Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors

Abstract

The clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC50]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC50). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PAN target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro. Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs.

Advances in respiratory virus therapeutics – A meeting report from the 6th isirv Antiviral Group conference

“…This high-throughput focus-forming assay was compared against the plaque assay for its ability to detect reduced susceptibility to baloxavir acid (the active form of baloxavir marboxil) among reference strains. The reference strains included viruses with and without the I38T substitution, which conferred a 40-to 50-fold reduction in susceptibility to baloxavir in both assays, consistent with the fold change observed for this substitution in other studies (Jones et al, 2018;Omoto et al, 2018). A panel of NAI-resistant viruses and their sensitive counterparts were also evaluated for their susceptibility in both assays, which showed similar results and validated the focus-forming assay as an adequate surveillance tool.…”

“…Selection studies in cell culture and analysis of viruses from drugtreated patients in clinical trials have revealed the principal pathways to reduced susceptibility to baloxavir Jones et al, 2018;Omoto et al, 2018) and pimodivir (Byrn et al, 2015;Trevejo et al, 2018). In baloxavir studies, treatment-emergent amino acid substitutions most frequently occurred at conserved position I38 (I38 F/ M/T) in the PA endonuclease active site and conferred reduced susceptibility by 10-57-fold in influenza A and 2-6-fold in influenza B. Substitutions at position S324 (S324C/I/N/R) in the cap-binding site of PB2 were recognized as the leading pathway for a 60-160-fold reduced susceptibility to pimodivir.…”

Aryl and Arylalkyl Substituted 3‐Hydroxypyridin‐2(1H)‐ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease

“…Baloxavir marboxil (Xofluza®) is a recently approved prodrug that targets the cap‐snatching endonuclease. However, resistance to Xofluza® has been reported in both clinical and in vitro systems, but with a significant decrease in viral fitness …”

Umifenovir susceptibility monitoring and characterization of influenza viruses isolated during ARBITR clinical study

“…Variants (I38T, I38F, and E23K) with reduced susceptibility to baloxavir marboxil were isolated from treated patients during phase II clinical trials . The same I38T PA substitution also caused resistance of influenza A(H1N1) viruses serially passaged in MDCK cells with RO‐7, an investigational endonuclease inhibitor that is similar but not identical to baloxavir marboxil …”

“…19 The same I38T PA substitution also caused resistance of influenza A(H1N1) viruses serially passaged in MDCK cells with an investigational endonuclease inhibitor that is similar but not identical to baloxavir marboxil. 20 Umifenovir (C 22 transition to its fusogenic state, and prevents HA-mediated membrane fusion during influenza virus infection. 21 Co-crystal structure of umifenovir with the influenza virus HA glycoprotein indicates that umifenovir binds in a hydrophobic cavity at the interface of the HA protomers in the upper region of the stem.…”