Resistance of human immunodeficiency virus type 1 to acyclic 6-phenylselenenyl- and 6-phenylthiopyrimidines

Schinazi, Raymond F.; Shi, Chaofu; Goudgaon, Ν. M.; McKenna, Philip M.; Mellors, John W.

doi:10.1128/aac.38.10.2409

Cited by 21 publications

(18 citation statements)

References 34 publications

(38 reference statements)

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“…Stock preparations of HIV-1 LAI were prepared as described previously (21). Briefly, MT-2 cells were electroporated with 10 g of plasmid DNA that encodes the HIV-1 LAI proviral clone.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Selection of Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Decrease Susceptibility to (−)-β- d -Dioxolane-Guanosine and Suppress Resistance to 3′-Azido-3′-Deoxythymidine

Bazmi

Hammond

Cavalcanti

et al. 2000

Antimicrob Agents Chemother

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Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (؊)-␤-D-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1 LAI in increasing drug concentrations (maximum concentration, 30 M). Two independent selection experiments were performed. Viral isolates for which the DXG median effective concentrations (EC 50 s) increased 7.3-and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1 LAI . The K65R mutation also conferred greater than threefold cross-resistance to 2,3-dideoxycytidine, 2,3-dideoxyinosine, 2,3-dideoxy-3-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3-azido-3-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2,3-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (؊)-␤-D-2,6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.

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“…Stock preparations of HIV-1 LAI were prepared as described previously (21). Briefly, MT-2 cells were electroporated with 10 g of plasmid DNA that encodes the HIV-1 LAI proviral clone.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Selection of Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Decrease Susceptibility to (−)-β- d -Dioxolane-Guanosine and Suppress Resistance to 3′-Azido-3′-Deoxythymidine

Bazmi

Hammond

Cavalcanti

et al. 2000

Antimicrob Agents Chemother

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“…WT and AZT R virus stocks, generated from plasmid DNA encoding an infectious proviral clone of HIV-1 LAI (19,20), were prepared in MT-2 cells and subjected to titer determination as previously described (21). Antiviral activity was determined by inoculating MT-2 cells with WT or AZT R virus stocks (multiplicity of infection, 0.01 50% tissue culture infective dose/cell) followed by incubation in the presence of 2-or 3-fold serial dilutions of 3Ј-azido-2Ј,3Ј-ddN.…”

Section: Electrophoretic Analysis Of Rt Polymerization Products Formementioning

confidence: 99%

The 3′-Azido Group Is Not the Primary Determinant of 3′-Azido-3′-deoxythymidine (AZT) Responsible for the Excision Phenotype of AZT-resistant HIV-1

Sluis‐Cremer

Arion

Parikh

et al. 2005

Journal of Biological Chemistry

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The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3-azido-3-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2,3-dideoxynucleoside-5-monophosphate (2,3-ddNMP) analogs that lack a 3-azido moiety. This led to the hypothesis that the 3-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZT R ) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3-azido-2,3-ddN including 3-azido-2,3-ddA (AZddA), 3-azido-2,3-ddC (AZddC), 3-azido-2,3-ddG (AZddG), AZT, and 3-azido-2,3-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3-azido group on the 3-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZT R RT for all 3-azido-ddNMP analogs. Thus, the 3-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations. The replication of human immunodeficiency virus (HIV)1 1 is dependent on the enzymatic activities of reverse transcriptase (RT), an RNA-and DNA-dependent DNA polymerase encoded by the viral pol gene. RT synthesizes the double-stranded proviral DNA precursor from the viral (ϩ) RNA genome (1). Because of its essential role in HIV-1 replication, RT is a major target for antiretroviral drug development and two structurally dissimilar classes of RT inhibitors, termed nucleoside RT inhibitors (NRTI) and nonnucleoside RT inhibitors, are routinely used for the clinical treatment of HIV-1-infected individuals (2). NRTI are 2Ј-deoxyribonucleoside analogs that usually lack a 3Ј-OH group on the ribose moiety. After intracellular conversion to the active 5Ј-triphosphate form, NRTI-TP inhibit DNA synthesis by competing with the natural nucleotides both for recognition by RT as a substrate and by incorporation into the nascent viral DNA chain (3). Incorporation of an NRTI into the nascent viral DNA chain by RT results in termination of DNA synthesis.As is the case with all antiretroviral agents, the emergence of drug-resistant HIV-1 variants limits the efficacy of NRTI. Most NRTI-resistant viruses isolated from patients treated with nucleoside analogs have mutations in the pol gene (4). To date, two major phenotypic mechanisms have been proposed to account for HIV-1 resistance to NRTIs (5, 6). One mechanism is NRTI discrimination in...

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“…Wild-type (WT) and mutant virus stocks were prepared in MT-2 cells and subjected to titer determination as previously described (31,37).…”

Section: Cells and Virusesmentioning

confidence: 99%

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Meyer

Matsuura

Zonarich

et al. 2003

J Virol

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Phosphonoformate (foscarnet) is a pyrophosphate (PP i ) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP i binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP i or a nucleotide such as ATP) which also interacts with the PP i binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3-azido-3-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP i , and ATP. Alternatively, the binding position or orientation of PP i , ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.Phosphonoformate (foscarnet) inhibits a wide variety of DNA and RNA polymerases including human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (40, 43). Foscarnet competes with pyrophosphate (PP i ) for binding in PP i exchange reactions, suggesting that foscarnet interacts with the site on the enzyme where PP i is formed (7, 38). On structural grounds, the site of interaction for PP i (and, presumably, for foscarnet) should lie within the deoxynucleoside triphosphate (dNTP) binding site since PP i is formed from the ␤ and ␥ phosphates of the dNTP substrate, but foscarnet inhibition of DNA synthesis in vitro is noncompetitive with dNTP substrates (58), indicating that foscarnet and dNTP bind to distinguishable forms of RT. In addition, median-effect analysis comparing foscarnet and 3Ј-azido-3Ј-deoxythymidine (AZT) triphosphate (AZTTP) alone or as mixtures has shown that inhibition by these compounds is mutually exclusive (10, 22, 50), implying that binding of either inhibitor prevents binding of the other to the same enzyme molecule.In intact cells, the inhibition of HIV-1 by foscarnet and AZT is synergistic (10,22). The mechanism of this synergy is unclear, but it has recently been demonstrated that AZT monophosphate (AZTMP) incorporation can be reversed by the primer-unblocking activity of RT, in which the chain-terminating residue is trans...

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Resistance of human immunodeficiency virus type 1 to acyclic 6-phenylselenenyl- and 6-phenylthiopyrimidines

Cited by 21 publications

References 34 publications

In Vitro Selection of Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Decrease Susceptibility to (−)-β- d -Dioxolane-Guanosine and Suppress Resistance to 3′-Azido-3′-Deoxythymidine

In Vitro Selection of Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Decrease Susceptibility to (−)-β- d -Dioxolane-Guanosine and Suppress Resistance to 3′-Azido-3′-Deoxythymidine

The 3′-Azido Group Is Not the Primary Determinant of 3′-Azido-3′-deoxythymidine (AZT) Responsible for the Excision Phenotype of AZT-resistant HIV-1

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

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