Nmr Screening Of Protein Kinases By Atpstd Method

The Schering-Plough group has recently reported on the use of ATP as a site-specific marker in STD-NMR experiments for screening of kinase inhibitors [50]. ATP-STD spectra detect the NMR signals from ATP in the active site of the kinase and reduction of that signal in the presence of competitive inhibitors allows measurement of the inhibitor Ki with respect to the natural substrate. Furthermore, by adding MnCl2 to the sample, ATP is converted into a paramagnetic probe, which can be used to elucidate the distance of non-competitive inhibitors from the ATP-binding site.

Four 1D experiments are performed. Initially a 1D spectrum of the kinase, ATP and TSP (trimethylsilylpropane sulfonate - as an internal reference) is recorded. Then an inhibitor (or compound of interest) is added to the sample and an additional spectrum is generated. Any compound degradation or side products are detectable by the NMR method and hydrolysis of ATP can be monitored by measuring chemical shift changes of the purine and sugar protons. In a third experiment, the ATP-STD signal is optimised. A typical STD spectrum of target-bound ATP gives peaks from the purine H8 and H2, and the sugar H1' protons. In the presence of an ATP-competitive binder, these ATP-STD signals are reduced and an inhibitor STD signal appears. This was demonstrated in an experiment in which the ATP-STD peak for the H2 proton of MgATP bound to extracellular signal-regulated kinase 2 (ERK2) was reduced by 54% by the addition of olomoucine. Olomoucine STD peaks were also observed. Competitive inhibitors can be distinguished from non-competitive inhibitors by the addition of a high-affinity inhibitor e.g. staurosporine. As staurosporine is a highly potent inhibitor and competitive with ATP for most serine/threonine kinases, its addition eliminates the STD peaks for ATP and for any other competitive inhibitors. The STD signals of non-competitive inhibitors are not affected by the addition of potent competitive inhibitors.

In a fourth experiment, a paramagnetic probe can be used to determine the proximity from the ATP-binding site of non-competitive inhibitors. An MnATP probe is generated by the addition of Mn2 + ions to ATP [51]. The use of the MnATP probe was demonstrated in an NMR-binding study of diarylamines to the serine/threonine kinase MEK1 (Map Erk kinase 1). The diarylamine, PD318088 is a non-ATP-competitive MEK1 inhibitor, known by X-ray crystallographic studies to bind proximally to the ATP in MEK1 [52]. NMR studies with a diarylamine analogue gave strong STD signals, indicating binding. However, these signals were not reduced by the addition of staurosporine suggesting non-competitive binding. The addition of an MnATP probe reduced the STD signals from the diarylamine compound, indicating that it was binding in close proximity to the ATP-binding site. Furthermore, proton signals on the two rings of the analogue were reduced to a different extent, allowing the orientation of the molecule to be deduced.

ATP-STD NMR is described as a simple, cost-effective and robust assay that can be used to identify inhibitors of virtually any nucleotide-binding target.

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