The influenza neuraminidase (NA) inhibitors zanamivir oseltamivir and peramivir were all designed based on the knowledge that this transition state analogue of the cleaved sialic acid 2 2 3 N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. of substrate and IC50s were calculated after LCI-699 each 10 min interval up to 60 min. Results showed that without preincubation IC50s for the wild type viruses started high and although they LCI-699 decreased constantly over the 60 min reaction time the final IC50s remained higher than LCI-699 for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast for viruses with decreased susceptibility preincubation had minimal effect on the IC50s consistent with fast binding. Therefore this altered assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC50 and critically demonstrates the differential effect of incubation occasions around the IC50 and Ki values of wild type and mutant viruses for each of the inhibitors. Introduction Two licensed neuraminidase (NA) inhibitors (NAIs) are currently approved globally for the treatment and prevention of influenza zanamivir (Relenza?) and oseltamivir (Tamiflu?). A third compound peramivir has recently received approval in Japan and had emergency authorisation for limited use during the pandemic outbreak [1]. All compounds were designed based on the knowledge of the structure of sialic acid bound in the NA active site [2]. The transition state analogue of sialic acid 2 LCI-699 2 3 N-acetyl neuraminic acid (DANA) was known to be a poor inhibitor of the NA. Addition of an amino group at the 4-position of DANA led to around 100-fold enhancement of the inhibitory activity whereas the addition of a guanidinium group (zanamivir) led to around a 10 0 enhancement [3]. Addition of the guanidinium group led to zanamivir being a time dependent or Bmp1 slow binding inhibitor [3] [4]. The hypothesis for the slow binding of zanamivir is usually that a water molecule has to be displaced before the guanidinium group can bind tightly in the active site [4]. While oseltamivir is also a slow binding inhibitor this is thought to be due to the need for the rotation of the E276 in the enzyme active site [5] to accommodate binding of its hydrophobic side chain [6]-[8]. Peramivir contains both the guanidinium group as in zanamivir and a hydrophobic side chain as in oseltamivir. Hence it is also a slow binding inhibitor LCI-699 possibly impacted by both mechanisms [6]. Some NAs with mutations conferring resistance to the NAIs appear to have lost this slow binding phenotype [6] [8]-[11]. Thus in addition to an increase in IC50 loss of slow binding can also be a phenotypic marker of reduced susceptibility. Sensitivity to influenza NAIs is determined by two types of enzyme inhibition assays a fluorescent based assay which uses 4-Methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) [12] and a chemiluminescent assay based on the NA-Star substrate [13] [14]. The inhibition assay includes preincubation of NA with its inhibitor initiation of the enzymatic reaction by addition of substrate and finally addition of a high pH answer which stops the reaction and enhances the fluorescent or chemiluminescent signal. Protocols for the fluorescent assay vary between different laboratories for the preincubation occasions and temperatures assay incubation time and buffers used all of which can impact on the IC50 [14]. Hence there is a need for a standardized assay to enable comparisons of results between different laboratories. There has been no study of how incubation occasions affect IC50s although Pegg et al. [4] reported that for binding of zanamivir to an N2 NA the apparent Ki varied by 10 0 depending on the incubation conditions. The availability of more sensitive fluorimeters with kinetics functions means we can continuously monitor changes in enzyme activity and therefore changes in IC50 with time. We have now modified the basic MUNANA assay to a real time assay and have developed what we term IC50 kinetics assays. This expands the information obtained from inhibition assays to also provide information about the slow or fast binding phenotype of an NA [11]. Thus this approach provides additional information about potential NAI resistance and the impacts of mutations on inhibitor binding. The chemiluminescent assay commercialised as NA-Star? (Applied Biosystems) is a rapid reaction with a substrate half life of around 15 min and a very low signal strength necessitating the addition of an enhancer. Hence this.