The rapid replication of HIV-1 and the errors made during viral replication, cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular drugs that are effective against the current drug-resistant strains of HIV-1.In the absence of an effective vaccine, drugs are the only therapeutic tools that can be used to treat HIV-1 infections. Unfortunately, HIV-1 infections cannot be cured, so that drug therapy, once initiated, must be continued for the life of the patient. This places a special burden on the design of anti-HIV drugs: They need to be relatively nontoxic so that they can be used in longterm therapy. HIV-1 replication is error prone (1) (and references within) and the errors that arise during the viral life cycle, together with the rapid replication of the virus in patients, allows the virus to escape the host's immune system and develop resistance to all of the available drugs (2). The virus evolves sufficiently rapidly that, unless the therapy is well-© 2008 Elsevier Ltd. All rights reserved.Correspondence: Professor Eddy Arnold, Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854-5627, USA, Email: arnold@cabm.rutgers.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. designed, resistance will develop in all treated patients. The only way to stop the development of resistance is to completely block viral replication; this, in turn, stops the evolution of resistance. It takes ...
The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.
Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.
Variants of human immunodeficiency virus type 1 that display 500- to 1,000-fold resistance to the (-) enantiomer of 2'-deoxy-3'-thiacytidine and approximately 4- to 8-fold resistance to 2',3'-dideoxycytidine and 2',3'-dideoxyinosine have been generated through in vitro selection with the former compound. The polymerase regions of several of these resistant viruses shared a codon alteration at site 184 (ATG-->GTG; methionine-->valine), a mutation previously associated with low-level resistance to 2',3'-dideoxycytidine. The biological relevance of this mutation for the (-) enantiomer of 2'-deoxy-3'-thiacytidine was confirmed by site-directed mutagenesis with the HXB2-D clone of human immunodeficiency virus type 1.
The technique of in vitro selection was used to generate variants of the human immunodeficiency virus type 1 that are resistant to 2',3'-dideoxycytidine (ddC). Most of the pol regions of such viruses, including the complete reverse transcriptase open reading frame and portions of flanking protease and integrase genes, were cloned and sequenced, using PCR-based procedures. Mutations were variously detected at amino acid site 65 (Lys-*Arg; AAA--AGA) and at a previously reported codon, site 184 (Met->Val; ATG->GTG). We introduced the site 65 mutation into the pol gene of infectious, cloned HxB2-D DNA by site-directed mutagenesis in order to confirm by viral replication assay the importance of this site in conferring resistance to ddC. The recombinant virus possessed greater than 10-fold resistance against this compound in conmparison with parental HxB2-D. Cross-resistance of approximately 20-and 3-fold, respectively, was detectable against the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine and 2',3'-dideoxyinosine but not against 3'-azido-3'-deoxythymidine. Combinations of the site 65 and 184 mutations did not yield levels of resistance higher than those attained with the site 65 mutation alone. The presence of the site 65 mutation was confirmed by PCR analysis of peripheral blood mononuclear cells from patients on long-term ddC therapy in 4 of 11 cases tested. Viruses that possessed a ddC resistance phenotype were isolated from subjects whose viruses contained the site 65 mutation in each of four instances. Four of these clinical samples were also demonstrated to possess the Met-184-Wal mutation, and one of them possessed both the Lys-65->Arg and Met-184-Wal substitutions.Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from these individuals.
Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), a nucleoside analog that retains a 3-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC 50 of 0.05 nM, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.Nucleoside reverse transcriptase inhibitors (NRTIs) 4 are central components of first line regimens for treatment of HIV infections (1-6). Currently, there are eight clinically approved NRTIs: AZT, 3TC, FTC, ABC, ddI, ddC, d4T, and the nucleotide tenofovir (TFV; reviewed in Refs. 7 and 8). A structural hallmark of these NRTIs is the lack of a 3Ј-OH; it has long been considered that the absence of the 3Ј-OH is essential for antiviral activity. However, the absence of the 3Ј-OH in NRTIs also imparts detrimental properties to the inhibitor, including reduced affinity for RT compared with the analogous dNTP substrate, as well as reduced intracellular conversion to the active nucleoside triphosphate (9).Previously we described a series of 4Ј-substituted NRTIs (10) that retain the 3Ј-OH group and have excellent antiviral properties and significantly improved selectivity indices (CC 50 / EC 50 ) compared with the approved NRTIs. Furthermore, these NRTIs efficiently suppress various NRTI-resistant HIV. The most potent of these 4Ј-substituted NRTIs are the adenosine analogs that have an ethynyl group at the 4Ј position of the ribose ring. Despite their high anti-HIV activity, 4Ј-substituted compounds are susce...
APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. 20/34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access substrate DNA cytosines.
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