RESUMO
Resistance to anti-HIV drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT) (zidovudine), and (-)-l-2',3'-dideoxy-3'-thiacytidine (3TC) (lamivudine). The RT had mutations at positions 64, 67, 70, 184, and 219 and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside RT inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by tenofovir.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Lamivudina/farmacologia , Mutação/genética , Inibidores da Transcriptase Reversa/química , Zidovudina/farmacologiaRESUMO
Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the "primer grip" region and could be influenced by the M230I mutation.IMPORTANCE Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.
Assuntos
Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Farmacorresistência Viral/genética , Células HEK293 , Infecções por HIV/virologia , Transcriptase Reversa do HIV/efeitos dos fármacos , HIV-1/genética , Humanos , Mutação/efeitos dos fármacos , Nucleosídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.
Assuntos
Replicação do DNA/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Naftiridinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Naftiridinas/química , Conformação Proteica , Inibidores da Transcriptase Reversa/químicaRESUMO
Although anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3' OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3' end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration.
Assuntos
Substituição de Aminoácidos , DNA Viral/química , Transcriptase Reversa do HIV/química , RNA Viral/química , Inibidores da Transcriptase Reversa/química , Zidovudina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Clonagem Molecular , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Cinética , Modelos Moleculares , Mutação , Fenilalanina/química , Fenilalanina/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade , Treonina/química , Treonina/metabolismo , Tirosina/química , Tirosina/metabolismo , Zidovudina/farmacologiaRESUMO
As anti-HIV therapy becomes more widely available in developing nations, it is clear that drug resistance will continue to be a major problem. The related viruses HIV-1 and HIV-2 share many of the same resistance pathways to nucleoside reverse transcriptase inhibitors (NRTIs). However, clinical data suggest that while HIV-1 reverse transcriptase (RT) usually uses an ATP-dependent excision pathway to develop resistance to the nucleoside analog zidovudine (AZT), HIV-2 RT does not appear to use this pathway. We previously described data that suggested that wild-type (WT) HIV-2 RT has a much lower ability to excise AZT monophosphate (AZTMP) than does WT HIV-1 RT and suggested that this is the reason that HIV-2 RT more readily adopts an exclusion pathway against AZT triphosphate (AZTTP), while HIV-1 RT is better able to exploit the ATP-dependent pyrophosphorolysis mechanism. However, we have now done additional experiments, which show that while HIV-1 RT can adopt either an exclusion- or excision-based resistance mechanism against AZT, HIV-2 RT can use only the exclusion mechanism. All of our attempts to make HIV-2 RT excision competent did not produce an AZT-resistant RT but instead yielded RTs that were less able to polymerize than the WT. This suggests that the exclusion pathway is the only pathway available to HIV-2.
Assuntos
Farmacorresistência Viral , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-2/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-2/efeitos dos fármacos , HIV-2/genética , Humanos , Inibidores da Transcriptase Reversa/metabolismo , Zidovudina/metabolismo , Zidovudina/farmacologiaRESUMO
Unlike orthoretroviruses, foamy retroviruses (FV) synthesize Pol independently of Gag. The FV Pol precursor is cleaved only once between reverse transcriptase (RT) and integrase (IN) by the protease (PR), resulting in a PR-RT and an IN protein. Only the Pol precursor, not the cleaved subunits, is packaged into virions. Like orthoretroviral PRs, FV PR needs to dimerize to be active. Previously, we showed that a Pol mutant lacking IN has defects in PR activity and Pol packaging into virions. We now show that introduction of a leucine zipper (zip) dimerization motif in an IN truncation mutant can restore PR activity, leading to Pol processing in cells. However, these zip mutants neither cleave Gag nor incorporate Pol into virions. We propose that IN is required for Pol dimerization, which is necessary for the creation of a functional PR active site.
Assuntos
Produtos do Gene pol/metabolismo , Integrases/química , Peptídeo Hidrolases/metabolismo , Vírus Espumoso dos Símios/enzimologia , Animais , Domínio Catalítico , Linhagem Celular , Ativação Enzimática , Produtos do Gene pol/química , Produtos do Gene pol/genética , Genes pol , Humanos , Integrases/genética , Integrases/metabolismo , Zíper de Leucina , Mutação , Multimerização Proteica , Vírus Espumoso dos Símios/genética , Vírus Espumoso dos Símios/metabolismoRESUMO
It is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3'-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4'-C-alkyl-deoxyadenosine (4'-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4'-C-methyl- and 4'-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4'-C-methyl- or 4'-C-ethyl-2'-deoxyadenosine. In vitro experiments with purified HIV-1 RT showed that 4'-C-methyl-2'-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4'-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.
Assuntos
Fármacos Anti-HIV/farmacologia , Replicação do DNA/efeitos dos fármacos , Desoxiadenosinas/farmacologia , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/química , Linhagem Celular , Desoxiadenosinas/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Humanos , Espectroscopia de Ressonância Magnética , Reação em Cadeia da PolimeraseRESUMO
We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.
Assuntos
Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , Modelos Biológicos , Mutação/genética , RNA/metabolismo , Zidovudina/metabolismo , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Humanos , MutagêneseRESUMO
In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.
Assuntos
Peptídeo Hidrolases/química , Poliproteínas/química , DNA Polimerase Dirigida por RNA/química , Spumavirus/química , Cristalização , Peptídeo Hidrolases/metabolismo , Poliproteínas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição ReversaRESUMO
K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg(72) were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg(72) were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations.
Assuntos
Adenina/análogos & derivados , Farmacorresistência Viral/fisiologia , Transcriptase Reversa do HIV , Mutação , Organofosfonatos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Adenina/química , Adenina/farmacologia , Arginina/genética , Arginina/metabolismo , Cristalização , Cristalografia por Raios X , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/efeitos dos fármacos , Transcriptase Reversa do HIV/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Organofosfonatos/química , Conformação Proteica , Inibidores da Transcriptase Reversa/química , TenofovirRESUMO
HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.0 A resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 A resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 A resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.
Assuntos
Cristalografia por Raios X , Transcriptase Reversa do HIV/química , Nitrilas/química , Engenharia de Proteínas/métodos , Pirimidinas/química , Inibidores da Transcriptase Reversa/química , Clonagem Molecular , Desenho de Fármacos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Modelos Moleculares , Mutagênese , RilpivirinaRESUMO
Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.
Assuntos
Adenina/análogos & derivados , Adenina/química , DNA Viral/química , Transcriptase Reversa do HIV/química , Organofosfonatos , Compostos Organofosforados/química , Inibidores da Transcriptase Reversa/química , Sequência de Bases , Primers do DNA , Modelos Moleculares , TenofovirRESUMO
Lys103Asn and Tyr181Cys are the two mutations frequently observed in patients exposed to various non-nucleoside reverse transcriptase inhibitor drugs (NNRTIs). Human immunodeficiency virus (HIV) strains containing both reverse transcriptase (RT) mutations are resistant to all of the approved NNRTI drugs. We have determined crystal structures of Lys103Asn/Tyr181Cys mutant HIV-1 RT with and without a bound non-nucleoside inhibitor (HBY 097, (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(1H)-thione) at 3.0 A and 2.5 A resolution, respectively. The structure of the double mutant RT/HBY 097 complex shows a rearrangement of the isopropoxycarbonyl group of HBY 097 compared to its binding with wild-type RT. HBY 097 makes a hydrogen bond with the thiol group of Cys181 that helps the drug retain potency against the Tyr181Cys mutation. The structure of the unliganded double mutant HIV-1 RT showed that Lys103Asn mutation facilitates coordination of a sodium ion with Lys101 O, Asn103 N and O(delta1), Tyr188 O(eta), and two water molecules. The formation of the binding pocket requires the removal of the sodium ion. Although the RT alone and the RT/HBY 097 complex were crystallized in the presence of ATP, only the RT has an ATP coordinated with two Mn(2+) at the polymerase active site. The metal coordination mimics a reaction intermediate state in which complete octahedral coordination was observed for both metal ions. Asp186 coordinates at an axial position whereas the carboxylates of Asp110 and Asp185 are in the planes of coordination of both metal ions. The structures provide evidence that NNRTIs restrict the flexibility of the YMDD loop and prevent the catalytic aspartate residues from adopting their metal-binding conformations.
Assuntos
Trifosfato de Adenosina/metabolismo , Fármacos Anti-HIV/metabolismo , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Cristalografia por Raios X , DNA/metabolismo , Farmacorresistência Viral , Transcriptase Reversa do HIV/antagonistas & inibidores , Humanos , Manganês/metabolismo , Modelos Moleculares , Conformação Molecular , Mutação , Conformação Proteica , Quinoxalinas/química , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
HIV-1 can become resistant to nucleoside analogs by developing an enhanced ability to excise the analogs after they have been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have describe nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional dNTPs have been added to the DNA. These nucleoside analogs are called "delayed chain terminators" and are relatively effective inhibitors of drug-resistant HIV-1 reverse transcriptases (RTs) that are excision proficient. However, the first delayed chain terminator that we characterized was poorly phosphorylated in cultured cells. We have examined other nucleoside analogs to determine whether these compounds also act as delayed chain terminators, but were more efficiently converted to the triphosphate form by cellular kinases. These analogs contain substitutions on the deoxyribose sugar ring at the 4' carbon (4'C-methyl dT and 4'C-ethyl dT). Unlike true delayed chain terminators, which terminate DNA synthesis in a spatial sense (DNA synthesis is halted only after additional dNTPs have been incorporated after the analog), 4'C-methyl dTTP causes a pause in DNA synthesis at the point of incorporation. However, HIV-1 RT can eventually extend the primer blocked by the 4' C-Me dTMP analog. 4'C-methyl dTTP blocks DNA synthesis in a temporal sense, rather than in a spatial sense. A primer blocked by 4'C-ethyl dTMP is not extended by HIV-1 RT, and this compound acts like a conventional chain terminator, despite the presence of a 3'-OH group. These compounds effectively block the replication of an HIV-1-based vector that replicates using wild-type HIV-1 RT, but only in the presence of herpes simplex virus thymidine kinase (HSV TK). These compounds are effective against many NRTI drug-resistant RT variants; however, the M184V mutant is relatively resistant.
Assuntos
DNA Viral/biossíntese , Farmacorresistência Viral/efeitos dos fármacos , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Timidina/análogos & derivados , Linhagem Celular , Vetores Genéticos/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Estrutura Molecular , Mutação/genética , Fosforilação , Inibidores da Transcriptase Reversa/química , Transcrição Reversa/genética , Timidina/farmacologia , Timidina Quinase/metabolismoRESUMO
The human immunodeficiency virus type 1 (HIV-1) develops resistance to all available drugs, including the nucleoside analog reverse transcriptase inhibitors (NRTIs) such as AZT. ATP-mediated excision underlies the most common form of HIV-1 resistance to AZT. However, clinical data suggest that when HIV-2 is challenged with AZT, it usually accumulates resistance mutations that cause AZT resistance by reduced incorporation of AZTTP rather than selective excision of AZTMP. We compared the properties of HIV-1 and HIV-2 reverse transcriptase (RT) in vitro. Although both RTs have similar levels of polymerase activity, HIV-1 RT more readily incorporates, and is more susceptible to, inhibition by AZTTP than is HIV-2 RT. Differences in the region around the polymerase active site could explain why HIV-2 RT incorporates AZTTP less efficiently than HIV-1 RT. HIV-1 RT is markedly more efficient at carrying out the excision reaction with ATP as the pyrophosphate donor than is HIV-2 RT. This suggests that HIV-1 RT has a better nascent ATP binding site than HIV-2 RT, making it easier for HIV-1 RT to develop a more effective ATP binding site by mutation. A comparison of HIV-1 and HIV-2 RT shows that there are numerous differences in the putative ATP binding sites that could explain why HIV-1 RT binds ATP more effectively. HIV-1 RT incorporates AZTTP more efficiently than does HIV-2 RT. However, HIV-1 RT is more efficient at ATP-mediated excision of AZTMP than is HIV-2 RT. Mutations in HIV-1 RT conferring AZT resistance tend to increase the efficiency of the ATP-mediated excision pathway, while mutations in HIV-2 RT conferring AZT resistance tend to increase the level of AZTTP exclusion from the polymerase active site. Thus, each RT usually chooses the pathway best suited to extend the properties of the respective wild-type enzymes.
Assuntos
Farmacorresistência Viral/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Zidovudina/farmacologia , Sequência de Aminoácidos , Glutamina , HIV-1/genética , HIV-2/genética , Metionina , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/genéticaRESUMO
An important mechanism of resistance to nucleoside analogs is the enhanced excision of the analog after it has been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have developed nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional normal dNTPs have been added to the DNA. Such "delayed chain terminators" should be relatively resistant to excision and therefore effective against drug-resistant HIV-1 reverse transcriptases (RTs) that are proficient at excision. We tested a class of nucleoside analogs in which a pseudosugar ring is locked in either the North or the South conformation. These analogs have a 3' OH present on the pseudosugar ring, which allows extension of the primer strand after the analog is incorporated. We asked whether these analogs would inhibit polymerization by HIV-1 RT in assays using purified HIV-1 RT and in cell-based assays. HIV-1 RT did not effectively incorporate the analogs in which the pseudosugar is in the South conformation. The North conformation analogs are readily incorporated into the primer; the primer can be extended for two or three additional nucleotides before extension is inhibited. This block to polymerization is not complete; larger extension products are detectable at longer incubation times. Experiments with purified excision-proficient HIV-1 RT mutants suggest that the North conformation analogs are relatively resistant to excision. These analogs can also block the replication of viruses containing excision-proficient RTs. Although the fixed-conformation nucleotides are probably not suitable for development as drugs, other nucleoside analogs that cause delayed chain termination may complement the nucleoside analogs already approved for HIV-1 therapy.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Nucleosídeos/química , Nucleosídeos/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Conformação de Ácido NucleicoRESUMO
As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond 'donor-acceptor-donor' pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond 'acceptor-donor-acceptor' pattern. This base pair fits the Watson-Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.
Assuntos
Pareamento de Bases , DNA/biossíntese , DNA/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Reação em Cadeia da Polimerase/métodos , Engenharia de Proteínas , Substituição de Aminoácidos/genética , Sítios de Ligação , DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Variação Genética/genética , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Ligação de Hidrogênio , Taq Polimerase/metabolismoRESUMO
Mutations in the thumb subdomain of reverse transcriptase (RT) of HIV-1 can cause this enzyme to be degraded in virions by the viral protease (PR). Many of these mutations confer a temperature-sensitive phenotype on RT and viral replication. The degradation of RT by PR appears to take place after Gag-Pol has been processed. We show here that mutations in other parts of RT, including the RNase H domain, can make RT PR-sensitive and temperature-sensitive. These data explain why some mutations in the RNase H domain, which had little or no effect on the polymerase activity of purified recombinant RT, had a profound effect on viral titer. Because the PR-sensitive phenotype significantly reduced viral titer, we previously suggested that these mutations would be selected against in patients. We also show that RT mutations that are known to confer a temperature sensitive phenotype are rarely found in the Stanford database.
Assuntos
Protease de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/crescimento & desenvolvimento , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Seleção Genética , Linhagem Celular , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Hidrólise , Proteínas Mutantes/genética , Carga ViralRESUMO
4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.
Assuntos
Alcinos/síntese química , Fármacos Anti-HIV/síntese química , Didesoxinucleosídeos/síntese química , Zalcitabina/análogos & derivados , Zalcitabina/síntese química , Alcinos/química , Alcinos/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , DNA Viral/química , Didesoxinucleosídeos/química , Didesoxinucleosídeos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Organofosfatos/síntese química , Organofosfatos/química , Organofosfatos/farmacologia , Fosforilação , Fosfotransferases/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Zalcitabina/química , Zalcitabina/farmacologiaRESUMO
The synthesis and biological evaluation of a bicyclo[3.1.0]hexene nucleoside designed as a conformational mimic of the anti-HIV agent stavudine (1, D4T) is described. The unsaturated methanocarbocyclic pseudosugar of N-MCD4T (2) was constructed from an iodo-substituted precursor by a DBU-catalyzed olefination reaction. Mitsunobu coupling with N(3)-benzoylthymine afforded the desired target after deprotection. Both D4T and N-MCD4T are in the North (N) hemisphere of the pseudorotational cycle but 70 degrees away from a perfect N (P = 0 degrees ) conformation toward the East and West hemispheres, respectively. Despite this large difference, the double bond reduces the puckering amplitude (nu(max)) of N-MCD4T to 6.81 degrees, and the superposition of both structures showed a RMS deviation of only 0.039 A. The combined structural analysis of P and nu(max) shows that while the value of P may differ substantially, the low nu(max) resolves the differences and becomes the dominant pseudorotational parameter. N-MCD4T is active against HIV-1 and HIV-2 in CEM, MT-2, and MT-4 cells, and while it is somewhat less potent than D4T, it also appears to be less toxic. The triphosphate (N-MCD4TTP) inhibits HIV reverse transcriptase with a 10-fold higher IC(50) than D4TTP. By virtue of its carbocyclic nature, N-MCD4T (2) is a more robust molecule stable to conditions that would cleave D4T.