RESUMO
Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.
Assuntos
Fosfoproteínas/ultraestrutura , RNA Polimerase Dependente de RNA/ultraestrutura , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/enzimologia , Proteínas Virais/ultraestrutura , Acetatos/química , Animais , Antivirais/química , Antivirais/uso terapêutico , Domínio Catalítico , Microscopia Crioeletrônica , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Quinolinas/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vacinas contra Vírus Sincicial Respiratório/química , Células Sf9 , Spodoptera , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Nucleoside analogues have excellent records as anti-HBV drugs. Chronic infections require long-term administration ultimately leading to drug resistance. Therefore, the search for nucleosides with novel scaffolds is of high importance. Here we report the synthesis of novel 2'-hydroxy- and 2'-hydroxymethyl-apionucleosides, 4 and 5, corresponding triphosphates and phosphoramidate prodrugs. Triphosphate 38 of 2'-hydroxymethyl-apionucleoside 5 exhibited potent inhibition of HBV polymerase with an IC50 value of 120 nM. In an HBV cell-based assay, the phosphoramidate prodrug 39 demonstrated potent activity with an EC50 value of 7.8 nM.
Assuntos
Antivirais , Pró-Fármacos , Antivirais/farmacologia , Vírus da Hepatite B , Nucleosídeos/farmacologia , Pró-Fármacos/farmacologiaRESUMO
Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.
Assuntos
Antivirais/farmacologia , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais/genéticaRESUMO
The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.
Assuntos
Norovirus/enzimologia , Norovirus/genética , Regiões Promotoras Genéticas , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Genoma Viral , Camundongos , Mutação , Ligação Proteica , Elementos Reguladores de Transcrição , Iniciação da Transcrição GenéticaRESUMO
Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3'-terminal CMP to generate the dinucleoside tetraphosphate products Ap(4)C, Up(4)C, and Cp(4)C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3'deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.
Assuntos
Hepacivirus/metabolismo , Nucleotídeos/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Monofosfato de Citidina/química , Monofosfato de Citidina/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/genética , Citidina Trifosfato/metabolismo , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Transcriptase Reversa do HIV/metabolismo , Hepacivirus/enzimologia , Hepacivirus/genética , Cinética , Modelos Químicos , Modelos Genéticos , Nucleotídeos/química , Nucleotídeos/genética , RNA Viral/genética , RNA Viral/metabolismo , Uridina Trifosfato/química , Uridina Trifosfato/genética , Uridina Trifosfato/metabolismoRESUMO
Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2'-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2'-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Citidina/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Pirazinas/farmacologia , Ribonucleotídeos/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Citidina/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Cinética , Camundongos , Norovirus/efeitos dos fármacos , Norovirus/enzimologia , Norovirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2'F-2'C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2'F-2'C-Me-UTP was efficiently incorporated by HCV polymerase with apparent Kd (equilibrium constant) and kpol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 µM and 0.67 ± 0.05 s(-1), respectively, giving an overall substrate efficiency (kpol/Kd) of 0.0059 ± 0.0015 µM(-1) s(-1). We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2' position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH2 > F-C-Me > C-Me > N3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2'F-2'C-Me-, 2'C-Me-, and 2'ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2'-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2'-modified NTP analogs against HCV polymerase.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/farmacologia , Algoritmos , Guanosina Trifosfato/metabolismo , Humanos , CinéticaRESUMO
Respiratory syncytial virus (RSV) is an RNA virus infecting the upper and lower respiratory tract and is recognized as a major respiratory health threat, particularly to older adults, immunocompromised individuals, and young children. Around 64 million children and adults are infected every year worldwide. Despite two vaccines and a new generation monoclonal antibody recently approved, no effective antiviral treatment is available. In this manuscript, we present the medicinal chemistry efforts resulting in the identification of compound 28 (JNJ-8003), a novel RSV non-nucleoside inhibitor displaying subnanomolar activity in vitro as well as prominent efficacy in mice and a neonatal lamb models.
RESUMO
Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.
Assuntos
Antivirais , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Replicação Viral , Antivirais/farmacologia , Antivirais/química , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Animais , Humanos , Replicação Viral/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Ovinos , Farmacorresistência Viral , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/genética , Pulmão/virologiaRESUMO
NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 µM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 µM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.
Assuntos
Hepacivirus/enzimologia , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Escherichia coli , Genoma Viral/fisiologia , Humanos , Cinética , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
Assuntos
Vírus Sincicial Respiratório Humano , RNA Polimerase Dependente de RNA/química , Ligação Proteica , RNA/metabolismo , Nucleotídeos/metabolismoRESUMO
A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.
Assuntos
Bacteriófago T7/enzimologia , Bacteriófago T7/genética , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA , Nucleotídeos/metabolismo , Domínio Catalítico/genética , Simulação por Computador , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Químicos , Mutagênese , Polimerização , Ligação Proteica/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato/genéticaRESUMO
Dengue virus (DENV) infects 50-100 million people worldwide per year, causing severe public health problems. DENV RNA-dependent RNA polymerase, an attractive target for drug development, catalyzes de novo replication of the viral genome in three phases: initiation, transition, and elongation. The aim of this work was to characterize the mechanism of nucleotide addition catalyzed by the polymerase domain of DENV serotype 2 during elongation using transient kinetic methods. We measured the kinetics of formation of the elongation complex containing the polymerase and a double-stranded RNA by preincubation experiments. The elongation complex assembly is slow, following a one-step binding mechanism with an association rate of 0.0016 ± 0.0001 µM(-1) s(-1) and a dissociation rate of 0.00020 ± 0.00005 s(-1) at 37 °C. The elongation complex assembly is 6 times slower at 30 °C and requires Mg(2+) during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a K(d) of 275 ± 52 µM and k(pol) of 18 ± 1 s(-1). The fidelity of the polymerase is 1/34,000, 1/59,000, 1/135,000 for misincorporation of UTP, ATP, and CTP opposite CMP in the template, respectively. The fidelity of DENV polymerase is comparable with HIV reverse transcriptase and the poliovirus polymerase. This work reports the first description of presteady-state kinetics and fidelity for an RNA-dependent RNA polymerase from the Flaviviridae family.
Assuntos
Vírus da Dengue/enzimologia , RNA de Cadeia Dupla/química , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Vírus da Dengue/genética , Genoma Viral/fisiologia , HIV/enzimologia , HIV/genética , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Cinética , Nucleotídeos/química , Nucleotídeos/metabolismo , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/fisiologiaRESUMO
Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.
Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotídeos de Guanina/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Animais , Antivirais/síntese química , Antivirais/toxicidade , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/toxicidade , Feminino , Nucleotídeos de Guanina/síntese química , Nucleotídeos de Guanina/toxicidade , Humanos , Masculino , Pró-Fármacos/síntese química , Pró-Fármacos/toxicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacosRESUMO
Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis- and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical-quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate with an immediate isomerization to an alternate fluorescent form and chemistry occurs, followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a seven-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment.
Assuntos
Hidrolases/metabolismo , Acrilatos/química , Acrilatos/metabolismo , Actinomycetales/enzimologia , Brometos/química , Brometos/metabolismo , Catálise , Cromatografia por Troca Iônica , Hidrolases/química , Cinética , Moraxellaceae/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Like most enzymes, DNA polymerases undergo a large conformational change on the binding of a correct nucleotide. To determine how the conformational change contributes to substrate specificity, we labeled the T7 DNA polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. Here we describe methods to document that only one site on the polymerase was labeled with the fluorophore based on mass spectral analysis of tryptic peptides. In addition, we show by equilibrium titrations of opposing signals that mismatches and correct bases compete for the same site. This analysis forms an essential basis for characterization of a fluorescently labeled enzyme intended for mechanistic studies. Finally, we show that the labeled enzyme can be used to identify single-nucleotide mutations in a procedure that could be automated.
Assuntos
Cumarínicos/química , DNA Polimerase Dirigida por DNA/química , Corantes Fluorescentes/química , Polimorfismo de Nucleotídeo Único , Bacteriófago T7/enzimologia , Cromatografia Líquida de Alta Pressão , Conformação Proteica , Coloração e Rotulagem/métodosRESUMO
We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.
Assuntos
Alanina/análogos & derivados , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleotídeos de Uracila/farmacologia , Uridina/análogos & derivados , Alanina/síntese química , Alanina/farmacologia , Animais , Antivirais/síntese química , Linhagem Celular Tumoral , Cães , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Humanos , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforamidas , Pró-Fármacos/síntese química , Replicon/efeitos dos fármacos , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Uracila/metabolismo , Uridina/síntese química , Uridina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Noroviruses belong to the Caliciviridae family of single-stranded positive-sense RNA viruses. The genus Norovirus includes seven genogroups (designated GI-GVII), of which GI, GII and GIV infect humans. Human noroviruses are responsible for widespread outbreaks of acute gastroenteritis and represent one of the most common causes of foodborne illness. No vaccine or antiviral treatment options are available for norovirus infection. The RNA-dependent RNA polymerase (RdRp) of noroviruses is a key enzyme responsible for transcription and replication of the viral genome. Here, we review the progress made in understanding the structures and functions of norovirus RdRp and its use as a target for small molecule inhibitors. Crystal structures of the RdRp at different stages of substrate interaction have been determined, which shed light on its multi-step catalytic cycle. The in vitro assays and in vivo animal models that have been developed to identify and characterize inhibitors of norovirus RdRp are also summarized, followed by an update on the current antiviral research targeting different regions of norovirus RdRp. In the future, structure-based drug design and rational optimization of known nucleoside and non-nucleoside inhibitors of norovirus RdRp may pave the way towards the next generation of direct-acting antivirals.
Assuntos
Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Norovirus/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Gênica , Replicação Viral , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Cristalografia por Raios X , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Testes de Sensibilidade Microbiana , Norovirus/efeitos dos fármacos , Conformação Proteica , RNA Polimerase Dependente de RNA/antagonistas & inibidoresRESUMO
Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.
Assuntos
Antivirais/toxicidade , Mitocôndrias/efeitos dos fármacos , Ribonucleosídeos/química , Ribonucleosídeos/toxicidade , Adenosina/análogos & derivados , Amidas/toxicidade , Linhagem Celular/efeitos dos fármacos , Citidina/análogos & derivados , Citidina/toxicidade , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/toxicidade , Humanos , Concentração Inibidora 50 , Proteínas Mitocondriais/metabolismo , Nucleosídeos/toxicidade , Pró-Fármacos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Pirazinas/toxicidade , RNA/metabolismo , RNA Mitocondrial , Sofosbuvir/toxicidade , Relação Estrutura-Atividade , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.