P 3 Further modification of inhibitor of CK 2 — 4 , 5 , 6 , 7 - tetrabromo - 1 H - benzotriazole ( TBBt )

Kinases that phosphorylate substrate proteins on tyrosine, serine and threonine, play critical roles in all aspects of cell homeostasis and are under intense scrutiny as potential drug targets in a clinical setting. Dysfunctions of protein kinases (and protein phosphatases) are associated with numerous severe pathological states and consequently it is not surprising that the pharmaceutical and biotechnology sectors are amongst the most active participants in the design of protein kinase inhibitors, primarily with an eye to potential therapeutic applications. Hence, these kinases are extensively studied and their importance in cell biology is unquestioned. Not so histidine kinases! Unlike the well-known protein kinases, histidine kinases (HK) phosphorylate proteins on the imidazole nitrogens of histidine [1] and have received scant attention in mammalian cells. Recently, significant progress in the development of experimental techniques to study histidine phosphorylation, has paved the way for a more comprehensive study of histidine kinases and their role in the cell [2]. Examples of histidine phosphorylation of proteins have been reported in relation to breast cancer metastasis [3], pancreatic islet cells involving diabetes [4] and airway epithelium [5]. In particular, histone H4 has been shown to be a substrate for mammalian histone H4 histidine kinases (HHKs) [6]. HHK activity is known to be markedly elevated in proliferating tissue from human foetal liver, regenerating liver, liver “oval cell” progenitor cells, hepatocellular carcinomas and other proliferating tissues [7]. Many unknowns exist with respect to HK research including, fundamental questions concerning the cellular localisation of HKs, their binding partners, substrates and how these all work in concert inside the cell. The implications from all of these findings will have broader ramifications in many areas of biomedical science and down-stream clinical outcomes with respect to kinase-drug targets. In this respect, mammalian HKs potentially represent an untapped novel kinase drug target resource. Recent advances in mass spectrometry [8] have made a fundamentally significant contribution toward the method development involving the purification and detection of histidine kinases and histidine phosphorylated proteins. Using mass spectrometry and other techniques specific for the study of histidine phosphorylation and histidine kinase specific assays, we have purified a liver histidine kinase to homogeneity. Initial characterisation of this kinase examined the effects of two reagents as potential histidine kinase inhibitors. Preliminary evidence indicates that the liver histidine kinase activity is inhibited by both cystamine and genistein (Fig. 1). There appeared to be complete abolition of in vitro histidine kinase activity for histone H4 at 50 μM cystamine, although to what level the inhibition occurs has yet to be determined by enzymatic inhibition studies. To further characterise this liver histidine kinase activity, we used the tyrosine/histidine kinase inhibitor genistein as demonstrated by Huang et al. [9]. They found the genistein IC50 for the yeast histidine kinase to be 110 μM. They also determined the Ki for genistein inhibition of histidine phosphorylation of histone H4 to be 270 μM. We found histidine kinase inhibition using 100 μM genistein, which was well within values determined previously [9]. The inhibition results for cystamine and genistein presented in this study are by no means exhaustive, and are presented as part of the preliminary characterisation of the recently purified liver histidine kinase.