Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

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...

Introduction Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers. Areas covered Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments. Expert Opinion We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.

Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 ( mda -7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda -7/IL-24–mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda -7/IL-24–induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda -7/IL-24–mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda -7/IL-24 (Ad.5/3- mda -7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda -7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3- mda -7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda -7/IL-24–mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.

Proapoptotic nuclear receptor family member Nur77 translocates from the nucleus to the mitochondria, where it interacts with Bcl-2 to trigger apoptosis. Nur77 translocation is induced by certain apoptotic stimuli, including the synthetic retinoid-related 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN)/ CD437 class. In this study, we investigated the molecular mechanism by which AHPN/CD437 analog (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces Nur77 nuclear export. Our results demonstrate that 3-Cl-AHPC effectively activated Jun N-terminal kinase (JNK), which phosphorylates Nur77. Inhibition of JNK activation by a JNK inhibitor suppressed 3-Cl-AHPC-induced Nur77 nuclear export and apoptosis. In addition, several JNK upstream activators, including the phorbol ester TPA, anisomycin and MAPK kinase kinase-1 (MEKK1), phosphorylated Nur77 and induced its nuclear export. However, Nur77 phosphorylation by JNK, although essential, was not sufficient for inducing Nur77 nuclear export. Induction of Nur77 nuclear export by MEKK1 required a prolonged MEKK1 activation and was attenuated by Akt activation. Expression of constitutively active Akt prevented MEKK1-induced Nur77 nuclear export. Conversely, transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor accelerated MEKK1-induced Nur77 nuclear export. Furthermore, mutation of an Akt phosphorylation residue Ser351 in Nur77 abolished the effect of Akt or the PI3-K inhibitor. Together, our results demonstrate that both activation of JNK and inhibition of Akt play a role in translocation of Nur77 from the nucleus to the cytoplasm.