3-9-bis((ethylthio)methyl)-k-252a and Nerve-Degeneration

There is growing evidence that the molecular pathways of programmed cell death play a role in neurodegenerative disease, including Parkinson's disease, so there has been increased interest in them as therapeutic targets for the development of neuroprotective strategies. One pathway of cell death that has attracted particular attention is the mixed lineage kinase (MLK) -c-jun N-terminal kinase (JNK) signaling cascade, which leads to the phosphorylation and activation of the transcription factor c-jun. There is much evidence, from in vitro and in vivo studies, that this cascade can mediate cell death. In addition, there is evidence that it is operative upstream in the death process. It is possible that abrogation of this pathway may forestall death before irreversible cellular injury. One class of compounds that has shown promise for their ability to block cell death by inhibiting this cascade are the inhibitors of the MLKs, which are upstream in the activation of c-jun. One of these compounds, CEP1347, is now in a Phase II/III clinical trial for neuroprotection in PD. Whether this trial is successful or not, this signaling cascade is likely to be a focus of future therapeutic development. This review, therefore, outlines the principles of signaling within this kinase pathway, and the evidence for its role in cell death. We review the evidence that inhibition of the MLKs can prevent dopamine neuron cell death and the degeneration of their axons. These studies suggest important future directions for the development of therapies that will target this important cell death pathway.

Topics: Animals; Brain; Carbazoles; Cell Death; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; Models, Biological; Nerve Degeneration; Neuroprotective Agents; Parkinson Disease; Proto-Oncogene Proteins c-jun; Signal Transduction

CEP-1347 inhibits mixed lineage kinases that activate apoptotic pathways implicated in the pathogenesis of Parkinson disease (PD). CEP-1347 enhances neuronal survival in a variety of nonclinical models and was found to be safe and well tolerated during 4 weeks in PD patients. We conducted the Parkinson Research Examination of CEP-1347 Trial (PRECEPT) to assess its disease-modifying potential in early PD.. Consenting PD patients not yet requiring dopaminergic therapy (n = 806) were randomized equally to CEP-1347 in dosages of 10 mg BID, 25 mg BID, or 50 mg BID, or matching placebo, and were evaluated blindly and prospectively. The primary clinical end point was time to the development of disability requiring dopaminergic therapy. Secondary end points included changes in the Unified Parkinson's Disease Rating Scale (UPDRS) and beta-CIT SPECT imaging of striatal dopamine transporters.. The study was concluded early, after an average of 21.4 months of follow-up, when a planned interim analysis demonstrated that it would be futile to continue experimental treatment. At that time, 108 of 191 subjects randomized to placebo (57%) had reached the primary end point of disability requiring dopaminergic therapy compared with active CEP-1347: 133 of 205 (65%) on 10 mg BID, 126 of 212 (59%) on 25 mg BID, and 127 of 198 (64%) on 50 mg BID. Changes in UPDRS scores and beta-CIT imaging showed similar patterns.. In contrast to research in animal models that predicted favorable disease-modifying outcomes, we found CEP-1347 to be an ineffective treatment in early Parkinson disease.

Topics: Aged; Apoptosis; Apoptosis Regulatory Proteins; Carbazoles; Corpus Striatum; Disease Progression; Dopamine Plasma Membrane Transport Proteins; Double-Blind Method; Enzyme Inhibitors; Female; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Signaling System; Middle Aged; Nerve Degeneration; Neuroprotective Agents; Parkinson Disease; Prospective Studies; Tomography, Emission-Computed, Single-Photon; Treatment Failure

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75(NTR) is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75(NTR)) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell-derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75(NTR)-engineered cells. Function-blocking galectin-1 antibodies prevented the degeneration of processes, and recombinant galectin-1 caused the processes of wild-type neurons to degenerate first, followed by the cell bodies. In vivo, the application of a glutamate receptor agonist, a maneuver known to upregulate p75(NTR), led to an increase in the amount of galectin-1 and to the degeneration of neurons and their processes in a galectin-1-dependent fashion. Section of the sciatic nerve also rapidly upregulated levels of p75(NTR) and galectin-1 in terminal Schwann cells, and the elimination of nerve endings was delayed at the neuromuscular junction of mice lacking Lgals1 (the gene encoding galectin-1). These results indicate that galectin-1 actively participates in the elimination of neuronal processes after lesion, and that engineered ES cells are a useful tool for studying relevant aspects of neuronal degeneration that have been hitherto difficult to analyze.

Topics: Animals; Antibodies; Axotomy; Carbazoles; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Galectin 1; Gene Expression Regulation; Indoles; Lactose; Mice; Nerve Degeneration; Neuroprotective Agents; Protein Engineering; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Stem Cell Transplantation; Stem Cells; tau Proteins

Models of Parkinson's disease (PD) based on selective neuronal death have been used to study pathogenic mechanisms underlying nigral cell death and in some instances to develop symptomatic therapies. For validation of putative neuroprotectants, a model is desirable in which the events leading to neurodegeneration replicate those occurring in the disease. We developed a human in vitro model of PD based on the assumption that dysregulated cytoplasmic dopamine levels trigger cell loss in this disorder. Differentiated human mesencephalic neuron-derived cells were exposed to methamphetamine (METH) to promote cytoplasmic dopamine accumulation. In the presence of elevated iron concentrations, as observed in PD, increased cytosolic dopamine led to oxidative stress, c-Jun N-terminal kinase (JNK) pathway activation, neurite degeneration, and eventually apoptosis. We examined the role of the mixed-lineage kinases (MLKs) in this complex degenerative cascade by using the potent inhibitor 3,9-bis[(ethylthio)methyl]-K-252a (CEP1347). Inhibition of MLKs not only prevented FeCl2+/METH-induced JNK activation and apoptosis but also early events such as neurite degeneration and oxidative stress. This broad neuroprotective action of CEP1347 was associated with increased expression of an oxidative stress-response modulator, activating transcription factor 4. As a functional consequence, transcription of the cystine/glutamate and glycine transporters, cellular cystine uptake and intracellular levels of the redox buffer glutathione were augmented. In conclusion, this new human model of parkinsonian neurodegeneration has the potential to yield new insights into neurorestorative therapeutics and suggests that enhancement of cytoprotective mechanisms, in addition to blockade of apoptosis, may be essential for disease modulation.

Topics: Animals; Apoptosis; Carbazoles; Cell Line; Cells, Cultured; Cystine; Cytosol; Dopamine; Enzyme Activation; Ferrous Compounds; Glutathione; Humans; Hydrogen Peroxide; Indoles; JNK Mitogen-Activated Protein Kinases; Lipid Peroxidation; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mesencephalon; Methamphetamine; Nerve Degeneration; Neurites; Neurons; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Superoxides

We have identified a bis-ethylthiomethyl analog of K-252a, CEP-1347/KT-7515, that promotes neuronal survival in culture and in vivo. The neuronal survival properties of CEP-1347/KT-7515 may be related to its ability to inhibit the activation of c-jun N-terminal kinase, a key kinase in some forms of stress-induced neuronal death and perhaps apoptosis. There is evidence that the selective nigrostriatal dopaminergic neurotoxin, MPTP, produces neuronal apoptosis in culture and in adult mice. Thus, our studies were designed to determine if CEP-1347/KT-7515 could protect dopaminergic neurons from MPTP-mediated neurotoxicity. CEP-1347/KT-7515 was assessed for neuroprotective activity in a low dose MPTP model (20 mg/kg) where there was a 50% loss of striatal dopaminergic terminals in the absence of substantia nigra neuronal loss, and a high dose (40 mg/kg) MPTP model where there was a complete loss of dopaminergic terminals and 80% loss of dopaminergic cell bodies. In the low dose MPTP model, CEP-1347/KT-7515 (0.3 mg/kg/day) attenuated the MPTP-mediated loss of striatal dopaminergic terminals by 50%. In the high dose model, CEP-1347/KT-7515 ameliorated the loss of dopaminergic cell bodies by 50% and partially preserved striatal dopaminergic terminals. CEP-1347/KT-7515 did not inhibit monoamine oxidase B or the dopamine transporter, suggesting that the neuroprotective effects of CEP-1347/KT-7515 occur downstream of the metabolic conversion of MPTP to MPP+ and accumulation of MPP+ into dopaminergic neurons. These data implicate a c-jun N-terminal kinase signaling system in MPTP-mediated dopaminergic degeneration and suggest that CEP-1347/KT-7515 may have potential as a treatment for Parkinson's disease.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Dopamine; Dopamine Agents; Dopamine Uptake Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Indoles; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Monoamine Oxidase; Monoamine Oxidase Inhibitors; MPTP Poisoning; Nerve Degeneration; Neurons; Signal Transduction; Substantia Nigra; Tyrosine 3-Monooxygenase

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

Topics: Animals; Apoptosis; Carbazoles; Chick Embryo; Choline O-Acetyltransferase; Enzyme Inhibitors; Female; Humans; Indole Alkaloids; Indoles; Motor Neurons; Nerve Degeneration; Nerve Growth Factors; Neurons; Prosencephalon; Protein Kinase C; Proto-Oncogene Proteins; Rats; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptors, Nerve Growth Factor; Spinal Cord; Substantia Innominata