Selection and characterization of HIV-1 with a novel S68 deletion in reverse transcriptase.

Resistance to human immunodeficiency virus type 1 (HIV-1) represents a significant problem in the design of novel therapeutics and the management of treatment regimens in infected persons. Resistance profiles can be elucidated by defining modifications to the viral genome conferred upon exposure to novel nucleoside reverse transcriptase (RT) inhibitors (NRTI). In vitro testing of HIV-1LAI-infected primary human lymphocytes treated with ß-D-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (DFC; Dexelvucitabine; Reverset) produced a novel deletion of AGT at codon 68 (S68Δ) alone and in combination with K65R that differentially affects drug response. Dual-approach clone techniques utilizing TOPO cloning and pyrosequencing confirmed the novel S68Δ in the HIV-1 genome. The S68Δ HIV-1 RT was phenotyped against various antiviral agents in a heteropolymeric DNA polymerase assay and in human lymphocytes. Drug susceptibility results indicate that the S68Δ displayed a 10- to 30-fold increase in resistance to DFC, lamivudine, emtricitabine, tenofovir, abacavir, and amdoxovir and modest resistance to stavudine, ß-d-2',3'-oxa-5-fluorocytidine, or 9-(ß-D-1,3-dioxolan-4-yl)guanine and remained susceptible to 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine (ddI), 1-(ß-D-dioxolane)thymine (DOT) and lopinavir. Modeling revealed a central role for S68 in affecting conformation of the ß3-ß4 finger region and provides a rational for the selective resistance. These data indicate that the novel S68Δ is a previously unrecognized deletion that may represent an important factor in NRTI multidrug resistance treatment strategies.

5'-O-Aliphatic and amino acid ester prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT): synthesis, anti-HIV activity, cytotoxicity and stability studies.

A series of (-)-beta-D-(2R,4R)-dioxolane-thymine-5'-O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1(LAI) in human peripheral blood mononuclear (PBM) cells as well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was observed for the compound 2-4 (5'-O-aliphatic acid esters) without increase in cytotoxicity in comparison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0 and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the half-lives from 12.3 to 48.0 min.

Synthesis and anti-HIV activity of 5-haloethynyl and 5-(1,2-dihalo)vinyl analogues of AZT and FLT.

In this paper, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleoside analogues of the anti-HIV AZT, and FLT drugs. The key step of those syntheses is a Pd(0) cross-coupling at C5 position under Sonogashira conditions. Finally, based on their in vitro anti-HIV activities and their cytotoxicity on PBM, CEM, and VERO cell lines, the best compounds were the 2',3'-dideoxy-3'-fluoro-5-(bromo-2-iodo)vinyluridine (10b, EC50 of 0.6 µM), and the 3'-azido-2',3'-dideoxy-5-(bromo-2-iodo)vinyluridine (16b, EC50 of 1.1 µM).

D- and L-2',3'-didehydro-2',3'-dideoxy-3'-fluoro-carbocyclic nucleosides: synthesis, anti-HIV activity and mechanism of resistance.

Introducing 2'-fluoro substitution on the 2',3'-double bond in carbocyclic nucleosides has provided biologically interesting compounds with potent anti-HIV activity. As an extension of our previous works in the discovery of anti-HIV agents, D- and L-2',3'-unsaturated 3'-fluoro carbocyclic nucleosides were synthesized and evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells. Among the synthesized L-series nucleosides, compounds 18, 19, 26 and 28 exhibited moderate antiviral activity (EC50 7.1 microM, 6.4 microM, 10.3 microM, and 20.7 microM, respectively), while among the D-series, the guanosine analogue (35, D-3'-F-C-d4G) exhibited the most potent anti-HIV activity (EC50 0.4 microM, EC90 2.8 microM). However, the guanosine analogue 35 was cross-resistant to the lamivudine-resistant variants (HIV-1M184V). Molecular modeling studies suggest that hydrophobic interaction as well as hydrogen-bonding stabilize the binding of compound 35 in the active site of wild type HIV reverse transcriptase (HIV-RT). In the case of L-nucleosides, these two effects are opposite which results in a loss of binding affinity. According to the molecular modeling studies, cross-resistance of D-3'-F-C-d4G (35) to M184V mutant may be caused by the realignment of the primer and template in the HIV-RTM184V interaction, which destabilizes the RT-inhibitor triphosphate complex, resulting in a significant reduction in anti-HIV activity of the D-guanine derivative 35.

Synthesis, antiviral activity, and mechanism of drug resistance of D- and L-2',3'-didehydro-2',3'-dideoxy-2'-fluorocarbocyclic nucleosides.

Carbocyclic nucleosides have received much attention due to their interesting biological activity and metabolic stability. Among nucleoside analogues, a fluorine substitution on the carbohydrate moiety or introduction of a 2',3'-unsaturated structure motif has been proven to be successful in producing effective antiviral agents. By combining these structural features, both D- and L-2',3'-dideoxy-2',3'-didehydro-2'-fluoro-carbocyclic nucleosides (D- and L-2'F-C-d4Ns) were synthesized as potential anti-HIV agents. The target D- and L-carbocyclic nucleosides were both stereospecifically synthesized from D-ribose. The structure-activity relationships of synthesized compounds against HIV-1 in activated human peripheral blood mononuclear (PBM) cells were studied, from which we found that the L-2',3'-dideoxy-2'3'-didehydro-2'-fluoroadenosine analogue (L-2'F-C-d4A) 46 showed potent anti-HIV activity (EC50 = 0.77 microM), although it is cross-resistant to the lamivudine-resistant variant (HIV-1M184V). Modeling studies demonstrated a good correlation between calculated relative binding energies and activity/resistance data. The modeling study also indicated that an additional hydrogen bond and a favorable van der Waals interaction contribute to the higher antiviral activity of L-2'F-C-d4A in comparison to its D-counterpart. Also, like other L-nucleosides, the unfavorable steric hindrance of the sugar moiety of L-2'F-C-d4A and the side chain of Val184 could explain the cross-resistance of L-2'F-C-d4A with the M184V mutant. The significant difference of antiviral activity between carbovir and its analogue L-2'F-C-d4G 25 may be due to distortion of the phenyl ring of Tyr115 in the L-2'F-C-d4G-TP/HIV-RT complex, which resulted in a poor pi-pi interaction.

Differential induction of interleukin-I beta mRNA in the brain parenchyma of Lewis and Fischer rats after peripheral injection of lipopolysaccharides.

The expression of interleukin-1 beta (IL-1beta) mRNA was compared in the brain of inflammatory susceptible LEW/N and resistant F344/N rats at 3, 6, and 12 h after peripheral administration of lipopolysaccharide (LPS) or saline. No differences between strains were observed in the circumventricular organs (CVOs) and choroid plexus. At 12 h after LPS administration, increased IL-1beta mRNA expression was detected in the hypothalamus of LEW/N rats. In contrast, increased IL-1beta mRNA expression was detected in the cerebral cortex of F344/N rats. These data show region-specific differences of IL-1beta mRNA expression in the brain of these rat strains that differ in their susceptibility to inflammation.

Synthesis, antiviral activity, and stability of nucleoside analogs containing tricyclic bases.

A series of 3,9-dihydro-9-oxo-5H-imidazo[1,2-A]purine nucleosides (tricylic nucleosides) were synthesized from 9-[4-alpha-(hydroxymethyl)cyclopent-2-ene-1-alpha-yl]guanine (CBV) 5, (-)-beta-D-(2R,4R)-1,3-dioxolane-guanosine (DXG) 6, 3'-azido-3'-deoxy-guanosine (AZG) 7, and 2'-C-methylguanosine 8. Their in vitro activity against HIV and HCV was evaluated and correlated to their ability to degrade to their purine counterpart.

Pre-steady-state kinetic studies establish entecavir 5'-triphosphate as a substrate for HIV-1 reverse transcriptase.

The novel 2'-deoxyguanosine analog Entecavir (ETV) is a potent inhibitor of hepatitis B virus (HBV) replication and is recommended for treatment in human immunodeficiency virus type 1 (HIV-1) and HBV-co-infected patients because it had been reported that ETV is HBV-specific. Recent clinical observations, however, have suggested that ETV may indeed demonstrate anti-HIV-1 activity. To investigate this question at a molecular level, kinetic studies were used to examine the interaction of 5'-triphosphate form of ETV with wild type (WT) HIV-1 reverse transcriptase (RT) and the nucleoside reverse transcriptase inhibitor-resistant mutation M184V. Using single turnover kinetic assays, we found that HIV-1 WT RT and M184V RT could use the activated ETV triphosphate metabolite as a substrate for incorporation. The mutant displayed a slower incorporation rate, a lower binding affinity, and a lower incorporation efficiency with the 5'-triphosphate form of ETV compared with WT RT, suggesting a kinetic basis for resistance. Our results are supported by cell-based assays in primary human lymphocytes that show inhibition of WT HIV-1 replication by ETV and decreased susceptibility of the HIV-1 containing the M184V mutation. This study has important therapeutic implications as it establishes ETV as an inhibitor for HIV-1 RT and illustrates the mechanism of resistance by the M184V mutant.

Synthesis, cytotoxicity, and antiviral activities of new neolignans related to honokiol and magnolol.

A series of new bisphenol derivatives bearing allylic moieties were synthesized as potential analogs of honokiol and/or magnolol. Certain compounds exhibited specific anti-proliferation activity against SVR cells and moderate anti-HIV-1 activity in primary human lymphocytes. Compound 5h was the most potent compound and its anti-tumor activity was evaluated in vivo.

Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil nucleosides: antiviral activity and cellular toxicity.

In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the 2'-deoxy, 3'-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-2-iodo)vinyluridine (SI > 2.8).