Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors, with good cell potency, and good brain penetration properties. The structure activity relationships (SAR) around a series of aminopyrimidines was evaluated utilizing biochemical and cell- based assays to measure JNK inhibition, and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC50 values < 100 nM. Moreover, compound 9l showed an IC50= 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rat, along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.

The accumulation of excess glutamate in the extracellular space as a consequence of CNS trauma, neurodegenerative diseases, infection, or deregulation of glutamate clearance results in neuronal damage by excessive excitatory neurotransmission. Glutamate excitotoxicity is thought to be one of several mechanisms by which HIV exerts neurotoxicity that culminates in HIV-associated neurocognitive disorders (HAND). Excess glutamate is released upon HIV infection of macrophage/microglial cells and has been associated with neurotoxicity mediated by gp120, transactivator of transcription (Tat) and other HIV proteins. Several strategies have been used over the years to try to prevent glutamate excitotoxicity. Since the main toxic effects of excess glutamate are thought to be due to excitotoxicity from over activation of glutamate receptors, antagonists of these receptors have been popular therapeutic targets. Early work to ameliorate the effects of excess extracellular glutamate focused on NMDA receptor antagonism, but unfortunately, potent blockade of this receptor has been fraught with side effects. One alternative to direct receptor blockade has been the inhibition of enzymes responsible for the production of glutamate such as glutaminase and glutamate carboxypeptidase II. Another approach has been to regulate the transporters responsible for modulation of extracellular glutamate such as excitatory amino acid transporters and the glutamate-cystine antiporter. There is preliminary experimental evidence that these approaches have potential therapeutic utility for the treatment of HAND. These efforts however, are at an early stage where the next steps are dependent on the identification of drug-like inhibitors as well as the development of predictive neuroAIDS animal models.

The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNKs interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-SabKIM1 selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-SabKIM1 prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation, but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.