RESUMEN
Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus de la Influenza A/enzimología , Gripe Aviar/tratamiento farmacológico , Neuraminidasa/antagonistas & inhibidores , Oseltamivir/análogos & derivados , Oseltamivir/farmacología , Animales , Antivirales/síntesis química , Sitios de Unión , Aves/virología , Subtipo H7N1 del Virus de la Influenza A/química , Subtipo H7N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N1 del Virus de la Influenza A/enzimología , Subtipo H7N3 del Virus de la Influenza A/química , Subtipo H7N3 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N3 del Virus de la Influenza A/enzimología , Virus de la Influenza A/química , Virus de la Influenza A/efectos de los fármacos , Gripe Aviar/enzimología , Modelos Moleculares , Neuraminidasa/química , Neuraminidasa/metabolismo , Oseltamivir/síntesis químicaRESUMEN
CONTEXT: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors. OBJECTIVE: Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay. MATERIALS AND METHODS: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor. RESULTS: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments. DISCUSSION AND CONCLUSIONS: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.