Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a ß-thujaplicinol analogue.

EFV (efavirenz) and ß-thujaplicinol [2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] have contrasting effects on the RNase H activity of HIV-1 RT (reverse transcriptase). EFV binds in the non-nucleoside inhibitor-binding pocket and accelerates this activity, whereas ß-thujaplicinol binds in the RNase H active site and inhibits it. We have used pre-steady-state kinetic analyses to gain an insight into the mechanism by which EFV and a ß-thujaplicinol analogue [19616 (2,7-dihydroxy-2,4,6-cyclo-heptatrien-1-one)] modulate RT RNase H activity. Our data show that EFV and 19616 have no effect on polymerase-dependent RNase H cleavages. However, both compounds significantly affected the rates of polymerase-independent RNase H cleavages. In regard to the latter, we found no evidence that the bound RNA/DNA template/primer substrate restricted 19616 from interacting with RT. In light of these data, we propose a model in which 19616 binds to the RNase H active site of RT after the primary polymerase-dependent RNase H cleavage has occurred and stabilizes the 3'-end of the DNA primer in the polymerase active site thus blocking the enzyme's ability to carry out the polymerase-independent cleavages. By contrast, EFV destabilizes the 3'-end of the DNA primer in the DNA polymerase active site and promotes RT-mediated polymerase-independent cleavages. Consistent with this model, we show antagonism between EFV and 19616.

N348I in HIV-1 reverse transcriptase counteracts the synergy between zidovudine and nevirapine.

The efficacy of regimens that include both zidovudine and nevirapine can be explained by the synergistic interactions between these drugs. N348I in HIV-1 reverse transcriptase confers decreased susceptibility to zidovudine and nevirapine. Here, we demonstrate that N348I reverses the synergistic inhibition of HIV-1 by zidovudine and nevirapine. Also, the efficiency of zidovudine-monophosphate excision in the presence of nevirapine is greater for N348I HIV-1 reverse transcriptase compared with the wild-type enzyme. These data help explain the frequent selection of N348I in regimens that contain zidovudine and nevirapine, and suggest that the selection of N348I should be monitored in resource-limited settings where these drugs are routinely used.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Synthesis, antiviral activity, cytotoxicity and cellular pharmacology of l-3'-azido-2',3'-dideoxypurine nucleosides.

Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified l-3'-azido-2',3'-dideoxypurine nucleosides. These l-nucleoside analogs were evaluated against HIV and hepatitis B virus. The l-3'-azido-2',3'-dideoxypurines nucleosides were metabolized to nucleoside 5'-triphosphates in primary human lymphocytes, but exhibited weak or no antiviral activity against HIV-1. The nucleosides were also inactive against HBV in HepG2 cells. Pre-steady state kinetic experiments demonstrated that the l-3'-azido-2',3'-dideoxypurine triphosphates could be incorporated by purified HIV-1 reverse transcriptase, although their catalytic efficiency (k(pol)/K(d)) of incorporation was low. Interestingly, a phosphoramidate prodrug of l-3'-azido-2',3'-dideoxyadenosine exhibited anti-HIV-1 activity without significant toxicity.

Synthesis and anti-HIV evaluation of 3'-triazolo nucleosides.

A series of hitherto unknown 3'-α-[1,2,3]-substituted triazolo-2',3'-dideoxypyrimidine nucleoside analogues of the anti-HIV 3'-azido-3'-deoxythymidine (AZT) were synthesized through catalyzed alkyne-azide 1,3-dipolar cycloaddition (Huisgen reaction). Those 3'-[1,2,3]-triazolo analogues bearing an azido alkyl chain were evaluated for their anti-HIV activity against HIV-1 in primary human lymphocytes as well as for their cytotoxicity in different cells. None of them inhibit HIV replication (EC(50) > 20 µM); two of them were converted to their triphosphate form to evaluate their HIV-RT inhibition.

The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization.

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

The human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitor resistance mutation I132M confers hypersensitivity to nucleoside analogs.

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice.

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.