RESUMEN
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
Asunto(s)
ADN/química , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/efectos de los fármacos , Oseltamivir/análogos & derivados , Ácidos Fosforosos/química , Antivirales/química , Antivirales/farmacología , Inhibidores Enzimáticos/química , Humanos , Virus de la Influenza A/enzimología , Virus de la Influenza A/fisiología , Gripe Humana/tratamiento farmacológico , Gripe Humana/enzimología , Gripe Humana/virología , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , Oseltamivir/química , Reproducibilidad de los Resultados , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismoRESUMEN
In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC50 values higher than 28µM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC50 values ranging from 3 to 60µM. The congeners with a fluoro substituent introduced at the 4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding to the C-terminal domain of the capsid protein.
