Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal

Abstract

The influenza virus neuraminidase inhibitors are normally slow binding inhibitors, but many mutations leading to resistance, also result in the loss of the slow binding phenotype. Mutations can also affect the rate of dissociation of the inhibitors from the neuraminidase, but the assays to measure this require large amounts of virus and are time consuming. To more fully understand the impacts of mutations on the binding and dissociation of the neuraminidase inhibitors we have developed a solid phase reactivation assay, which can use small amounts of crude virus sample bound to an ELISA plate. Multiple viruses can be assayed simultaneously against multiple inhibitors. Using this assay we have demonstrated differences in the relative rates of dissociation of the inhibitors and reactivation of enzyme activity among different influenza A and B viruses for zanamivir, oseltamivir and peramivir. In general oseltamivir dissociated the fastest, and dissociation of peramivir was much slower than both the other inhibitors. Viruses with H274Y, E119V and E119G mutations demonstrated faster dissociation of the inhibitor to which they were resistant. Dissociation of zanamivir and oseltamivir were faster from the D197E mutant, but not of peramivir.

Structural and Functional Analysis of Anti-Influenza Activity of 4-, 7-, 8- and 9-Deoxygenated 2,3-Difluoro-N-acetylneuraminic Acid Derivatives

“…Despite large sequence differences between the (H1N1)pdm09 and (H5N1) NAs, their kinetics profiles were very similar for each of the inhibitors (Figure 2B, Figure 2C), but both were different to the seasonal (H1N1) NA and its H274Y mutant (Figure 2A). Consistent with previous findings, clade 1 (H5N1) NA showed faster dissociation of 1 than clade 2 NA 16,19 (Figure 2C and Supporting Information Table S1).…”

“…The IC 50 kinetics assay monitors the changes in IC 50 values for each 10 min over 60 min, in two separate reactions with and without preincubation. This provides information on not only relative affinity (IC 50 ), but the relative rates of binding and dissociation of each of the compounds 16,19 A solid phase reactivation assay monitors the time to recover activity after removal of inhibitor over several hours. 19 We have previously used these assays to demonstrate slow binding kinetics and rates of dissociation of the NAIs consistent with those observed using purified NA in the classic assays.…”

“…This provides information on not only relative affinity (IC 50 ), but the relative rates of binding and dissociation of each of the compounds 16,19 A solid phase reactivation assay monitors the time to recover activity after removal of inhibitor over several hours. 19 We have previously used these assays to demonstrate slow binding kinetics and rates of dissociation of the NAIs consistent with those observed using purified NA in the classic assays. 16,19−22 Additionally, we have shown that while wild type viruses generally demonstrate slow binding of the known NAIs, the NAs of resistant mutants demonstrate rapid binding and dissociation of the NAIs.…”

“…16,19−22 Additionally, we have shown that while wild type viruses generally demonstrate slow binding of the known NAIs, the NAs of resistant mutants demonstrate rapid binding and dissociation of the NAIs. 11,15,16,19,23,24 We have used these assays here to compare relative inhibition of the NAs of a large panel of wild type and drug resistant mutants by each of the deoxy derivatives 3−6. For the sake of simplicity, we will refer to inactivation as binding and reactivation as dissociation as we are also comparing them to the binding and dissociation of competitive NAIs.…”

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

“…For example, there has been rapid emergence of IAV strains that are resistant to amantadine and rimantadine, and these antivirals are thus no longer recommended for anti-influenza treatment (Barr et al, 2007;Bright et al, 2005). Resistance against neuraminidase inhibitors, such as oseltamivir and zanamivir as well as newly developed peramivir and laninamivir, has also been reported (Barrett & McKimm-Breschkin, 2014;Hurt et al, 2009;Kamali & Holodniy, 2013;Orozovic, Orozovic, Jarhult, & Olsen, 2014). Therefore, it is increasingly urgent to develop drugs that target host factors rather than viral proteins, which is less likely to cause drug resistance.…”