Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors

A Novel Pim-1 Kinase Inhibitor Targeting Residues That Bind the Substrate Peptide

“…We obtained the percentage ratio of the amount of the Pim-1-binding form of the fluoroPimtide incubated with a compound as compared to that without a compound (control). The results showed that Pimtide produced 87% inhibition of fluoro-Pimtide binding, while quercetin, which is known to interact with only the ATP-binding site residues, 11,19 exhibited no inhibition (Fig. 3b).…”

“…It was shown to be a suitable fluoroprobe for the FCS-based competitive binding assay to screen for kinase inhibitors that bind to the ATP-binding site. 19 Thus, we measured the competitive binding of fluoro-staurosporine to Pim-1. As expected from the structural results, the addition of 10 μM compound 1 to the mixture of 3 μM Pim-1 and 3 nM fluoro-staurosporine indeed inhibited 79% of the fluoro-staurosporine binding.…”

Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

“…As such, the acyl‐based chemical modification may result in some loss of affinity of the probe relative to staurosporine itself. This has indeed been suggested as one of the reasons why, compared with staurosporine, a probe obtained by acylation of the secondary amine showed decreased affinity to the kinase ASK1 . However, acylated tools have been shown to retain kinase binding and in some cases even showed a slight increase in affinity (e.g., Ref.…”

“…Staurosporine itself is not used as a therapeutic agent, but has proven invaluable in the discovery of novel anticancer drugs based on kinase inhibition . Furthermore, it has been modified to generate functionalised molecular tools such as immobilised staurosporine to capture kinases, a staurosporine‐based photoaffinity probe, a cell‐permeable affinity‐based probe for kinome labelling, a highly selective bivalent staurosporine‐tethered peptide ligand, and a fluorescent staurosporine conjugate …”

“…[4,5] Furthermore, it has been modified to generate functionalised molecular tools such as immobilisedstaurosporine to capture kinases, [6] astaurosporine-based photoaffinity probe, [7] ac ell-permeable affinitybased probe for kinome labelling, [8] ah ighly selective bivalent staurosporine-tethered peptide ligand, [9] and af luorescent staurosporine conjugate. [10] The molecular interactions between staurosporine and numerous protein kinase catalytic domains have been determined in great detail in co-crystallisation experiments. [11][12][13][14][15][16] The main polar interaction points on staurosporine involvet he lactam oxygen and nitrogen atoms, the tetrahydropyran moiety,a sw ell as the 3'-methoxy and 4'-methylamine side groups (see Supporting Information for numbering).…”