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...

Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy.

SUMMARY Paneth cells are a highly specialized population of intestinal epithelial cells located in the crypt adjacent to Lgr5+ stem cells, from which they differentiate through a process that requires downregulation of the Notch pathway. Their ability to store and release antimicrobial peptides protects the host from intestinal pathogens and controls intestinal inflammation. Here we show that PKCλ/ι is required for Paneth cell differentiation at the level of Atoh1 and Gfi1, through the control of EZH2 stability by direct phosphorylation. The selective inactivation of PKCλ/ι in epithelial cells results in the loss of mature Paneth cells, increased apoptosis and inflammation, and enhanced tumorigenesis. Importantly, PKCλ/ι expression in human Paneth cells decreases with progression of Crohn’s disease. Kaplan-Meier survival analysis of CRC patients revealed that low PRKCI levels correlated with significantly worse patient survival. Therefore, PKCλ/ι is a negative regulator of intestinal inflammation and cancer through its role in Paneth cell homeostasis.

The development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this manuscript, we used a combination of NMR-guided structure activity relationships along with biochemical and cellular studies to derive a novel tumor homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.

The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of novel Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry and functional data demonstrate that these peptide-mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in cell. The strategy proposed may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice. Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Recent studies identified the receptor tyrosine kinase EphA4 as a disease-modifying gene that is critical for the progression of motor neuron degeneration. We report on the design and characterization of a family of EphA4 targeting agents that bind to its ligand binding domain with nanomolar affinity. The molecules exhibit excellent selectivity and display efficacy in a SOD1 mutant mouse model of ALS. Interestingly, the molecules appear to act as agonists for the receptor in certain surrogate cellular assays. While the exact mechanisms responsible for the therapeutic effect of the new agonists remain to be elucidated, we believe that the described agent represents both an invaluable pharmacological tool to further decipher the role of the EphA4 in ALS and potentially other human diseases, and a significant stepping stone for the development of novel treatments.