Michael S. Saporito

MPTP Activates c-Jun NH2-Terminal Kinase (JNK) and Its Upstream Regulatory Kinase MKK4 in Nigrostriatal Neurons In Vivo

Abstract: The neuropathology of Parkinsons disease is reflected in experimental animals treated with the selective nigrostriatal dopaminergic neurotoxin MPTP. Neurons exposed to MPTP (MPP+) express morphological features of apoptosis, although the intracellular pathways that produce this morphology have not been established. The c‐Jun NH2‐terminal kinase (JNK) signaling cascade has been implicated as a mediator of MPTP‐induced apoptotic neuronal death based on the ability of CEP‐1347/KT‐7515, an inhibitor of JNK activation, to attenuate MPTP‐induced nigrostriatal dopaminergic degeneration. In these studies, MPTP‐mediated activation of the JNK signaling pathway was assessed in the nigrostriatal system of MPTP‐treated mice. MPTP elevated levels of phosphorylated JNK and JNK kinase (MKK4; also known as SEK1 or JNKK), by 2.5‐ and fivefold, respectively. Peak elevations occurred soon after administration of MPTP and coincided with peak CNS levels of MPP+. Increased MKK4 phosphorylation, but not JNK phosphorylation, was found in the striatum, suggesting that activation of MKK4 occurs in injured dopaminergic terminals. Both JNK and MKK4 phosphorylations were attenuated by pretreatment with l‐deprenyl, indicating that these phosphorylation events were mediated by MPP+. Moreover, CEP‐1347/KT‐7515 inhibited MPTP‐mediated MKK4 and JNK signaling at a dose that attenuates MPTP‐induced dopaminergic loss. These data implicate this signaling pathway in MPTP‐mediated nigrostriatal dopaminergic death and suggest that it may be activated in the degenerative process in Parkinsons disease.

Mixed lineage kinase activity of indolocarbazole analogues

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.

Discovery of CEP-1347/KT-7515, an inhibitor of the JNK/SAPK pathway for the treatment of neurodegenerative diseases.

Apoptosis has been proposed as a mechanism of cell death in Alzheimers, Huntingtons and Parkinsons diseases and the occurrence of apoptosis in these disorders suggests a common mechanism. Events such as oxidative stress, calcium toxicity, mitochondria defects, excitatory toxicity, and deficiency of survival factors are all postulated to play varying roles in the pathogenesis of the diseases. However, the transcription factor c-jun may play a role in the pathology and cell death processes that occur in Alzheimers disease. Parkinsons disease (PD) is also a progressive disorder involving the specific degeneration and death of dopamine neurons in the nigrostriatal pathway. In Parkinsons disease, dopaminergic neurons in the substantia nigra are hypothesized to undergo cell death by apoptotic processes. The commonality of biochemical events and pathways leading to cell death in these diseases continues to be an area under intense investigation. The current therapy for PD and AD remains targeting replacement of lost transmitter, but the ultimate objective in neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss. This chapter will describe a novel approach for the treatment of neurodegenerative diseases through the development of kinase inhibitors that block the active cell death process at an early transcriptional independent step in the stress activated kinase cascade. In particular, preclinical data will be presented on the c-Jun Amino Kinase pathway inhibitor, CEP-1347/KT-7515, with respect to its properties that make it a desirable clinical candidate for treatment of various neurodegenerative diseases.

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D3 resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D3. In rats, 1,25-dihydroxyvitamin D3 (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increases observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D3 did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D3 resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D3-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D3. On the basis of NGFs ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.

Pharmacological Induction of Nerve Growth Factor mRNA in Adult Rat Brain

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimers disease.

Targeting the JNK pathway for therapeutic benefit in CNS disease.

The c-Jun N-terminal Kinase (JNK) pathway leading to c-Jun phosphorylation plays a causal role in apoptosis of isolated primary embryonic neurons and of multiple neuronal cell lines following a wide variety of stimuli. Activation of this pathway may also contribute to the neuronal atrophy and death that is associated with neurodegenerative pathological conditions including Alzheimers, Parkinsons, Huntingtons Diseases and stroke. Here, the data that providelinks between the activation of the JNK pathway and its potential to play an operative role in CNS disease are reviewed. Also included is the progress on development of inhibitors targeting the JNK pathway for therapeutic benefit.

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells

The neuropathology of Parkinsons Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

Preservation of cholinergic activity and prevention of neuron death by CEP-1347/KT-7515 following excitotoxic injury of the nucleus basalis magnocellularis

We have identified a class of small organic molecules, derived from the indolocarbazole K-252a, that promote the survival of cultured neurons. However, many of these indolocarbazoles inhibit protein kinase C and neurotrophin-activated tyrosine kinase receptors. These kinase inhibitory activities may limit the utility of these compounds for neurological disorders. A bis-ethyl-thiomethyl analogue of K-252a, CEP-1347/KT-7515, has been identified that lacks protein kinase C and tyrosine kinase receptor inhibitory activities, yet retains the ability to promote survival of cultured neurons, including cholinergic neurons derived from the basal forebrain. In the present studies, CEP-1347/KT-7515 was assessed for neurotrophic activity on basal forebrain neurons of in vivo rats following excitotoxic insult. Ibotenate infusion into the nucleus basalis magnocellularis reduced levels of choline acetyltransferase activity in the cortex, as well as reduced numbers of choline acetyltransferase-immunoreactive and retrogradely (FluoroGold)-labelled cortically-projecting neurons in the nucleus basalis. Systemically administered CEP-1347/KT-7515 attenuated the loss of cortical choline acetyltransferase activity and the loss of the number of choline acetyltransferase-immunoreactive and retrogradely-labelled FluoroGold neurons in the nucleus basalis. Moreover, CEP-1347/KT-7515 ameliorated the loss of cortical choline acetyltransferase if administration was initiated one day, but not seven days post-lesion. Together, these results demonstrate that CEP-1347/KT-7515 protects damaged cortically-projecting basal forebrain neurons from degeneration. Thus, CEP-1347/KT-7515 may have therapeutic potential in neurodegenerative diseases, such as Alzheimers disease, in which basal forebrain cholinergic neurons degenerate.

Mixed-lineage kinase 1 and mixed-lineage kinase 3 subtype-selective dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-ones: optimization, mixed-lineage kinase 1 crystallography, and oral in vivo activity in 1-methyl-4-phenyltetrahydropyridine models.

The optimization of the dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-one R(2) and R(12) positions led to the identification of the first MLK1 and MLK3 subtype-selective inhibitors within the MLK family. Compounds 14 (CEP-5104) and 16 (CEP-6331) displayed good potency for MLK1 and MLK3 inhibition with a greater than 30- to 100-fold selectivity for related family members MLK2 and DLK. Compounds 14 and 16 were orally active in vivo in a mouse MPTP biochemical efficacy model that was comparable to the first-generation pan-MLK inhibitor 1 (CEP-1347). The MLK1 structure-activity relationships were supported by the first-reported X-ray crystal structure of MLK1 bound with 16.

MLR-1023 Is a Potent and Selective Allosteric Activator of Lyn Kinase In Vitro That Improves Glucose Tolerance In Vivo

2(1H)-pyrimidinone,5-(3-methylphenoxy) (MLR-1023) is a candidate for the treatment of type 2 diabetes. The current studies were aimed at determining the mechanism by which MLR-1023 mediates glycemic control. In these studies, we showed that MLR-1023 reduced blood glucose levels without increasing insulin secretion in vivo. We have further determined that MLR-1023 did not activate peroxisome proliferator-activated α, δ, and γ receptors or glucagon-like peptide-1 receptors or inhibit dipeptidyl peptidase-4 or α-glucosidase enzyme activity. However, in an in vitro broad kinase screen MLR-1023 activated the nonreceptor-linked Src-related tyrosine kinase Lyn. MLR-1023 increased the Vmax of Lyn with an EC50 of 63 nM. This Lyn kinase activation was ATP binding site independent, indicating that MLR-1023 regulated the kinase through an allosteric mechanism. We have established a link between Lyn activation and blood glucose lowering with studies showing that the glucose-lowering effects of MLR-1023 were abolished in Lyn knockout mice, consistent with existing literature linking Lyn kinase and the insulin-signaling pathway. In summary, these studies describe MLR-1023 as a unique blood glucose-lowering agent and show that MLR-1023-mediated blood glucose lowering depends on Lyn kinase activity. These results, coupled with other results (J Pharmacol Exp Ther 342:23–32, 2012), suggest that MLR-1023 and Lyn kinase activation may be a new treatment modality for type 2 diabetes.