| Summary: | Protein phosphorylation, the most prevalent post-translational modification, is critical in the regulation of cellular events. Mapping the kinome is important in understanding how these processes occur and could provide a basis for new cancer therapies, but current methods are insufficient for this task. This research here was aimed at developing a new substrate identification strategy by combining two existing methods; steric complementation and cross-linking. Steric complementation was employed to generate a pyrazolo[3,4-d]pyrimidine ATP analogue that binds selectively to an analogue-sensitive (as-) kinase over the wild-type kinase and other intracellular kinases. The synthesis of C3-substituted pyrazolo[3,4-d]pyrimidines was extended to incorporate both alkyl and alkynyl substituents. A construct for the expression of PKCα (M417A) was generated and both PKCα and PKCα (M417A) expressed after lengthy optimisation. In vitro kinase radioassays were used to identify the most potent and selective inhibitor for PKCα (M417A). In order to allow late-stage introduction of photoactivatable groups into the pyrazolo[3,4-d]pyrimidine scaffold, various functionalised hydrazines were synthesised. The most potent C3-unsubstituted photoaffinity probes were selected for inhibition of PKCα and optimisation of both cross-linking and [3+2] Huisgen cyclisation to a fluorophore-containing azide allowed visualisation of the cross-linked products, although as yet, no substrate-selective labelling has been detected. The application of steric complementation to the photoaffinity probes was briefly investigated with a view to initially allowing improved detection of substrate-selective labelling, and ultimately achieving selective cross-linking to as-kinase substrates in intact cells.
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