An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease

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 center of this enzyme 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. There is large interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We here focused on a series of β-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, to build a 3D pharmacophore model. Besides, we developed a molecular beacon (MB)-based PA-Nter assay which enables to compare the inhibition versus Mn(2+) or Mg(2+), the latter probably being the biologically relevant cofactor. This real-time MB assay allows to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter with IC50 values comparable to that of the prototype L-742,001 (i.e. below 2 μM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg(2+) or Mn(2+). Three compounds ( 10: , with a pyrrole scaffold, and 40: and 41: , with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values: 64-95 μM), and were proven to affect viral RNA synthesis. Our approach to integrate complementary enzymatic, cellular and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.