casein-kinase-ii and 2--deoxyuridylic-acid

Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent in vitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinase IIα as a control and tested a series of its inhibitors against the TS template. Ellagic acid and apigenin were identified as TS inhibitors, and various flavonoids were selected and synthesized in a second-round selection. The compounds were demonstrated to be active in the low-micromolar range.

Topics: Algorithms; Apigenin; Binding Sites; Casein Kinase II; Deoxyuracil Nucleotides; Drug Repositioning; Ellagic Acid; Humans; Hydrogen Bonding; Ligands; Molecular Docking Simulation; Protein Kinase Inhibitors; Protein Structure, Tertiary; Thymidylate Synthase; Water

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E(act) and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.

Topics: Amino Acid Motifs; Casein Kinase II; Circular Dichroism; Deoxyuracil Nucleotides; Diphosphonates; Humans; Ligands; Mutation; Phosphorylation; Protein Binding; Protein Structure, Secondary; Quinazolines; Structure-Activity Relationship; Thiophenes; Thymidylate Synthase