Selective mutation in ATP-binding site reduces affinity of drug to the kinase: a possible mechanism of chemo-resistance

Excerpt

Mutation in protein kinases is very common in cancer and causes constitutive activation of protein kinases resulting in hyper activation of survival pathways. Recent studies suggest that mutation in ATP-binding site of kinases confers resistance to the chemotherapy [1]. Thus, understanding how mutations reverse to the drug will be beneficial for effective drug development against cancer. We explored this issue using a family of protein serine/threonine kinases as a model. The protein kinase C (PKC) family of protein serine/threonine kinases consists of 10 proteins encoded by 9 genes, which is known to involve in many cancers [2]. We first modeled kinase domain structure of all 10 PKC isoforms using SWISS-MODEL and further verified using ProSA. Modeled structures were processed for energy minimization in a water cube using GROMACS. Autodock4 was used for docking inhibitors in kinase domains. Initially, we used PDK1 kinase domain with LY333531 to validate our system. We observed that our modeled structure docked with LY333531 perfectly overlapped with X-ray structure of PDK1 kinase domain and LY333531 complex (Fig. 1a) suggesting that our method is reliable. Furthermore, we docked LY333531 in PKCβ2 kinase domain and observed perfect docking of inhibitor in ATP-binding site (Fig. 1b–d). Three residues (K, D, and D) of VAIK, HRD, and DFG motifs are catalytically important and highly conserved in eukaryotic protein kinases. Threonine in activation loop is also highly conserved in PKC and also some AGC kinases. Though PKC family is divided into three sub-families [2], we asked the question that whether conserved residues are also structurally conserved within the family. To address this question, we determined torsion angles of respective residues and observed that catalytically important residues are also structurally well conserved (Fig. 1e). Though we found that catalytically important residues are structurally well conserved across the family, we set out to determine inhibitor-binding residues across the 10 kinase domains of PKC isoforms. LY333531 was used as a ligand to determine inhibitor-interacting residues. Docking studies identified 14 residues which are critical for the interactions (Table S1), and these residues are also well conserved within the family (Fig. 1f). Then, we determined whether inhibitor-interacting residues are common for other kinase inhibitors. PKCβ2 was used as a macromolecule to dock nine different kinase inhibitors. We observed that most of residues are common for the interaction (Table S2) suggesting similar mechanism is involved in inhibition. Thus, we suggest that conventional kinase inhibitors are designed to interact within the similar binding pocket. Since all 10 isoforms share similar residues for interaction with inhibitors, we used PKCβ2 kinase domain to determine importance of individual residues involved in interaction with inhibitors. We replaced eight individual residues with glycine and determined binding energy with LY333531 using Autodock4. We observed that mutation in any of those residues decreases binding energy and increases inhibitory concentration (Table S3). Taken together, our results suggest that conventional kinase inhibitors target similar binding pocket in ATP-binding site of kinase domain and mutation in this pocket results in resistance to the inhibitor which is often observed in cancer patients [3].

…