a-705253 and Alzheimer-Disease

Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer's disease (AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.

Topics: Alzheimer Disease; Aminobutyrates; Animals; Calpain; Cathepsins; Cysteine Proteinase Inhibitors; Dogs; Hippocampus; Humans; Inhibitory Concentration 50; Macaca fascicularis; Male; Microsomes, Liver; Niacinamide; Pyrazoles; Rats, Inbred F344; Rats, Sprague-Dawley; Rats, Wistar; Sleep, REM; Spectrin; Stereoisomerism; Structure-Activity Relationship

Calcium-mediated pathologic activation of the cysteine protease calpain has been linked to neurodegenerative disorders such as Alzheimer's disease (AD) through the cleavage of proteolytic substrates that negatively affect neuronal function. Hyperphosphorylation of the microtubule-associated protein tau and the subsequent aggregation of tau filaments resulting in the intracellular formation of neurofibrillary tangles are recognized as key etiological factors in AD pathology. Cyclin-dependent kinase 5 (Cdk5), a major kinase responsible for tau hyperphosphorylation in the AD brain, becomes hyperactivated through calpain-mediated cleavage-conversion of the Cdk5 regulatory protein p35 to p25. In the present study, we examined the effects of the novel small-molecule calpain inhibitor A-705253 in acute models of tau hyperphosphorylation in vitro and in vivo. In hippocampal slices in vitro, lowering medium temperature to 33 °C increased tau phosphorylation in which incubation with A-705253 blocked low temperature-induced tau phosphorylation as measured by Western blot analysis. Pentobarbital-induced hypothermia or acute systemic LPS treatment in normal mice increased tau phosphorylation in hippocampal CA3 mossy fibers, as measured by immunohistochemistry, whereas acute A-705253 pretreatment prevented the stress-induced tau hyperphosphorylation in both models. In support of a Cdk5-mediated mechanism, A-705253 administered for two weeks in the drinking water of six month-old prepathogenic 3x Tg-AD mice resulted in decreased expression of the calpain proteolytic p25 fragment. Taken together, results of these studies suggest that calpain inhibition has potential utility in reducing tau hyperphosphorylation and may represent a novel disease-modifying approach in the treatment of AD.

Topics: Alzheimer Disease; Animals; Benzamides; Calpain; Cold Temperature; Cysteine Proteinase Inhibitors; Disease Models, Animal; Hippocampus; In Vitro Techniques; Male; Mice; Mice, Inbred Strains; Mice, Transgenic; Molecular Targeted Therapy; Mossy Fibers, Hippocampal; Neurons; Peptide Fragments; Phosphorylation; Protein Processing, Post-Translational; Stress, Physiological; tau Proteins

Calpains are cysteine proteinases that selectively cleave proteins in response to calcium signals. Exacerbated activation of calpain has been implicated as a major component in the signaling cascade that leads to β-amyloid (Aβ) production and tau hyperphosphorylation in Alzheimer's disease (AD). In this study, we analyzed the potential therapeutic efficacy of inhibiting the activation of calpain by a novel calpain inhibitor in aged 3xTgAD mice with well-established cognitive impairment, plaques, and tangles. The administration of a novel inhibitor of calpain, A-705253, attenuated cognitive impairment and synaptic dysfunction in a dose-dependent manner in 3xTgAD mice. Inhibition of calpain lowered Aβ(40) and Aβ(42) levels in both detergent-soluble and detergent-insoluble fractions and also reduced the total number and size of thioflavin S-positive fibrillar Aβ deposits. Mechanistically, these effects were, in part, explained by a down-regulation of β-secretase 1 (BACE1) and an up-regulation of ATP-binding cassette transporter A1 (ABCA1) expression, which, in turn, contributed to reduced production and increased clearance of Aβ, respectively. Moreover, A-705253 decreased the activation of cyclin-dependent kinase 5 (CDK5) and thereby diminished the hyperphosphorylation of tau. Finally, blockage of calpain activation reduced the astrocytic and microglial responses associated with AD-like pathological characteristics in aged 3xTgAD mice. Our data provide relevant functional and molecular insights into the beneficial therapeutic effects of inhibiting calpain activation for the management of AD.

Topics: Aging; Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Benzamides; Cognition; Cyclin-Dependent Kinase 5; Enzyme Activation; Glycoproteins; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nervous System; Phosphorylation; tau Proteins

Topics: Aging; Alzheimer Disease; Animals; Benzamides; Cognition; Glycoproteins; Humans

We have previously shown that beta-amyloid (Abeta) oligomers induced dynamin 1 and tau cleavage in cultured hippocampal neurons. As a result of this cleavage, dynamin 1 levels decreased and a toxic tau fragment was generated. Abeta-induced cleavage of these proteins was calpain-mediated and impacted both synaptic vesicle recycling and the integrity of neuronal processes [Kelly, B.L., Vassar, R., Ferreira, A., 2005. Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease. J. Biol. Chem. 280, 31746-31753; Park, S.Y., Ferreira, A., 2005. The generation of a 17kDa neurotoxic fragment: an alternative mechanism by which tau mediates beta-amyloid-induced neurodegeneration. J. Neurosci. 25, 5365-5375; Kelly, B.L., Ferreira, A., 2006. Beta-amyloid-induced dynamin 1 degradation is mediated by N-methyl-d-aspartate receptors in hippocampal neurons. J. Biol. Chem. 281, 28079-28089, Kelly, B.L., Ferreira, A., 2007. Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons. Neuroscience 147, 60-70]. Building on previous reports, these results identified calpain as a potential target for therapeutic intervention in Alzheimer's disease. In the present study, we tested the ability of A-705253, a novel water-soluble calpain inhibitor with oral availability and enhanced metabolic stability, to prevent Abeta-induced dynamin 1 and tau cleavage in cultured hippocampal neurons. Quantitative Western blot analysis indicated that the incubation of these cells with A-705253 prior to the addition of oligomeric Abeta reduced both dynamin 1 and tau cleavage in a dose-dependent manner. In addition, our results showed that this calpain inhibitor significantly ameliorated the cleavage of these proteins when added simultaneously with oligomeric Abeta. Furthermore, our data indicated that the use of this calpain inhibitor could have some beneficial effects even when added after the cleavage of these proteins have been triggered by Abeta. Collectively, these results suggest that, indeed, specific calpain inhibitors could play an important role in the treatment of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzamides; Blotting, Western; Calpain; Cells, Cultured; Dimerization; Dose-Response Relationship, Drug; Dynamin I; Enzyme Inhibitors; Hippocampus; Neurons; Peptide Fragments; Rats; tau Proteins