RESUMEN
We report on the discovery of norbornyl moiety as a novel structural motif for cyclin-dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC50 of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics-based scoring was used to rationalize the affinities. In conclusion, the discovered 9-hydroxymethylnorbornyl moiety was shown by joint experimental-theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Norbornanos/farmacología , Nucleósidos/análogos & derivados , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
The nucleoside/nucleotide derived antiviral agents have been the most important components of antiviral therapy used in clinics. Recently, the focus of the medicinal chemists within this exciting research field has been affected mainly by the lack of effective therapies for the Hepatitis C virus (HCV) infection and several other "neglected" diseases caused by viruses such as Zika or Dengue. 2'-Methyl modified nucleosides and their monophosphate prodrugs (ProTides) have revolutionized the therapies for HCV in the last few years and, according to the latest research efforts, have also brought a promise for treatment of diseases caused by other members of Flaviviridae family. Here, we report on the design and synthesis of 5'-N and S modified ProTides derived from 2'-methyladenosine. We studied potential applicability of these derivatives as prodrugs of this archetypal antiviral compound.
Asunto(s)
Antivirales/química , Nucleótidos/química , Profármacos/química , Adenosina/análogos & derivados , Adenosina/química , Antivirales/síntesis química , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Nitrógeno/química , Nucleótidos/metabolismo , Nucleótidos/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Azufre/química , Virus Zika/efectos de los fármacosRESUMEN
We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.
Asunto(s)
Técnicas de Química Sintética/métodos , ARN de Transferencia/química , Ribonucleótidos/química , Ribosa/análogos & derivados , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN de Transferencia/síntesis química , ARN de Transferencia/metabolismo , Ribonucleótidos/síntesis química , Ribonucleótidos/metabolismo , Ribosa/síntesis química , Ribosa/metabolismo , Weissella/enzimología , Weissella/metabolismoRESUMEN
We describe a novel stereoselective synthesis of nucleoside derivatives with the ribose ring locked in the South conformation by a bridge between C2' and C5'. Despite the intrinsic constraints of the bicyclic structure, we demonstrate that their synthesis can be achieved by ring closing metathesis of readily accessible precursors. The obtained ribose derivatives are, however, very poor substrates for further installation of the nucleobases, and even simple nucleophiles, such as azido or cyano anions, react with unexpected stereo- or regioselectivity under standard glycosylation conditions. Here we explain this behavior by employing density functional theory (DFT) computations and devise an alternative approach resulting in isomers with the desired orientation of the nucleobase.
RESUMEN
We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano B/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Rhinovirus/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Purinas/síntesis química , Purinas/química , Relación Estructura-ActividadRESUMEN
Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIß and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIß and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9â Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/química , Diseño de Fármacos , Nucleósidos/química , Inhibidores de Proteínas Quinasas/química , 1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Nucleósidos/farmacología , Conformación Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 µM for 7-deaza-2'-CMA, 7.1 ± 1.2 µM for 2'-CMA, and 14.2 ± 1.9 µM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of â¼50 µM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 µM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacología , Animales , Línea Celular , Citidina/análogos & derivados , Citidina/química , Citidina/farmacología , Humanos , Porcinos , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
We describe here the preparation of conformationally locked cyclohexane nucleic acids designed as hybrids between locked nucleic acids (LNAs) and cyclohexene nucleic acids (CeNAs), both of which excel in hybridization with complementary RNAs. We have accomplished the synthesis of these adenine derivatives starting from a simple ketoester and installed all four chiral centres by means of total synthesis. The acquired monomers were incorporated into nonamer oligonucleotides.
Asunto(s)
Adenosina/química , Ciclohexanos/síntesis química , Ácidos Nucleicos/síntesis química , Adenosina/síntesis química , Ciclohexanos/química , Estructura Molecular , Ácidos Nucleicos/químicaRESUMEN
We prepared a novel series of conformationally restricted bicyclonucleosides and nucleotides. The synthetic approach employed a ring closing metathesis to provide access to both 6 and 7 membered saturated and unsaturated rings linking the 3' to 5' methylene groups of the sugar. The bicyclonucleosides were also transformed to the corresponding phosphoramidate prodrugs by an innovative one-pot protocol of boronate ester protection, coupling of the phosphoryl chloridate and deprotection of the boronate. A similar strategy was also employed for the synthesis of the corresponding monophosphates as crucial intermediates for the synthesis of selected triphosphates. The biological properties of the nucleosides and monophosphate prodrugs were assessed for antiviral and cytostatic activities in cell based assays whilst the triphosphates were evaluated in enzymatic assays. The lack of significant effects suggests that the linkage of the 3' to 5'via a ring system and the subsequent conformational restriction of the ribose ring to the South conformation are incompatible with the kinases and polymerases that recognize nucleosides and their metabolites.
Asunto(s)
Adenina/química , Antivirales/síntesis química , Antivirales/farmacología , Nucleótidos/síntesis química , Nucleótidos/farmacología , Antivirales/metabolismo , Conformación de Carbohidratos , Línea Celular Tumoral , Técnicas de Química Sintética , Diseño de Fármacos , Hepacivirus/efectos de los fármacos , Humanos , Modelos Moleculares , Nucleótidos/metabolismo , Profármacos/metabolismoRESUMEN
We report on the synthesis of novel conformationally locked nucleoside and nucleotide derivatives, which are structurally closely related to clinically used antivirals such as didanosine and abacavir. As a suitable conformationally rigid substitute of the sugar/pseudosugar ring allowing a permanent stabilization of the nucleoside in North conformation we employed bicyclo[2.2.1]heptane (norbornane) substituted in the bridgehead position with a hydroxymethyl group and in the C-3 position with a nucleobase. Prepared nucleoside derivatives were also converted into appropriate phosphoramidate prodrugs (ProTides) in order to increase delivery of the compounds in the cells. All target compounds were evaluated in a broad antiviral and cytostatic assay panel.
Asunto(s)
Antivirales/síntesis química , Norbornanos/química , Nucleósidos/química , Nucleótidos/química , Humanos , Norbornanos/síntesis química , Conformación de Ácido Nucleico , Nucleósidos/síntesis química , Nucleótidos/síntesis química , EstereoisomerismoRESUMEN
6-Chloropurines substituted at the position 9 with variously modified bicyclic skeletons represent promising antiviral and anticancer agents. This work aimed to investigate the transport mechanisms of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP) and their relationship to the metabolism and biological activity of the compound. Transport experiments were conducted in CCRF-CEM cells using radiolabeled compound ([(3)H]NCP). The pattern of the intracellular uptake of [(3)H]NCP in CCRF-CEM cells pointed to a combination of passive and facilitated diffusion as prevailing transport mechanisms. NCP intracellular metabolism was found to enhance its uptake by modifying NCP concentration gradient. The transport kinetics reached steady state under the conditions of MRP and MDR proteins blockade, indicating that NCP is a substrate for these efflux pumps. Their inhibition also increased the cytotoxicity of NCP. Our findings suggest that the novel nucleoside analog NCP has potential to become a new orally available antileukemic agent due to its rapid membrane permeation.
Asunto(s)
Antineoplásicos/farmacología , Purinas/farmacología , Linfocitos T/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Transporte Biológico , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dibenzocicloheptenos/farmacología , Ácido Etacrínico/farmacología , Difusión Facilitada , Expresión Génica , Humanos , Cinética , Propionatos/farmacología , Purinas/síntesis química , Purinas/metabolismo , Quinolinas/farmacología , Linfocitos T/metabolismo , Linfocitos T/patología , TritioRESUMEN
A synthetic route toward a series of unique cyclic nucleoside phosphonates locked in South conformation is described. The desired conformation is stabilized by a substitution of the sugar moiety by bicyclo[2.2.1]heptane (norbornane) bearing a purine or pyrimidine nucleobase in the bridgehead position. Although the final phosphonate derivatives are devoid of any significant antiviral activity probably due to the unfavorable conformational properties, several intermediates and their analogs exhibit surprising activity against feline herpes virus. Since these compounds do not possess an appropriate hydroxymethyl function allowing phosphorylation and subsequent incorporation into the polynucleotide chain, it seems to be likely that these compounds act by a novel unknown mechanism of action and may represent a new possible alternative for nucleoside and nucleotide therapeutics of this widely spread feline infection. A number of derivatives exerted also a significant antiviral activity against Coxsackievirus B3 and B4.
Asunto(s)
Antivirales/farmacología , Calicivirus Felino/efectos de los fármacos , Norbornanos/farmacología , Nucleótidos/farmacología , Antivirales/síntesis química , Antivirales/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Norbornanos/síntesis química , Norbornanos/química , Nucleótidos/síntesis química , Nucleótidos/química , Relación Estructura-ActividadRESUMEN
The synthesis of a novel library of purine derivatives bearing various bicyclic and polycylic substituents at the N-9 position is described. The series includes norbornanes, bicyclo[2.2.2]octanes, and bicyclo[3.2.1]octanes attached at the bridgehead position as well as bicyclo[3.1.1]heptanes, tetrahydro-1-naphthalenes, and adamantanes bonded either directly or via a linear chain to the 6-chloropurine nucleobase. A number of prepared derivatives exerted significant activity against the enterovirus. Despite attempts to correlate the activity against picornaviruses with their phosphatidylinositol 4-kinase KIIIß inhibitory activity, it is clear that the inhibition of this host factor cannot explain the observed antiviral potency.
Asunto(s)
Antivirales/síntesis química , Hidrocarburos Aromáticos con Puentes/síntesis química , Enterovirus/efectos de los fármacos , Norbornanos/síntesis química , Purinas/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Antivirales/toxicidad , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Efecto Citopatogénico Viral , Enterovirus/fisiología , Estructura Molecular , Norbornanos/química , Norbornanos/farmacología , Norbornanos/toxicidad , Purinas/química , Purinas/farmacología , Purinas/toxicidad , Replicación Viral/efectos de los fármacosRESUMEN
We report on the synthesis and the study of the structure-activity relationship of novel 9-norbornyl-6-chloropurine derivatives, which exert selective antiviral activity on the replication of Coxsackievirus B3. In particular, the synthetic approaches towards norbornyl derivatives bearing diverse side chains were studied. The main goal of the study was to determine the influence of the norbornane moiety substitution at positions 5' and 6' on selective antiviral activity with special regard to the liphophilicity profile of the substituent.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Enterovirus Humano B/efectos de los fármacos , Norbornanos/química , Norbornanos/farmacología , Purinas/química , Purinas/farmacología , Animales , Antivirales/síntesis química , Chlorocebus aethiops , Infecciones por Coxsackievirus/tratamiento farmacológico , Humanos , Norbornanos/síntesis química , Purinas/síntesis química , Células VeroRESUMEN
Coxsackievirus and related enteroviruses are important human pathogens that cause various diseases with clinical manifestations ranging from trivial flu-like syndromes to dangerous or even fatal diseases such as myocarditis, meningitis and encephalitis. Here, we report on our continuous SAR study focused on 9-(bicyclo[2.2.1]hept-2-yl)-9H-purines as anti-enteroviral inhibitors. The purine moiety was modified at positions 2, 6 and 8. Several analogues inhibited Coxsackievirus B3 as well as other enteroviruses at low-micromolar concentrations. The 6-chloropurine derivative was confirmed as the most active compound in this series.
Asunto(s)
Antivirales/química , Enterovirus/efectos de los fármacos , Purinas/química , Animales , Antivirales/síntesis química , Antivirales/farmacología , Chlorocebus aethiops , Purinas/síntesis química , Purinas/farmacología , Relación Estructura-Actividad , Células VeroRESUMEN
The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66-2muM). Minimal or no cytotoxicity was observed.
Asunto(s)
Antivirales/síntesis química , Enterovirus Humano B/efectos de los fármacos , Purinas/química , Animales , Antivirales/química , Antivirales/farmacología , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacología , Chlorocebus aethiops , Diseño de Fármacos , Humanos , Purinas/síntesis química , Purinas/farmacología , Relación Estructura-Actividad , Células VeroRESUMEN
Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Flavivirus/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Antivirales/química , Betacoronavirus/efectos de los fármacos , Betacoronavirus/enzimología , COVID-19 , Flavivirus/enzimología , Humanos , Concentración 50 Inhibidora , Pandemias , Virus ARN/efectos de los fármacos , Virus ARN/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.
Asunto(s)
Inhibidores Enzimáticos/química , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Peso Corporal/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Exosomas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Piridazinas/química , Piridazinas/metabolismo , Piridazinas/uso terapéutico , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/farmacología , Relación Estructura-ActividadRESUMEN
Thymidine phosphorylase plays an important role in angiogenesis, which is an attractive target for therapy of cancer and other diseases. In our continuous effort to develop novel inhibitors of thymidine phosphorylase, we have discovered that 6-halouracils substituted at position C5 by certain hydrophobic groups exhibit significant inhibitory activity against this enzyme. The most potent compounds bear a five- or six-membered cyclic substituent containing a pi-electron system at C5 and a chlorine atom attached at C6. 6-Chloro-5-cyclopent-1-en-1-yluracil 7a is the most efficient derivative in this study, with Ki = 0.20 +/- 0.03 microM (Ki/dThdKm = 0.0017) for thymidine phosphorylase expressed in V79 cells and Ki = 0.29 +/- 0.04 microM (Ki/dThdKm = 0.0024) for the enzyme purified from placenta.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Timidina Fosforilasa/antagonistas & inhibidores , Uracilo/análogos & derivados , Uracilo/síntesis química , Inhibidores de la Angiogénesis/química , Humanos , Cinética , Modelos Moleculares , Relación Estructura-Actividad , Uracilo/químicaRESUMEN
We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus.