De Algemene Inhibitie van Proteïne Kinase D

Cancer remains one of the leading causes of death globally, with approximately 10 million deaths each year. This horrifying statistic underscores the need for improved treatment methods. This thesis contributes to the ongoing effort to develop effective anti-cancer therapies by designing and developing all-round protein-kinase D (PKD) inhibitors. PKD has been identified as a promising drug target within the human kinome. This work seeks to design multi-purpose PKD inhibitors that offer versatility in their different mechanism of action (MOA), designed to provide researchers with a toolkit that allows for the selection of inhibitors tailored to specific experimental or therapeutic needs. In first instance, previous research from this research group led to the discovery of novel pyrazolo[3,4-d]pyrimidine-based pan PKD inhibitors, 17m (IC50: 17-35 nM) and 18m (IC50: 55-78 nM). These inhibitors exhibit an ATP-competitive mechanism by targeting PKD's active site. While these inhibitors show excellent potency against PKD, they are expected to be less selective across the human kinome, as was previously observed for the currently known PKD inhibitors. These all show excellent potency against PKD, however, they lack selectivity and are, therefore, hampered from entering clinical trials. To address these selectivity issues, a second group of PKD inhibitors was developed to exploit the presence of a cysteine residue in PKD's active site—a feature found in only about 10% of kinases. The covalent inhibitors were developed by incorporating an electrophilic warhead (acryl amide) onto the pyrazolo[3,4-d]pyrimidine scaffold of 18m and derivatives thereof. A library of six compounds was synthesised and evaluated for biological activity. The compounds showed to be potent PKD2 inhibitors, with compound 2.23c showing the highest inhibition out of all the tested potential covalent inhibitors. These inhibitors have the potential to provide increased selectivity, but the covalent behaviour still needs to be confirmed through additional biological experiments. Upon confirmation, these compounds could serve as an excellent starting point in the further optimisation towards potent covalent PKD inhibitors. The design and development of PKD inhibitors face, next to kinome selectivity, another big challenge. All known PKD inhibitors are pan inhibitors - inhibiting all three isoforms of PKD (PKD1, PKD2 and PKD3). Strikingly, these isoforms have opposing roles in cancer cell signalling. Recent studies showed that PKD1 generally exhibits tumour-suppressive properties, while PKD2 and PKD3 are more frequently linked to pro-oncogenic signalling. These understandings are the foundations for the third group of PKD inhibitors, specifically PKD2-specific inhibitors. Using computer-aided drug design, the project aimed to develop allosteric PKD2-selective inhibitors that uniquely bind to and stabilise the interface of PKD2 in its auto-inhibited state. The results obtained through virtual screening were combined with a synthetic optimisation, hereby expanding the library for biological analysis. A set of five isoform-selective allosteric inhibitors demonstrated modest potency. Further biological experiments confirmed the envisioned stabilising effect of the inhibitors. Finally, in a novel approach, the thesis also explored the development of PROTACs (proteolysis-targeting chimeras) based on both the active site-directed inhibitors and the PKD2-selective allosteric inhibitors. The goal was to design and develop two PROTACs that induce the degradation of PKD, rather than merely inhibit its activity. While they showed low inhibition potential, PROTAC strategies don't rely on high affinity with the protein of interest and further studies should elucidate whether the developed PROTACs are able to degrade PKD2 in cellulo. If successful, these PROTACs pave the way for further optimisation towards a novel approach in PKD inhibition.