Monitoring signaling by the p75(NTR) receptor utilizing a caspase-3 activation assay amenable to small-molecule screening.

p75(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75(NTR) is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75(NTR), no p75(NTR) signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75(NTR) modulators and studying p75(NTR) function. Stimulation of p75(NTR)-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75(NTR) mutant could not substitute for wild-type p75(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75(NTR) as well as further characterizing downstream signaling events.

Quantifying amyloid beta (Aß)-mediated changes in neuronal morphology in primary cultures: implications for phenotypic screening.

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aß) in the brain. Herein, the authors quantify Aß-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aß caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aß-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aß-mediated cytotoxicity. Finally, we showed that Aß challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aß-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

Design, synthesis and structure-activity relationship of novel [3.3.1] bicyclic sulfonamide-pyrazoles as potent γ-secretase inhibitors.

The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.