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1.
Molecules ; 27(8)2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35458620

RESUMO

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/farmacologia
2.
PLoS Pathog ; 11(6): e1004995, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26098424

RESUMO

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/genética
3.
Antimicrob Agents Chemother ; 59(12): 7504-16, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26392512

RESUMO

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 fármacos
4.
Antimicrob Agents Chemother ; 58(7): 3636-45, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24733478

RESUMO

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 , Cinética
5.
J Med Chem ; 63(18): 10380-10395, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32816483

RESUMO

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 fármacos
6.
J Med Chem ; 62(9): 4555-4570, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-30951311

RESUMO

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 & inibidores
7.
ACS Chem Biol ; 12(1): 83-91, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28103684

RESUMO

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.


Assuntos
Ativação Metabólica , Antivirais/metabolismo , Desoxicitidina/análogos & derivados , Pró-Fármacos/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/enzimologia , Antivirais/farmacologia , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Desoxicitidina Quinase/metabolismo , Descoberta de Drogas , Humanos , Fosforilação , Pró-Fármacos/farmacologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/fisiologia , Replicação Viral/efeitos dos fármacos
8.
Antiviral Res ; 143: 151-161, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28412183

RESUMO

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 fármacos
9.
PLoS One ; 11(5): e0154097, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27163448

RESUMO

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.


Assuntos
Antivirais/farmacologia , Benzazepinas/farmacologia , Citidina Trifosfato/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Farmacorresistência Viral/genética , Niacinamida/análogos & derivados , Mutação Puntual , Proteínas Virais/antagonistas & inibidores , Linhagem Celular Tumoral , Citidina Trifosfato/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Combinação de Medicamentos , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Humanos , Niacinamida/farmacologia , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
J Med Chem ; 59(10): 4611-24, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27120583

RESUMO

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.


Assuntos
Antivirais/farmacologia , Nucleosídeos/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Piridazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Piridazinas/síntese química , Piridazinas/química , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
11.
J Med Chem ; 58(4): 1862-78, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25667954

RESUMO

Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 µM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 µM without appreciable inhibition of human DNA and RNA polymerases at 100 µM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.


Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Pró-Fármacos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/enzimologia , Animais , Antivirais/administração & dosagem , Antivirais/química , Cricetinae , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Haplorrinos , Humanos , Masculino , Conformação Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Infecções por Vírus Respiratório Sincicial/virologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
12.
J Med Chem ; 45(4): 805-17, 2002 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-11831893

RESUMO

A new series of short pyrrole tetraamides are described whose submicromolar DNA binding affinity is an essential component for their strong antibacterial activity. This class of compounds is related to the linked bis-netropsins and bis-distamycins, but here, only one amino-pyrrole-carboxamide unit and an amidine tail is connected to either side of a central dicarboxylic acid linker. The highest degree of DNA binding, measured by compound-induced changes in UV melting temperatures of an AT-rich DNA oligomer, was observed for flat, aromatic linkers with no inherent bent, i.e., terephthalic acid or 1,4-pyridine-dicarboxylic acid. However, the antibacterial activity is critically linked to the size of the N-alkyl substiutent of the pyrrole unit. None of the tetraamides with the commonly used methyl-pyrrole showed antibacterial activity. Isoamyl- or cyclopropylmethylene-substituted dipyrrole derivatives have the minimum inhibitory concentrations in the submicromolar range. In vitro toxicity against human T-cells was studied for all compounds. The degree to which compounds inhibited cell growth was neither directly correlated to DNA binding affinity nor directly correlated to antibacterial activity but seemed to depend strongly on the nature of the N-alkyl pyrrole substituents.


Assuntos
Amidas/síntese química , Antibacterianos/síntese química , DNA/química , Enterococcus/efeitos dos fármacos , Pirróis/síntese química , Staphylococcus aureus/efeitos dos fármacos , Amidas/química , Amidas/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Humanos , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Modelos Moleculares , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas , Resistência a Vancomicina
13.
Nucleic Acids Symp Ser (Oxf) ; (52): 643-4, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776544

RESUMO

In our search for improved therapeutic agents against HCV we synthesized 7-deaza-7-ethynyl-2'-C-methyladenosine (1) and its 2'-deoxy-2'-fluoro analogue 2. The corresponding nucleoside triphosphates were efficient chain terminators of the HCV NS5b polymerase with IC(50)'s of 0.75 microM and 0.4 microM respectively. However, only the ribo-nucleoside 1 exhibited activity in a Huh7 cell based replicon assay with an EC(50) of 0.09 microM. In order to overcome the lack of activity of the fluoro analogue 2 we synthesised several phosphoroamidate prodrugs.


Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Tubercidina/análogos & derivados , Antivirais/química , Antivirais/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Tubercidina/síntese química , Tubercidina/química , Tubercidina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores
14.
J Biol Chem ; 278(13): 11072-7, 2003 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-12538585

RESUMO

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in approximately 20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.


Assuntos
Ciclo Celular , DNA/fisiologia , Marcação de Genes , Animais , Células CHO , Cricetinae , Hipoxantina Fosforribosiltransferase/genética
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