Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity, was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase and retain potency against drug-resistance mutations. Our discovery that water molecules play an essential role in the drug binding is reported. Femtosecond experiments and theory expose molecular level dynamics of rilpivirine bound to HIV-1 reverse transcriptase. The two nitrile substituents (-CN), one on each arm of the drug, have vibrational spectra consistent with their protein environments being similar in crystals and in solutions. Two-dimensional vibrational-echo spectroscopy reveals a dry environment for one nitrile while unexpectedly the other is hydrogen-bonded to a mobile water molecule, not identified in earlier X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure indeed reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond, despite the enlargement and structural variation of the binding pocket, may help retain the potency of rilpivirine against the pocket mutations.