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...

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

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.

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization—a key readout and driver of cell behaviour3,4—at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the ‘wiring’ of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.