The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K m for ATP. In addition, profiling at a physiological ATP concentration (1 mM) was carried out, and the IC 50 values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C trough and C max values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K m and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors.
MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitro myristoylation of selected predictions. Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.
AbstractWe evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitro myristoylation of selected predictions. Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.
Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.
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