Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase

Abstract

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

Peering Into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness

“…The PB2 (polymerase basic protein 2) subunit is one of the major virulence and host transmission determinants [34][35][36][37][38], forming the ribonucleoprotein (RNP) complex with PB1 and PA (see Figure 3A). PB2 is proposed as a drug target due to its cap-snatching mechanism for mRNA transcription (see Figure 3B, [39][40][41]). Primarily localized in the nucleus, the PB2 subunit can also accumulate in the mitochondria, interacting with the mitochondrial antiviral signaling protein (MAVS) while inhibiting MAVS-mediated beta interferon (IFN-β) expression [42].…”

“…Notably, only PB2 proteins of seasonal human influenza viruses associate with the mitochondria given their asparagine residue at amino acid residue 9 while the PB2 proteins of other avian influenzas viruses with aspartic acid do not [42]. [39]) modified with QuteMol [43].…”

“…One example of such rational drug designs is JNJ7918, an oral small molecule mimicking broadly neutralizing antibodies, that was improved for binding and virus neutralization, and further refined for stability and oral bioavailability [111]. Another oral inhibitor is Pimodivir (VH-787, Figure 3B), a novel inhibitor of influenza virus replication, inhibits cap binding to the PB2 subunit [113] and recently entered the third phase of clinical trials [39]. However, several major drug-mutations in the PB2 subunit has already been observed (such as F404Y and M431I and H357N, [39]) to show resistance.…”

“…Another oral inhibitor is Pimodivir (VH-787, Figure 3B), a novel inhibitor of influenza virus replication, inhibits cap binding to the PB2 subunit [113] and recently entered the third phase of clinical trials [39]. However, several major drug-mutations in the PB2 subunit has already been observed (such as F404Y and M431I and H357N, [39]) to show resistance. Thus, the mapping of PB2 single-amino-mutations could prepare against Pimodivir resistance [114].…”

Comprehensive Profiling of Mutations to Influenza Virus PB2 That Confer Resistance to the Cap-Binding Inhibitor Pimodivir

“…Identifying resistance mutations can guide the clinical deployment of appropriate antivirals and aid in developing new antivirals that are less susceptible to resistance. Known resistance mutations to pimodivir, as with previous influenza antivirals, have typically been identified one at a time as they arose over the course of antiviral treatment either in patients or in the laboratory in cell culture [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. These mutations are located primarily in the PB2 7-methyl GTP cap-binding pocket [ 17 ], as well as the mid-link domain where additional contacts with pimodivir are made [ 24 , 25 ].…”