiency than natural TTP. Further, the anti-HIV effect of the Rp-D4T-TPalphaB isomer was investigated in HIV-infected cells using Nanogel delivery systems. An increased inhibition of drug resistant HIV strains was again observed with the Rp-D4T-TPalphaB isomer as compared to D4T-TP.;Lastly, because nucleoside analog toxicity has been linked to nucleoside analog triphosphate incorporation into mitochondrial DNA, the incorporation by human DNA Pol gamma was studied. It was shown that alpha-P -borano substituted TTP analogs were incorporated into DNA with similar efficiencies as their parent analogs. These studies demonstrate the potential of alpha-P-borano nucleotide analogs as a useful class of HIV-RT inhibitors to specifically target drug resistant HIV-RT.