Drug Discovery


Figure 1


Binding of Dasatinib (DAS) and Ibrutinib (IB) to Bruton’s Tyrosine Kinase (BTK)


Nanoluc. HEK293 cells were transiently transfected with a


Nanoluc-BTK construct. Cells were incubated with a fixed concentration of tracer for two hours then treated with


DAS or IB as a dose response. The degree of loss of luminescence signal is


proportional to the binding of DAS or IB to the BTK receptor. Each curve is a


separate replicate on the same 96-well plate


A survey of the scientific and patent literature


(ChEMBL, SciFinder and PubMed) revealed that many of the 243 clinical kinase inhibitors exam- ined in this study are poorly characterised in terms of their selectivity. The process of assessing selec- tivity is both costly (often outsourced to CROs) and time-consuming and therefore not often accomplished in a comprehensive manner during drug development. Rarely is a complete profile of activity of the compound studied in detail across many kinases at many compound concentrations. In an effort to bring a degree of clarity to this area, Klaeger et al profiled the activity of numerous inhibitors in a dose-dependent manner on many kinases at near thermodynamic equilibrium in material isolated from cellular lysates using a kinobead approach. This binding data enabled the development of a new selectivity metric termed CATDS (concentration- and target-dependent selectivity), a metric capturing aspects of both tar- get binding and drug mechanism of action. “CATDS measures the reduction of the binding of a particular protein to kinobeads at a particular compound concentration relative to the summed reduction of binding of all proteins at that concen- tration”. CATDS values close to one or zero indi- cate either selective or non-selective compounds respectively. The group has published this data in an interactive database called ProteomicsDB (www.proteomicsdb.org) or as a PDF on Pro- teomeXchange (www.proteomexchange.org). This represents a great resource for kinase hunters and candidate drug reprofilers. The kinobead approach is a competition assay


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followed by a ‘pull down’ then analysis of bead bound material using mass spectrometry. Briefly, beads labelled with kinase binding compounds are incubated with cell lysates in the presence of test compounds at different concentrations to allow competition to occur between the test compound and the bead bound compound for binding to the kinase derived from the lysate. Kinase captured by the bead is precipitated, eluted from the bead and analysed by LC-MS/MS. Specific compound bind- ing to a specific kinase will sequestrate that kinase, preventing it from binding to the bead and thereby decreasing the amount of that kinase being pulled down in the assay. Non-specific compound binding will generally inhibit the binding of kinases to the beads and result in a more non-specific pull down result. A disadvantage of this technique is that it utilises the promiscuity of the ATP binding pocket to pull down kinases and therefore has limited use as this approach is aimed at Type I compounds binding active enzyme. More traditional screening assays designed to


detect compound binding to protein kinases tend to be biochemical in nature using recombinant kinases, either full length or partial sequence. While these assays can give highly accurate, reproducible binding kinetics, they fail to predict how a kinase may function inside a cell. Differences in localised ATP concentration inside the cell, the use of truncated kinase domains, absence of appropriate cellular co-factors and dif- ferences in kinase activation states can result in the development of compounds that appear potent in a biochemical assay but this activity


Drug Discovery World Spring 2018


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