RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.
Assuntos
Endonucleases/química , Endonucleases/metabolismo , Herpesvirus Humano 8/enzimologia , Sarcoma de Kaposi/virologia , Varredura Diferencial de Calorimetria , Domínio Catalítico , DNA Viral/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Ativação Enzimática/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , Herpesvirus Humano 8/genética , Integrases/genética , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta/genética , Estrutura Secundária de Proteína , Ribonuclease H/genéticaRESUMO
The natural product α-hydroxytropolones manicol and ß-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an "RNase H-like" fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein-nucleic acid contacts over a region of â¼ 14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme-substrate complex to the enzyme-product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site.
Assuntos
Inibidores Enzimáticos/farmacologia , Herpesvirus Humano 1/química , Nucleotidiltransferases/antagonistas & inibidores , Proteínas Virais/química , FluorescênciaRESUMO
Two new sesquiterpenoid tropolone glycosides, liriosmasides A (1) and B (2), along with two known compounds, secoxyloganin and oplopanpheside C, were isolated from a methanol extract of the roots of Liriosma ovata. The structures of 1 and 2 were elucidated by spectroscopic methods including 1D and 2D NMR and by high-resolution mass spectrometry involving an ultra-high-performance liquid chromatography-quadrupole-orbital ion trap mass spectrometric (UHPLC-Q-Orbitrap MS) method. Compound 1 showed weak inhibitory activity against HIV RNase H.
Assuntos
Glicosídeos/isolamento & purificação , Olacaceae/química , Sesquiterpenos/isolamento & purificação , Tropolona/análogos & derivados , Tropolona/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Glicosídeos/química , Glicosídeos/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peru , Raízes de Plantas/química , Ribonuclease H/antagonistas & inibidores , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Tropolona/química , Tropolona/farmacologiaRESUMO
Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteômica/métodos , Transporte Ativo do Núcleo Celular , Sistema Livre de Células , Análise Mutacional de DNA , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas/química , Células Germinativas/citologia , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Métodos , Fosforilação , Especificidade por Substrato , Triticum/química , Triticum/citologiaRESUMO
α-Hydroxytropolones are established inhibitors of several therapeutically relevant binuclear metalloenzymes, and thus lead drug targets for various human diseases. We have leveraged a recently-disclosed three-component oxidopyrylium cycloaddition in the first solid-phase synthesis of α-hydroxytropolones. We also showed that, while minor impurities exist after cleavage and aqueous wash, the semi-crude products display activity in HIV RT-associated RNaseH enzymatic and cell-based assays consistent with pure molecules made in solution phase. These proof-of-principle studies demonstrate the feasibility of solid-phase α-hydroxytropolone synthesis and its potential to serve as a powerful platform for α-hydroxytropolone-based drug discovery and development.
RESUMO
The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 µM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Hidrazonas/farmacologia , Isatina/análogos & derivados , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Alcinos , Benzoxazinas/antagonistas & inibidores , Benzoxazinas/farmacologia , Sítios de Ligação , Ciclopropanos , HIV-1/enzimologia , Humanos , Isatina/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Replicação Viral/efeitos dos fármacosRESUMO
HIV Reverse Transcriptase-associated ribonuclease H activity is a promising enzymatic target for drug development that has not been successfully targeted in the clinic. While the α-hydroxytropolone-containing natural products ß-thujaplicinol and manicol have emerged as some of the most potent leads described to date, structure-function studies have been limited to the natural products and semi-synthetic derivatives of manicol. Thus, a library of α-hydroxytropolones synthesized through a convenient oxidopyrylium cycloaddition/ring-opening sequence have been tested in in vitro and cell-based assays, and have been analyzed using computational support. These studies reveal new synthetic α-hydroxytropolones that, unlike the natural product leads they are derived from, demonstrate protective antiviral activity in cellular assays.
RESUMO
Despite the significant progress achieved with combination antiretroviral therapy in the fight against human immunodeficiency virus (HIV) infection, the difficulty to eradicate the virus together with the rapid emergence of multidrug-resistant strains clearly underline a pressing need for innovative agents, possibly endowed with novel mechanisms of action. In this context, owing to its essential role in HIV genome replication, the reverse transcriptase associated ribonucleaseâ H (RNaseâ H) has proven to be an appealing target. To identify new RNaseâ H inhibitors, an in-house cycloheptathiophene-3-carboxamide library was screened; this led to compounds endowed with inhibitory activity, the structural optimization of which led to the catechol derivative 2-(3,4-dihydroxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (compound 33) with an IC50 value on the RNaseâ H activity in the nanomolar range. Mechanistic studies suggested selective inhibition of the RNaseâ H through binding to an innovative allosteric site, which could be further exploited to enrich this class of inhibitors.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Ribonuclease H/antagonistas & inibidores , Tiofenos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Benzamidas/síntese química , Benzamidas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , HIV-1/enzimologia , Modelos Moleculares , Estrutura Molecular , Ribonuclease H/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Darunavir (DRV) is one of the most powerful protease inhibitors (PIs) for treating human immunodeficiency virus type-1 (HIV-1) infection and presents a high genetic barrier to the generation of resistant viruses. However, DRV-resistant HIV-1 infrequently emerges from viruses exhibiting resistance to other protease inhibitors. To address this resistance, researchers have gathered genetic information on DRV resistance. In contrast, few structural insights into the mechanism underlying DRV resistance are available. To elucidate this mechanism, we determined the crystal structure of the ligand-free state of a protease with high-level DRV resistance and six DRV resistance-associated mutations (including I47V and I50V), which we generated by in vitro selection. This crystal structure showed a unique curling conformation at the flap regions that was not found in the previously reported ligand-free protease structures. Molecular dynamics simulations indicated that the curled flap conformation altered the flap dynamics. These results suggest that the preference for a unique flap conformation influences DRV binding. These results provide new structural insights into elucidating the molecular mechanism of DRV resistance and aid to develop PIs effective against DRV-resistant viruses.
RESUMO
Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.
RESUMO
OBJECTIVE: The human APOBEC3 family of proteins potently restricts HIV-1 replication APOBEC3B, one of the family genes, is frequently deleted in human populations. Two previous studies reached inconsistent conclusions regarding the effects of APOBEC3B loss on HIV-1 acquisition and pathogenesis. Therefore, it was necessary to verify the effects of APOBEC3B on HIV-1 infection in vivo. METHODS: Intact (I) and deletion (D) polymorphisms of APOBEC3B were analyzed using PCR. The syphilis, HBV and HCV infection rates, as well as CD4(+) T cell counts and viral loads were compared among three APOBEC3B genotype groups (I/I, D/I, and D/D). HIV-1 replication kinetics was assayed in vitro using primary cells derived from PBMCs. RESULTS: A total of 248 HIV-1-infected Japanese men who have sex with men (MSM) patients and 207 uninfected Japanese MSM were enrolled in this study. The genotype analysis revealed no significant differences between the APOBEC3B genotype ratios of the infected and the uninfected cohorts (p = 0.66). In addition, HIV-1 disease progression parameters were not associated with the APOBEC3B genotype. Furthermore, the PBMCs from D/D and I/I subjects exhibited comparable HIV-1 susceptibility. CONCLUSION: Our analysis of a population-based matched cohort suggests that the antiviral mechanism of APOBEC3B plays only a negligible role in eliminating HIV-1 in vivo.
Assuntos
Citidina Desaminase/genética , Predisposição Genética para Doença , Infecções por HIV/genética , HIV-1 , Polimorfismo Genético , Adulto , Povo Asiático , Estudos de Coortes , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade MenorRESUMO
Despite the wealth of information available for the reverse transcriptase (RT)-associated ribonuclease H (RNaseH) domain of lentiviruses, gammaretroviruses and long terminal repeat containing retrotransposons, exploiting this information in the form of an RNaseH inhibitor with high specificity and low cellular toxicity has been disappointing. However, it is now becoming increasingly evident that the two-subunit HIV-1 RT is a highly versatile enzyme, undergoing major structural alterations in order to interact with, position and ultimately hydrolyze the RNA component of an RNA/DNA hybrid. Thus, in addition to targeting the RNaseH active site, identifying small molecules that bind elsewhere and disrupt catalysis allosterically by impairing conformational flexibility is gaining increased attention. This review summarizes current progress towards development of both active site and allosteric RNaseH inhibitors.
Assuntos
Transcriptase Reversa do HIV/metabolismo , Ribonuclease H/antagonistas & inibidores , Regulação Alostérica , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Hidrólise , Ribonuclease H/metabolismoRESUMO
The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3',4'-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy.
Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Pirimidinonas/farmacologia , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Antivirais/química , Antivirais/farmacologia , Farmacorresistência Viral/genética , Inibidores Enzimáticos/química , Estabilidade Enzimática/efeitos dos fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Pirimidinonas/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H do Vírus da Imunodeficiência Humana/química , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , TemperaturaRESUMO
Yeast tRNA (m(7)G46) methyltransferase contains two protein subunits (Trm8 and Trm82). The enzyme catalyzes a methyl-transfer from S-adenosyl-L-methionine to the N(7) atom of guanine at position 46 in tRNA. We deviced synthesis of active Trm8-Trm82 heterodimer in a wheat germ cell-free translation system. When Trm8 or Trm82 mRNA were used for a synthesis, Trm8 or Trm82 protein could be synthesized. Upon mixing the synthesized Trm8 and Trm82 proteins, no active Trm8-Trm82 heterodimer was produced. Active Trm8-Trm82 heterodimer was only synthesized under conditions, in which both Trm8 and Trm82 mRNAs were co-translated. To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA(Phe) transcripts. In this meeting, we demonstrate that yeast Trm8-Trm82 has stricter recognition requirements for the tRNA molecule as compared to the bacterial enzyme, TrmB.