HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

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The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

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