Achim Moeller

The novel calpain inhibitor A-705253 potently inhibits oligomeric beta-amyloid-induced dynamin 1 and tau cleavage in hippocampal neurons

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 Alzheimers 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 Alzheimers disease.

The novel calpain inhibitor A-705253 prevents stress-induced tau hyperphosphorylation in vitro and in vivo.

Calcium-mediated pathologic activation of the cysteine protease calpain has been linked to neurodegenerative disorders such as Alzheimers 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.

Anticonvulsant effects of structurally diverse GABAB positive allosteric modulators in the DBA/2J audiogenic seizure test: Comparison to baclofen and utility as a pharmacodynamic screening model

The GABA(B) receptor has been indicated as a promising target for multiple CNS-related disorders. Baclofen, a prototypical orthosteric agonist, is used clinically for the treatment of spastic movement disorders, but is associated with unwanted side-effects, such as sedation and motor impairment. Positive allosteric modulators (PAM), which bind to a topographically-distinct site apart from the orthosteric binding pocket, may provide an improved side-effect profile while maintaining baclofen-like efficacy. GABA, the major inhibitory neurotransmitter in the CNS, plays an important role in the etiology and treatment of seizure disorders. Baclofen is known to produce anticonvulsant effects in the DBA/2J mouse audiogenic seizure test (AGS), suggesting it may be a suitable assay for assessing pharmacodynamic effects. Little is known about the effects of GABA(B) PAMs, however. The studies presented here sought to investigate the AGS test as a pharmacodynamic (PD) screening model for GABA(B) PAMs by comparing the profile of structurally diverse PAMs to baclofen. GS39783, rac-BHFF, CMPPE, A-1295120 (N-(3-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide), and A-1474713 (N-(3-(4-(4-chlorobenzyl)-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide) all produced robust, dose-dependent anticonvulsant effects; a similar profile was observed with baclofen. Pre-treatment with the GABA(B) antagonist SCH50911 completely blocked the anticonvulsant effects of baclofen and CMPPE in the AGS test, indicating such effects are likely mediated by the GABA(B) receptor. In addition to the standard anticonvulsant endpoint of the AGS test, video tracking software was employed to assess potential drug-induced motor side-effects during the acclimation period of the test. This analysis was sensitive to detecting drug-induced changes in total distance traveled, which was used to establish a therapeutic index (TI = hypoactivity/anticonvulsant effects). Calculated TIs for A-1295120, CMPPE, rac-BHFF, GS39783, and A-1474713 were 5.31x, 5.00x, 4.74x, 3.41x, and 1.83x, respectively, whereas baclofen was <1. The results presented here suggest the DBA/2J mouse AGS test is a potentially useful screening model for detecting PD effects of GABA(B) PAMs and can provide an initial read-out on target-related motor side-effects. Furthermore, an improved TI was observed for PAMs compared to baclofen, indicating the PAM approach may be a viable therapeutic alternative to baclofen.

Discovery of Novel and Highly Selective Inhibitors of Calpain for the Treatment of Alzheimer’s Disease: 2-(3-Phenyl-1H-pyrazol-1-yl)-nicotinamides

Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimers 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.