Drug Discovery


does not translate to cellular activity. Biochemical assays often do not recapitulate the physiological complexity of kinase biology encountered inside an intact cell, be it a normal or diseased cell. There is therefore a need to examine the proper- ties of kinase inhibitors in a more physiological cellular environment rather than an isolated bio- chemical system.


Biology is digital, pharmacology is analogue… In a cell, it costs ‘money’ (ATP) to phosphorylate proteins. This process is thermodynamically expen- sive, therefore it is unlikely that a cell will generate concentrations of phosphorylated proteins. A cell will manufacture a protein, phosphorylate it, trans- mit the signal and await a digital return signal – a feedback loop. If no feedback signal is forthcoming then the cell will manufacture and phosphorylate another protein and continue this process until the system returns a digital stop signal. Cellular kinase pathways are constantly switching on and off in this digital manner controlling cell functions. Proteins are dephosphorylated and rephosphorylat- ed or degraded and remade. Each phosphorylation event is a digital signal, on or off, one or zero. However, in pharmacological we think in terms of concentrations of proteins and concentrations of compounds. Who hasn’t run a Western blot to cor- relate a cellular response to a change in concentra- tion of a specific protein only to find the correlation was poor or non-existent? As demonstrated in this paper, digital signals are much more complex. The authors analysed the phosphoproteome (all phos-


phorylated proteins) of BT-474 cancer cells after treatment with the EGFR/HER2 inhibitors lapa- tinib, afatinib, cancertinib, dacomitinib and sapi- tinib to a depth of approximately 15,000 phospho- rylation sites. Analysis revealed a large number of statistically significant regulated phosphorylation events for each drug with more than 200 common phosphorylation events across all five compounds. Arguably this ‘shared’ commonality profile between the compounds could equate to the ‘thera- peutic footprint’ of these compounds represented in BT-474 cells, but what about the other 14,800 phosphorylation events, what do they tell us? Furthermore, the experiment literally represents a snap shot in time and drug concentration, the cellu- lar environment at the exact moment in time when the experiment ended and the cells were lysed. When developing compounds in the pre-clinical


phase of a kinase drug discovery programme, both biologists and chemists tend to drive structure- activity relationships (SAR) through league tables of kinetic binding data in an attempt to increase the potency of compounds and drive selectivity through affinity. This is followed by testing of a few chosen compounds in a selectivity panel of representative members of kinase families tested at selected doses of compounds to give a generalised selectivity profile. Subsequently, cellular penetra- tion in vitro is followed by drug disposition, phar- macokinetics and metabolism in a well plate based assay followed by in vivo studies. Techniques such as Surface Plasmon Binding (SPR) are often used to study the interaction of compound and kinase. In addition, biochemical kinase activity assays are


Figure 2 Binding of Dasatinib (DAS) to Dbf-4 Dependent Kinase (DDK) Nanoluc. HEK293 cells were transiently transfected with a Nanoluc-DDK construct. Cells were incubated with a fixed concentration of tracer for two hours then treated with DAS as a dose response. The degree of loss of luminescence signal is proportional to the binding of DAS to the DDK receptor. Each curve is a separate replicate on the same 96 well plate. Ibrutinib was also tested but showed no competition with the tracer on DDK (data not shown)


Drug Discovery World Spring 2018


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