Development of PKN2 chemical probes to enable drug discovery

Kinase drug discovery has been a relatively successful endeavour: since the approval of imatinib in 2001, another 51 kinase inhibitors have been approved for treating patients by targeting these signalling proteins when they are implicated in disease. That said, more than 80% of the near 600 human protein kinases lack suitably potent and selective compounds to validate their role in biology. The limited availability of such compounds is reflected in research output where a lot of the published work on kinases has been done on a relatively small number of targets. This thesis work is part of a collaborative effort with the Structural Genomics Consortium to provide suitable chemical probes for the, so-called, “dark kinome” to find the drug targets of the future. The aim of this PhD project was to develop a suitable chemical probe for a relatively understudied kinase, protein kinase N2 (PKN2). PKN2 is an AGC kinase that has reported roles in cell cycle, adhesion and transport amongst others. It has also been postulated as a target of interest in several cancers, heart failure and inflammation, but currently lacks selective inhibitors to convincingly validate its role in these various diseases. The European Molecular Biology Laboratory (EMBL) ChEMBL database literature screen has record of 1200 compounds from across the literature with biological activity data against PKN2. Following a triage of the data, three relatively small but potent PKN2 inhibitors were identified (10, 11 and 12). The Structure Activity Relationships of these compounds were explored by synthesis of a chemical library resulting in three series of compounds. The potencies and selectivities of these compounds within the PKN kinase family were evaluated using TR-FRET assay experiments. Selectivity within the wider kinome was investigated using the commercial DiscoverX KINOMEscan® panel containing nearly 500 kinases. This work showed it is possible to potently and selectively drug PKN2 within the PKN kinase family and the wider kinome. One compound (187) shows Ki = 2.7 nM in vitro potency towards PKN2, 5-fold selectivity for PKN2 over PKN1 and inhibits just 9/400 wild-type kinases in the DiscoverX screen above 70%. This compound was further optimised to yield a compound (312) with PKN2 Ki = 6.7 nM with 18-fold selectivity over PKN1 – with wider selectivity data pending at time of writing. This is a major step towards delivering a suitable probe for this relatively neglected kinase to validate its potential as a new drug target.