JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. JNKs are serine threonine protein kinases and members of the MAPK family (1-3). JNKs can be expressed as 10 different isoforms by mRNA alternative splicing of three highly related genes, JNK1, JNK2, and JNK3 (4). Although JNK1 and JNK2 are ubiquitous, JNK3 is principally present in the brain, cardiac muscle, and testis (4, 5). JNK activation by extracellular stimuli, such as stress or cytokines, leads to the phosphorylation of several transcription factors, and cellular substrates implicated in cell survival and proliferation, insulin receptor signaling, and mRNA stabilization (6-9). Because these pathways are related to the pathogenesis of several diseases, including diabetes, cancer, atherosclerosis, stroke, and Alzheimer's and Parkinson's diseases, JNKs represent valuable targets in the development of new therapies (10).JNKs bind to scaffold proteins and substrates containing a D-domain, consensus sequence of which is R/KXXXXLXL (11, 12). JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases (13). The JNK-JIP1 interaction is mediated by a specific, high affinity D-domain on JIP1, the critical features of which were elucidated by Barr and colleagues (14). Overexpression of either the D-domain of JIP1 or the full-length protein potently inhibits JNK signaling in the cell (15). The minimal region of JIP1, consisting of a single D-domain, has been identified as a JNK inhibitor (14, 16). A peptide corresponding to the D-domain of JIP1 (amino acids 153-163; pepJIP1), inhibits JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 and displays remarkable selectivity with little inhibition of the closely related Erk and p38 MAPKs (17).The mechanism of JNK1 inhibition by pepJIP1 is mainly because of the competition of pepJIP1 with the D-domains of substrat...

A chelator fragment library based on a variety of metal binding groups was screened against a metalloproteinase. Lead hits were identified and an expanded library of select compounds was synthesized, resulting in numerous high-affinity hits against several metalloprotein targets. The findings clearly demonstrate that chelators can be used to generate libraries suitable for fragmentbased lead design (FBLD) directed at important metalloproteins. Keywordschelators; fragment-based lead design; libraries; metalloproteins; zinc Fragment-based lead design (FBLD), sometimes referred to as fragment-based drug discovery (FBDD), is an increasingly important strategy for the discovery of biologically active compounds.[1] FBLD generally uses libraries consisting of modest collections (100-1000 compounds) of small molecular fragments (MW <300 amu) that are screened against targets of interest.[2] Although such fragments do not bind as tightly (K d values in the micro-to millimolar range) as more complex molecules, including those used in high-throughput screening (HTS) approaches, they can provide 'hits' that serve as efficient starting structures for the development of potent inhibitors. Fragments that bind to a target are identified, after which one of two approaches is generally pursued: a) a single fragment can be elaborated in order to obtain a tight binder; or b) multiple fragments binding at adjacent and distinct sites can be connected by an appropriate linker to obtain a potent inhibitor. Compared to HTS, FBLD is purported to have several advantages, including a more efficient exploration of chemically diverse space and higher ligand efficiencies.Although the application of FBLD to metalloprotein targets of medicinal interest has been described, [3] Matrix metalloproteinases (MMPs) represent one of the most well-established targets in the realm of metalloproteins. These zinc(II)-dependent enzymes have been extensively studied, and the development of MMP inhibitors (MMPi) has played an important role in the discovery of inhibitors for other zinc(II)-dependent metalloproteins, such as anthrax lethal factor (LF), histone deacetylases (HDACs), and others.[10] Indeed, the earliest application of FBLD to a metalloprotein target was directed at MMP-3.[3] Therefore, we focused our preliminary library screening efforts on MMPs, as a representative system for identifying fragments that would bind zinc(II) metalloproteins. Based on widely-reported criteria for fragment libraries, [2] an initial chelator fragment library (CFL-1) was assembled. A modest library containing 96 structural cores was prepared from chelators with two to four donor atoms for binding metal ions and sufficient solubility for screening ( Figure 1). The chelating groups included picolinic acids, hydroxyquinolones, pyrimidines, hydroxypyrones, hydroxypyridinones and salicylic acids, in addition to other compounds that are well-established components of metalloprotein inhibitors, such as hydroxamic acids and sulfonamides ( Figure 1). The CFL-1 librar...

We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.