Design, Synthesis, and in Vitro Biological Evaluation of 1H-1,2,3-Triazole-4-carboxamide Derivatives as New Anti-influenza A Agents Targeting Virus Nucleoprotein

Abstract

The influenza virus nucleoprotein (NP) is an emerging target for anti-influenza drug development. Nucleozin (1) and its closely related derivatives had been identified as NP inhibitors displaying anti-influenza activity. Utilizing 1 as a lead molecule, we successfully designed and synthesized a series of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents. One of the most potent compounds, 3b, inhibited the replication of various H3N2 and H1N1 influenza A virus strains with IC(50) values ranging from 0.5 to 4.6 μM. Compound 3b also strongly inhibited the replication of H5N1 (RG14), amantidine-resistant A/WSN/33 (H1N1), and oseltamivir-resistant A/WSN/1933 (H1N1, 274Y) virus strains with IC(50) values in sub-μM ranges. Further computational studies and mechanism investigation suggested that 3b might directly target influenza virus A nucleoprotein to inhibit its nuclear accumulation.

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

“…Nucleozin-induced cytoplasmic aggregates of NP have been noted before but interpreted to be newly synthesized NP on the way into the nucleus (13,16,17). However, the timing of the effect we observed, as well as the inclusion of Rab11 in the aggregates (Rab11 interacts with RNPs but not non-RNP NP [6,9]), suggested that the drug was altering the trafficking of RNPs.…”

“…Nucleozin aggregates outgoing viral RNPs (vRNPs) and Rab11 in the cytoplasm. Previous work has suggested two mechanisms of action for nucleozin: direct inhibition of RNP transcription, or a block to NP trafficking, postulated to be prior to nuclear import and RNP assembly (13,(15)(16)(17). However, neither mechanism seemed a plausible explanation of the late-acting drug effect, since vRNA synthesis, and therefore, by extrapolation, RNP formation, was near normal.…”

“…One attractive strategy is to target NP, since this protein plays many essential roles in vRNA synthesis, genome trafficking, and virus assembly (4,12). Recently, several groups identified NP-interacting molecules able to inhibit virus replication (13)(14)(15)(16)(17). These compounds, nucleozin or related derivatives, bind to at least two sites on NP and appear to act as "molecular staples" that promote the formation of higher-order oligomers or aggregates by stabilizing monomer interactions.…”

“…These compounds, nucleozin or related derivatives, bind to at least two sites on NP and appear to act as "molecular staples" that promote the formation of higher-order oligomers or aggregates by stabilizing monomer interactions. Evidence for this comes from various in vitro biophysical analyses of purified NP, cocrystallization studies with the inhibitor, and fluorescence microscopy of infected, drug-treated cells (13,(15)(16)(17). Further support for this model comes from the identification of several resistance mutations in NP that map to the separate drug-binding pockets (15)(16)(17).…”

“…Further support for this model comes from the identification of several resistance mutations in NP that map to the separate drug-binding pockets (15)(16)(17). The drug-induced aggregation evidently interferes with NP function, because the compounds potently inhibit virus replication in tissue culture and infected animals (13,(15)(16)(17). However, the precise mechanism(s) of action has not been investigated in detail.…”