Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

SAR156497, an exquisitely selective inhibitor of aurora kinases.

The Aurora family of serine/threonine kinases is essential for mitosis. Their crucial role in cell cycle regulation and aberrant expression in a broad range of malignancies have been demonstrated and have prompted intensive search for small molecule Aurora inhibitors. Indeed, over 10 of them have reached the clinic as potential anticancer therapies. We report herein the discovery and optimization of a novel series of tricyclic molecules that has led to SAR156497, an exquisitely selective Aurora A, B, and C inhibitor with in vitro and in vivo efficacy. We also provide insights into its mode of binding to its target proteins, which could explain its selectivity.

Discovery of the first non-ATP competitive IGF-1R kinase inhibitors: advantages in comparison with competitive inhibitors.

A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS. Their noncompetitive property as well as their slow binding characteristics provided a series of compounds with unique selectivity and excellent cellular activities.

Polymer-supported approach for solution-phase synthesis of cysteine trap protease inhibitors: procedure for straightforward optimization of the P1-P1' pocket.

Peptide-based reversible and irreversible cysteine proteases inhibitors are well reported in the literature. Many of these compounds have an electrophilic carbonyl group as a cysteine trap in the place of a scissile amide moiety of the natural substrate. As a common mechanism strategy, we have designed a probe library of a cysteine trap for rapid optimization of P1-P1' pockets of different cysteine proteases. The synthesis of this library using a straightforward methodology based on polymer-supported reagents and scavengers to avoid tedious purification steps has been achieved. For the selective monobromination of diazo ketones, preparation of a new supported reagent, piperidinoaminomethylpolystyrene hydrobromide, is also described.