Drug Discovery


Figure 3 Bruton’s Tyrosine Kinase


(BTK) and Dbf-4 Dependent Kinase (DDK) Nanoluc constructs were transient


expressed in HEK-293 cells in 96-well adherent format. Cells were treated for two hours with Dasatinib (DAS) and


Ibrutinib (IB) (30nM and 1uM) then washed to remove the


compounds. Respective tracer for each kinase were added


and the rate of dissociation of the compounds measured every five minutes. Data


represents mean +/- standard deviation for n=4 replicate wells per treatment


often run using radiolabelled material in the pres- ence of physiological levels of ATP. More recently, techniques such as affinity-based chemopro- teomics use cell lysates as a source of kinases fol- lowed by treatment with compound then LC-MS analysis of material bound to the kinobeads. Cellular kinase-based assays include cellular ther- mal shift assays (CETSAs) that do not require cell lysis during compound incubation but are depen- dent on the compound stabilising the kinase pro- tein prior to heat treatment and analysis. However, CETSA does not provide a quantitative measure of drug affinity. Other assay technology providers have kits designed to detect kinase activ- ity in cell lysates isolated from pre-treated cells. These provide a snap shot in time of activity inside the cell at the point of lysis but do not provide kinetic data. All of the above assay formats generate data on


binding, either reversible or irreversible, and potency of a molecule against the kinase of interest either as a recombinant protein or within a cell lysate, either before or after treatment of the cells with a fixed concentration of compound. None of these approaches can determine the duration of time the compound was in contact with the target kinase. Consider you are part of a kinase project development team and were presented with the option of choosing between two compounds that bind the target kinase, one compound has binding affinity of 1nM and residency time (eg the duration the compound occupies the binding site of the tar- get kinase) of, say, 10 minutes, versus a second compound with binding affinity of 100nM and res- idency time of five hours. Both have equivalent cel-


58


lular penetration and DMPK properties, which one would you choose to develop? My choice would be the compound with the longer residency time. Why? In a digital world the inhibition of the digital signal for a longer duration may have a more pro- found effect on the kinase pathway than simply the binding affinity. In this digital world what are the implications for selectivity versus affinity versus residency time? Is residency time and selectivity all part of the same puzzle? For example, if a kinase inhibitor is relatively non-selective but has a resi- dency time favouring the inhibition of the target kinase does this make it a better drug? This target engagement profile defined by both the on-rate (binding) and, equally important, the off-rate (res- idency) of the compound, for both the target kinase and other kinases inside and outside the tar- get kinase family may represent a more clinically efficacious measurement of the activity of the com- pound. Furthermore the target engagement profile may, in part, explain the mechanism of action asso- ciated with off target safety concerns. Target engagement parameters are fundamental


to drug efficacy. It is the affinity and residence time at primary and secondary target kinase that can often underlie the therapeutic potential of lead can- didates. However, it is debatable if target potency or binding kinetics are most relevant to establish intracellular SAR for a lead series of compounds. Drug efficacy is significantly influenced by drug res- idency time – ie the associative on rate (kon) and dissociative off rate (koff) of the compound inter- acting with the target kinase. It would be desirable to perform target engagement in live cells in a kinet- ic manner, using full-length kinase proteins without


Drug Discovery World Spring 2018


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