In Vitro Induction of Human Immunodeficiency Virus Type 1 Variants Resistant to Phosphoralaninate Prodrugs of Z-Methylenecyclopropane Nucleoside Analogues

Two methylenecyclopropane nucleoside analogues with a phenylphosphoralaninate moiety, QYL-685 and QYL-609, exert potent and specific activities against human immunodeficiency virus type 1 strain LAI (HIV-1(LAI)) and HIV-2 in vitro. In this study, we induced HIV-1 variants resistant to QYL-685 by exposing HIV-1(LAI) to increasing concentrations of QYL-685. After 16 passages, the virus (HIV-1(P16)) was less sensitive to QYL-685 (104-fold), QYL-609 (>41-fold), and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) (>1, 100-fold) than was HIV-1(LAI) and contained an M184I mutation. Two infectious clones, HIV-1(M184I) and HIV-1(M184V), were resistant to QYL-685, QYL-609, and 3TC, confirming that the M184I mutation was responsible for the observed resistance. Viral-fitness analyses (competitive HIV-1 replication assays) revealed that in the absence of drugs, M184I and M184V conferred a replication disadvantage on the virus compared to the replication efficiency of the wild-type infectious clone (HIV-1(wt)). However, in the presence of QYL-685 (4 microM), HIV-1(M184I) and HIV-1(M184V) showed greater fitness than HIV-1(wt). These data may provide structural and virological relevance with regard to the emergence of M184I and M184V substitutions in HIV-1.     

Anti-human immunodeficiency virus type 1 activity and in vitro toxicity of 2''-deoxy-3''-thiacytidine (BCH-189), a novel heterocyclic nucleoside analog.

We describe a novel nucleoside analog, 2'-deoxy-3'-thiacytidine (BCH-189), in which the 3' carbon of the ribose ring of 2'-deoxycytidine has been replaced by a sulfur atom. In MT-4 T cells, this compound had significant time- and dose-dependent antiviral activity against five different strains of human immunodeficiency virus type 1 (HIV-1) (mean 50% inhibitory dose, 0.73 microM); known 3'-azido-3'-deoxythymidine (AZT)-resistant HIV-1 variants did not exhibit cross-resistance to it. BCH-189 also suppressed HIV-1 replication in the U937 monocytoid cell line as well as in primary cultures of human peripheral blood mononuclear cells; in these latter systems, suppression was fuller and longer lasting than that induced by AZT. Moreover, BCH-189 was less toxic than AZT in cell culture. BCH-189 may be a promising drug for the treatment of HIV-1-associated disease.     

N4-acyl-modified D-2',3'-dideoxy-5-fluorocytidine nucleoside analogues with improved antiviral activity

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.     

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.