Assessing Kinase Selectivity

Kinase Profiling in Cell Lysates and Tissue Homogenates 

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We also assessed the effect of phosphorylation of the ERp AF-1 in regulating interaction with SRC-1 in vivo using transfection experiments. Treatment of transfected cells with PD98059, a selective inhibitor of MAPK activation, completely abrogated the SRC-1-mediated activation of unliganded ER 3, whereas use of staurosporin, which inhibits protein kinase C, had no significant effect (Tremblay et al., 1999). The interaction between ER 3 and SRC-1 in the absence of hormone was also demonstrated in vivo and shown to be influenced by factors known to change the phosphorylation status of nuclear receptors. 

Tomlinson B, Cruickshank JM, Hayes Y, Renondin JC, Lui JB, Graham BR, Jones A, Lewis AD, Prichard BN. Selective beta-adrenoceptor partial agonist effects of pindolol and xamoterol on skeletal muscle assessed by plasma creatine kinase changes in healthy subjects. Br J Clin Pharmacol 1990 30(5) 665-72. 

The safety of less selective dmgs, and especially that of promiscuous kinase inhibitors, is often addressed by assessing the full extent of their cross-reactivity [19], This assessment has been facilitated by novel high-throughput screening... 

The tetrasodium salt of carbonylbisphosphonate was originally synthesized by Quimby et al. [76], using hydrolysis of a tetraalkyl dichloromethylenebisphos-phonate in aqueous NaOH. In aqueous solution, the yellow ketone form reversibly converts to its colorless hydrate at acidic pH [76], a process which can be assessed by a combination of 31P-NMR and uv-visible spectroscopy at pH 7, the ketone predominates [93]. The salt moderately inhibits HIV reverse transcriptase in a p24 assay, whereas the parent methylene compound is inactive [64], displays some activity vs the pyrophosphate-dependent phosphofructose kinase of the parasite T. gondii [94] and has found use as a selective inhibitor of PCNA-independent DNA polymerase 6, allowing its enzyme activity to be distinguished from that of DNA polymerase a [95]. 

The sizes of these panels vary from about 200 kinases at Caliper to over 400 at Ambit. The assay counts include the lipid kinases, mutant kinases and kinases that are available in more than one assay condition (the number of wild type kinases in any of the panels is less than the total). These assay panels enable assessment of broad selectivity and liability information needed for lead and candidate characterization. They also enable broad screening for chemical starting points for kinases across the kinome in parallel. Indeed, we reported doing this in 2008 against the 203 kinases available in the Ambit panel at that time, which will be discussed elsewhere in this review. 

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