Assessing protein kinase target similarity: Comparing sequence, structure, and cheminformatics approaches.

In just over two decades, structure based protein kinase inhibitor discovery has grown from trial and error approaches, using individual target structures, to structure and data driven approaches that may aim to optimize inhibition properties across several targets. This is increasingly enabled by the growing availability of potent compounds and kinome-wide binding data. Assessing the prospects for adapting known compounds to new therapeutic uses is thus a key priority for current drug discovery efforts. Tools that can successfully link the diverse information regarding target sequence, structure, and ligand binding properties now accompany a transformation of protein kinase inhibitor research, away from single, block-buster drug models, and toward "personalized medicine" with niche applications and highly specialized research groups. Major hurdles for the transformation to data driven drug discovery include mismatches in data types, and disparities of methods and molecules used; at the core remains the problem that ligand binding energies cannot be predicted precisely from individual structures. However, there is a growing body of experimental data for increasingly successful focussing of efforts: focussed chemical libraries, drug repurposing, polypharmacological design, to name a few. Protein kinase target similarity is easily quantified by sequence, and its relevance to ligand design includes broad classification by key binding sites, evaluation of resistance mutations, and the use of surrogate proteins. Although structural evaluation offers more information, the flexibility of protein kinases, and differences between the crystal and physiological environments may make the use of crystal structures misleading when structures are considered individually. Cheminformatics may enable the "calibration" of sequence and crystal structure information, with statistical methods able to identify key correlates to activity but also here, "the devil is in the details." Examples from specific repurposing and polypharmacology applications illustrate these points. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Thermodynamics and structure of a salmon cold active goose-type lysozyme.

Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adaptation as well as renaturation following heat treatment. In this study differential scanning calorimetry (DSC) was carried out to investigate unfolding and potential refolding, while X-ray crystallography was used to study structural factors contributing to the temperature-related characteristics. The recombinant SalG has a melting temperature (T(m)) of 36.8 degrees C under thermal denaturation conditions and regains activity after returning to permissive (low) temperature. Furthermore, refolding is dramatically reduced in solutions with high SalG concentrations, coupled with significant protein precipitation. The structural features of SalG closely resemble those of other g-type lysozymes. However, the N-terminal region of SalG is less anchored to the rest of the molecule due to the absence of disulphide bonds, thus, contributing significantly to the low T(m) of SalG. The absence of disulphide bonds and the distribution of salt bridges may at the same time ease refolding leading to renaturation.

Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism.

Crystal structures of Atlantic cod lysozyme have been solved with and without ligand bound in the active site to 1.7 and 1.9 A resolution, respectively. The structures reveal the presence of NAG in the substrate binding sites at both sides of the catalytic Glu73, hence allowing the first crystallographic description of the goose-type (g-type) lysozyme E-G binding sites. In addition, two aspartic acid residues suggested to participate in catalysis (Asp101 and Asp90) were mutated to alanine. Muramidase activity data for two single mutants and one double mutant demonstrates that both residues are involved in catalysis, but Asp101 is the more critical of the two. The structures and activity data suggest that a water molecule is the nucleophile completing the catalytic reaction, and the roles of the aspartic acids are to ensure proper positioning of the catalytic water.

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme.

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein-protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the beta domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein-protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.