In search of novel metal-chelating influenza virus PA inhibitors: diverse scaffolds and even more diverse mechanistic profiles

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic centre resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg 2+ or Mn 2+ ions which are critical for its catalytic function. The flexible PA active site can be targeted by compounds with metal-chelating scaffolds. We designed new molecules from diverse chemical classes (i.e. ?-diketo acid derivatives, 2-hydroxybenzamides, thiosemicarbazones, N-acylhydrazones and dihydroxyindole-2-carboxamides) and found several to cause strong inhibition of influenza PA-Nter in enzymatic assays. Our SAR analysis provided a relevant starting point for development of more potent and selective PA inhibitors (PAIs). In addition, we demonstrated that the setup of the enzymatic assay (i.e. substrate, metal cofactor and type of readout) should be carefully chosen during PAI evaluation. Whereas most enzymatic studies with isolated PA-Nter have indicated that the enzyme is considerably more active in the presence of Mn 2+ compared to Mg 2+ , our novel molecular beacon based assay works equally well with Mg 2+ as with Mn 2+ . Since the intracellular concentration of free Mg 2+ is at least 1000 fold higher than that of Mn 2+ , magnesium may be more biologically relevant, and evaluation of potential PAIs against both metals (as possible with our molecular beacon assay) seems recommended. Some of the metal-chelating compounds displayed anti-influenza virus activity combined with favourable selectivity in cellular assays. However, although conceived as PAIs, for most of the inhibitors, the antiviral target in cell culture seems unrelated to PA, but rather associated with an early (virus entry) or late (maturation or release) event in the influenza virus life cycle. Clarification of the precise mode of action of these early lead compounds is currently ongoing.