Soluble fragments of influenza virus PB2 protein capable of binding RNA-cap

摘要

The present invention relates to soluble fragments of the Influenza virus RNA dependent RNA polymerase subunit PB2 and variants thereof, and crystallized complexes thereof comprising an RNA cap analog. This invention also relates to computational methods using the structural coordinates of said complex to screen for and design compounds that interact with the RNA cap binding pocket. In addition, this invention relates to methods identifying compounds that bind to PB2 polypeptide fragments comprising the RNA cap binding pocket, preferably inhibit the interaction with RNA caps or analogs thereof, by using said PB2 polypeptide fragments, preferably in a high-throughput setting. This invention also relates to compounds and pharmaceutical compositions comprising the identified compounds for the treatment of disease conditions due to viral infections caused by negative-sense single stranded RNA viruses.

主权项

1. A complex comprising a soluble polypeptide fragment of Influenza virus RNA-dependent RNA polymerase subunit PB2 and an RNA cap analog, wherein said soluble polypeptide fragment is capable of binding to a RNA cap or analog thereof and remains in the supernatant after centrifugation for 30 min at 100,000×g in an aqueous buffer under physiologically isotonic conditions at a protein concentration of 5 mg/ml in the absence of denaturants in effective concentrations and is selected from the group consisting of
(i) a polypeptide fragment between residues 220 and 510 of SEQ ID NO: 1; (ii) a polypeptide fragment between residues 222 and 511 of SEQ ID NO: 2; (iii) a polypeptide fragment between residues 227 and 528 of SEQ ID NO: 3; and (iv) variants thereof having at least 80% sequence identity to polypeptide fragment according to (i), (ii) or (iii); and wherein the RNA cap analog is selected from the group consisting of m7G, m7GMP, m7GTP, m7 GpppG, m7 GpppGm, m7 GpppA, m7 GpppAm, m7 GpppC, m7 GpppCm, m7 GpppU, and m7 GpppUm.