Last week I was presenting my DPhil work. In one of my projects I address the reasons for inhibitor selectivity in PIM protein kinase family. PIM kinases play key roles in signalling pathways and have been identified as oncogenes long time ago. Slightly unusual for protein kinases ATP-binding sites and cancer roles have prompted the investigation of potential PIM-selective inhibitors for anticancer therapy. Due to overlapping functions of the three PIM isoforms, efficacious inhibitors should bind to all three isozymes. However, most reported inhibitors show considerable selectivity for PIM1 and PIM3 over PIM2 and the mechanisms leading to this selectivity remain unclear.

Figure 1. Workflow of the sequence and structure analysis of PIM kinases

To establish the sequence determinants of inhibitor selectivity we investigated the phylogenetic relationships of PIM kinases and their structural conformations upon ligand binding (Figure 1). Together with my OPIG supervisor Charlotte Deane we predicted a set of candidates for site-directed mutagenesis as illustrated in Figure 2. The mutants were designed to convert PIM1 residues into analogous PIM2 residues at the same positions.

I then moved to the wetlab to test the hypotheses experimentally. Under guidance of Oleg Fedorov, I screened the SGC library of kinase inhibitors using differential scanning fluorimetry (DSF). After comparing melting temperature shift values across the PIM kinases and mutants, a set of potent inhibitors with different chemical scaffolds have been selected for quantitative binding analysis. I worked with Peter Drueker’s team at Novartis on PIMs enzymology, where I measured activities, Km values for ATP and IC50s using mobility shift assay. For my final set of measurements I performed isothermal titration calorimetry (ITC) experiments back at the SGC and determined binding constants and enthalpic/entropic contributions to the total free energy of ligand binding.

Figure 2. An overlay of PIM1 and PIM2 structures (P-loop and hinge regions), the mutated residues are shown as sticks

The data are yet to be published, I only briefly state the results here. The hinge mutant E124L demonstrated reduced thermal stability probably due to removal of E124-R122 salt bridge. The P-loop mutants had intermediate Km ATP values between PIM1 and PIM2, indicating that those residues could be responsible for stronger ATP binding in PIM2. As shown in Figure 2, the residues are located at the tip of the P-loop and might have involvement in the P-loop movement. Importantly, three mutants have shown reduced affinity to inhibitors validating my initial hypotheses.

Ideally having PIM1 and PIM2 co-crystal structures with the same inhibitors would allow direct comparison of the binding modes. So far I was able to solve apo-PIM2 structure in addition to the single PIM2 pdb, which will be deposited shortly.

I will update you soon about on my second project which involves more mutants, type II inhibitors, equilibrium shifts and speculations about conformational transitions. Keep visiting us!