In vitro anti-human immunodeficiency virus activities of Z- and E-methylenecyclopropane nucleoside analogues and their phosphoro-L-alaninate diesters.

Nucleoside analogues with a Z- or an E-methylenecyclopropane moiety were synthesized and examined for activity against human immunodeficiency virus type 1 (HIV-1) in vitro. The addition of a methyl phenyl phosphoro-L-alaninate moiety to modestly active analogues resulted in potentiation of their anti-HIV-1 activity. Two such compounds, designated QYL-685 (with 2,6-diaminopurine) and QYL-609 (with adenine), were most potent against HIV-1 in vitro, with 50% inhibitory concentrations of 0.034 and 0.0026 microM, respectively, in MT-2 cell-based assays. Both compounds were active against zidovudine-resistant, didanosine-resistant, and multi-dideoxynucleoside-resistant infectious clones in vitro. Further development of these analogues as potential therapies for HIV-1 infection is warranted.     

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.     

GRL-02031, a novel nonpeptidic protease inhibitor (PI) containing a stereochemically defined fused cyclopentanyltetrahydrofuran potent against multi-PI-resistant human immunodeficiency virus type 1 in vitro

We generated a novel nonpeptidic protease inhibitor (PI), GRL-02031, by incorporating a stereochemically defined fused cyclopentanyltetrahydrofuran (Cp-THF) which exerted potent activity against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) isolates, including multidrug-resistant HIV-1 variants. GRL-02031 was highly potent against laboratory HIV-1 strains and primary clinical isolates, including subtypes A, B, C, and E (50% effective concentration [EC(50)] range, 0.015 to 0.038 microM), with minimal cytotoxicity (50% cytotoxic concentration, >100 microM in CD4(+) MT-2 cells), although it was less active against two HIV-2 strains (HIV-2(EHO) and HIV-2(ROD)) (EC(50), approximately 0.60 microM) than against HIV-1 strains. GRL-02031 at relatively low concentrations blocked the infection and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to 5 microM of saquinavir, amprenavir, indinavir, nelfinavir, or ritonavir and 1 microM of lopinavir or atazanavir (EC(50) range, 0.036 to 0.14 microM). GRL-02031 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to the conventional antiretroviral regimens that then existed, with EC(50)s ranging from 0.014 to 0.042 microM (changes in the EC(50)s were less than twofold the EC(50) for wild-type HIV-1). Upon selection of HIV-1(NL4-3) in the presence of GRL-02031, mutants carrying L10F, L33F, M46I, I47V, Q58E, V82I, I84V, and I85V in the protease-encoding region and G62R (within p17), L363M (p24-p2 cleavage site), R409K (within p7), and I437T (p7-p1 cleavage site) in the gag-encoding region emerged. GRL-02031 was potent against a variety of HIV-1(NL4-3)-based molecular infectious clones containing a single primary mutation reported previously or a combination of such mutations, although it was slightly less active against HIV-1 variants containing consecutive amino acid substitutions: M46I and I47V or I84V and I85V. Structural modeling analysis demonstrated a distinct bimodal binding of GRL-02031 to protease, which may provide advantages to GRL-02031 in blocking the replication of a wide spectrum of HIV-1 variants resistant to PIs and in delaying the development of resistance of HIV-1 to GRL-02031. The present data warrant the further development of GRL-02031 as a potential therapeutic agent for the treatment of infections with primary and multidrug-resistant HIV-1 variants.     

Nucleoside and Nucleotide Analogs Select in Culture for Different Patterns of Drug Resistance in Human Immunodeficiency Virus Types 1 and 2

Recent findings suggest bidirectional antagonisms between the K65R mutation and thymidine analogue mutations in human immunodeficiency virus type 1 (HIV-1)-infected, treatment-experienced patients, yet little is known about HIV-2 in this regard. This study addressed the effects of innate polymorphisms in HIV-2 on emergent resistance to nucleoside/nucleotide analogues. Emergent drug resistance profiles in HIV-2 subtypes A (n = 3) and B (n = 1) were compared to those of HIV-1 subtypes B and C. Drug resistance was evaluated with cord blood mononuclear cells (CBMCs) and MT2 cells, using selective pressure with tenofovir (TFV), zidovudine (ZDV), stavudine (d4T), didanosine (ddI), abacavir (ABC), lamivudine (3TC), emtricitabine (FTC), or various dual-drug combinations. Resistance was evaluated using conventional and ultrasensitive sequencing approaches. In agreement with our previous findings, dual-drug combinations of TFV, ddI, ABC, d4T, ZDV, and 3TC preferentially selected for K65R in HIV-1 subtype C isolates. In HIV-1 subtype B, TFV-3TC and ZDV-3TC selected for M184I and D67N, respectively. In contrast, selections with all four HIV-2 cultures favored the development of M184I in dual-drug combinations that included either 3TC or FTC. Since HIV-2 cultures did not develop K65R, an ultrasensitive allele-specific real-time PCR assay was developed to distinguish the presence of 65R from wild-type K65 after 16 cycles with a discriminatory ability of 0.1% against a population of wild-type virus. These results underscore potential differences in emergent drug resistance pathways in HIV-1 and HIV-2 and show that polymorphisms may influence the development of the resistance pathways that are likely to emerge.