minocycline and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Despite the increasing implication of the permeability transition pore (PTP) in the pathophysiology of neurodegenerative diseases, few selective PTP inhibitors have been reported so far. Here, we evaluate the pharmacological properties of a novel PTP inhibitor, BBMP (5-(benzylsulfonyl)-4-bromo-2-methyl-3(2H)-pyridazinone). This drug was discovered from the screening of a compound library against the PTP using a functional assay with isolated mitochondria. Similarly to cyclosporin A, the drug prevented Ca2+-induced permeability transition and mitochondrial depolarization. BBMP appeared more potent that minocycline in both swelling and membrane potential assays displaying pIC50 values of 5.5+/-0.1 and 5.6+/-0.0, respectively. Unlike minocycline, BBMP dose-dependently prevented DNA fragmentation induced by KCl 25/5 mM shift and serum deprivation in cerebellar granule neurons with a pIC50 of 5.7+/-0.6. The inhibition of PTP-mediated cytochrome c release observed in isolated mitochondria at 10 and 100 microM may explain its neuroprotective properties in vitro. These data suggest that the mitochondrial PTP is potentially involved in neuronal cell death and that PTP inhibitors, like BBMP, may possess a therapeutic potential in neurodegenerative disorders.

Topics: Amino Acid Chloromethyl Ketones; Animals; Benzyl Compounds; Caspase Inhibitors; Cells, Cultured; Cerebellar Cortex; Culture Media, Serum-Free; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Intracellular Membranes; Ion Channels; Membrane Potentials; Minocycline; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Molecular Structure; Neurons; Potassium Chloride; Pyridazines; Rats; Rats, Sprague-Dawley; Time Factors

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimer's disease.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Anthracenes; Anti-Bacterial Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Bromodeoxyuridine; Carrier Proteins; Caspase 3; Caspases; CDC2-CDC28 Kinases; Cell Count; Cell Survival; Cells, Cultured; Cerebellum; Chromatin; Colchicine; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Flavonoids; Flow Cytometry; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Kainic Acid; MAP Kinase Kinase 4; Microtubules; Minocycline; Mitogen-Activated Protein Kinase Kinases; Neurons; Neuroprotective Agents; Piperidines; Purines; Rats; Rats, Sprague-Dawley; Roscovitine; Time Factors; Tubulin