Yong Jia

Current in vitro kinase assay technologies: the quest for a universal format.

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-α production

COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-alpha and other pro-inflammatory cytokines such as IL-1beta via the ERK/MAP kinase pathway. As TNF-alpha and IL-1beta are clinically validated targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT provides a potential avenue for amelioration of disease. Herein we describe identification of a cellular active selective small molecule inhibitor of COT kinase.

Utilization of Microarrayed Compound Screening (μARCS) to Identify Inhibitors of p56lck Tyrosine Kinase

Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied μARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE™) format in 96-well microplates and then converted into the gel-based μARCS format. The μARCS methodology is a well-less screening format in which 8640 compounds are arrayed on a microplate-sized piece of polystyene and subsequently assayed by placing reagents cast in agarose gels in contact with these compound sheets. A blotting paper soaked with adenosine triphosphate is applied on the gel to initiate the kinase reaction in the gel. Using this screening methodology, 300,000 compounds were screened in less than 40 h. Substantial reagent reduction was achieved by converting this tyrosine kinase assay from a 96-well plate assay to μARCS, resulting in significant cost savings. (Journal of Biomolecular Screening 2004: 12-21)

Designing in vitro assays for drug discovery: a study with the human MAP3 kinase COT

In drug discovery research, developing a validated and robust in vitro assay is crucial for high-throughput screening and subsequent hit characterization. There are many kinase assay technologies, so an assay developer has several decisions to make prior to undertaking experiments: which assay technology should be used? What are the advantages and disadvantages of a cascade assay compared with a direct assay format? Should the substrate be a peptide or a protein? When using a protein substrate, should it be physiologically relevant? In this perspective article, these questions will be addressed using examples from a study with the human mitogen-activated protein kinase kinase kinase COT.