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Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

Izzo NJ, Staniszewski A, To L, Fa M, Teich AF, Saeed F, Wostein H, Walko T, Vaswani A, Wardius M, Syed Z, Ravenscroft J, Mozzoni K, Silky C, Rehak C, Yurko R, Finn P, Look G, Rishton G, Safferstein H, Miller M, Johanson C, Stopa E, Windisch M, Hutter-Paier B, Shamloo M, Arancio O, LeVine H, Catalano SM - PLoS ONE (2014)

Bottom Line: We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers.The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking.These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect.

View Article: PubMed Central - PubMed

Affiliation: Cognition Therapeutics Inc., Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

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Small molecule therapeutic candidates can prevent or displace Abeta oligomer binding to mature primary hippocampal and cortical cultures (21DIV).Abeta synthetic oligomers were added to neuronal cultures (0.5 µM total Abeta concentration) for 40 min prior to (A–F) or following (G–L) addition of compounds and bound Abeta was detected by immunofluorescence. A, G Vehicle controls with no Abeta show background fluorescence. C, I, 15 µM CT0109, D, J, CT0093, E, K, CT01344, or F, L, CT01346 show that immuno-fluorescence for punctate binding of Abeta oligomers to neurites is blocked by all four compounds. Accompanying MAP2 panel shows similar density of neurons from right-hand side for each Abeta image. Scale bar  = 20 µm. M, Quantification of Abeta immunofluorescence shows that pretreatment with CT0093 prevents Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.79, N = 8, EC50  = 2.2±2.4 µM). N, CT01344 displaces pre-bound Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.92, N = 8, EC50  = 3.9±1.8 µM). *  =  statistically different than control, P<0.05, Student's t-test.
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pone-0111898-g010: Small molecule therapeutic candidates can prevent or displace Abeta oligomer binding to mature primary hippocampal and cortical cultures (21DIV).Abeta synthetic oligomers were added to neuronal cultures (0.5 µM total Abeta concentration) for 40 min prior to (A–F) or following (G–L) addition of compounds and bound Abeta was detected by immunofluorescence. A, G Vehicle controls with no Abeta show background fluorescence. C, I, 15 µM CT0109, D, J, CT0093, E, K, CT01344, or F, L, CT01346 show that immuno-fluorescence for punctate binding of Abeta oligomers to neurites is blocked by all four compounds. Accompanying MAP2 panel shows similar density of neurons from right-hand side for each Abeta image. Scale bar  = 20 µm. M, Quantification of Abeta immunofluorescence shows that pretreatment with CT0093 prevents Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.79, N = 8, EC50  = 2.2±2.4 µM). N, CT01344 displaces pre-bound Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.92, N = 8, EC50  = 3.9±1.8 µM). *  =  statistically different than control, P<0.05, Student's t-test.

Mentions: We examined whether these compounds could reduce Abeta oligomer binding to hippocampal/cortical cultures in vitro. Concentrations in this study represent the maximum dose of the compound in the membrane trafficking assay (Fig. 9 E–H) and an Abeta concentration equal to the Ki in the binding assay (Fig. 6). The addition of 15 µM CT0109, CT0093, CT01344 and CT01346 one hour prior to addition of synthetic Abeta oligomers (0.5 µM total Abeta concentration) all prevented the binding of Abeta oligomers to neuronal cultures by 99%±7%, 93%±2%, 94%±4% and 93%±4%, respectively (Fig. 10A–F). When the same concentration of these compounds were added 1 hour after addition of oligomers, all four compounds significantly displaced Abeta oligomers within 1 hour by 44%±3%, 49±5%, 66±14% and 93±13%, respectively (Fig. 10G–L). These compounds displace Abeta in a concentration dependent manner as shown for CT0093 in prevention (IC50 = 2.2 µM, Fig. 10M) and for CT01344 in treatment (IC50 = 3.9 µM, Fig. 10N). Compounds did not reduce the intensity of prebound Abeta oligomers as completely as they did when added to cultures before the oligomers. These displacement studies were performed after 1 hour treatment and it is possible that longer treatment with compounds can achieve a greater level of displacement. However, incubation with Abeta oligomers for longer times results in internalization of a portion of the Abeta oligomer labeling (27±8% internalization at 2 hr, Fig. S2), complicating the quantification of the compound treatment effects. In separate biochemical assays using an oligomer-specific ELISA, these small molecules do not appear to directly interact with oligomers and do not disrupt preformed oligomers or block formation of oligomers (Fig. 11). Since glial cell body binding at the oligomer concentrations used in these compound binding prevention and displacement experiments is so low (<10% of puncta binding intensity), we did not analyze the compound's ability to displace Abeta binding.


Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

Izzo NJ, Staniszewski A, To L, Fa M, Teich AF, Saeed F, Wostein H, Walko T, Vaswani A, Wardius M, Syed Z, Ravenscroft J, Mozzoni K, Silky C, Rehak C, Yurko R, Finn P, Look G, Rishton G, Safferstein H, Miller M, Johanson C, Stopa E, Windisch M, Hutter-Paier B, Shamloo M, Arancio O, LeVine H, Catalano SM - PLoS ONE (2014)

Small molecule therapeutic candidates can prevent or displace Abeta oligomer binding to mature primary hippocampal and cortical cultures (21DIV).Abeta synthetic oligomers were added to neuronal cultures (0.5 µM total Abeta concentration) for 40 min prior to (A–F) or following (G–L) addition of compounds and bound Abeta was detected by immunofluorescence. A, G Vehicle controls with no Abeta show background fluorescence. C, I, 15 µM CT0109, D, J, CT0093, E, K, CT01344, or F, L, CT01346 show that immuno-fluorescence for punctate binding of Abeta oligomers to neurites is blocked by all four compounds. Accompanying MAP2 panel shows similar density of neurons from right-hand side for each Abeta image. Scale bar  = 20 µm. M, Quantification of Abeta immunofluorescence shows that pretreatment with CT0093 prevents Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.79, N = 8, EC50  = 2.2±2.4 µM). N, CT01344 displaces pre-bound Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.92, N = 8, EC50  = 3.9±1.8 µM). *  =  statistically different than control, P<0.05, Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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pone-0111898-g010: Small molecule therapeutic candidates can prevent or displace Abeta oligomer binding to mature primary hippocampal and cortical cultures (21DIV).Abeta synthetic oligomers were added to neuronal cultures (0.5 µM total Abeta concentration) for 40 min prior to (A–F) or following (G–L) addition of compounds and bound Abeta was detected by immunofluorescence. A, G Vehicle controls with no Abeta show background fluorescence. C, I, 15 µM CT0109, D, J, CT0093, E, K, CT01344, or F, L, CT01346 show that immuno-fluorescence for punctate binding of Abeta oligomers to neurites is blocked by all four compounds. Accompanying MAP2 panel shows similar density of neurons from right-hand side for each Abeta image. Scale bar  = 20 µm. M, Quantification of Abeta immunofluorescence shows that pretreatment with CT0093 prevents Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.79, N = 8, EC50  = 2.2±2.4 µM). N, CT01344 displaces pre-bound Abeta binding in a dose-dependent manner fitting a single site binding model (r2 = 0.92, N = 8, EC50  = 3.9±1.8 µM). *  =  statistically different than control, P<0.05, Student's t-test.
Mentions: We examined whether these compounds could reduce Abeta oligomer binding to hippocampal/cortical cultures in vitro. Concentrations in this study represent the maximum dose of the compound in the membrane trafficking assay (Fig. 9 E–H) and an Abeta concentration equal to the Ki in the binding assay (Fig. 6). The addition of 15 µM CT0109, CT0093, CT01344 and CT01346 one hour prior to addition of synthetic Abeta oligomers (0.5 µM total Abeta concentration) all prevented the binding of Abeta oligomers to neuronal cultures by 99%±7%, 93%±2%, 94%±4% and 93%±4%, respectively (Fig. 10A–F). When the same concentration of these compounds were added 1 hour after addition of oligomers, all four compounds significantly displaced Abeta oligomers within 1 hour by 44%±3%, 49±5%, 66±14% and 93±13%, respectively (Fig. 10G–L). These compounds displace Abeta in a concentration dependent manner as shown for CT0093 in prevention (IC50 = 2.2 µM, Fig. 10M) and for CT01344 in treatment (IC50 = 3.9 µM, Fig. 10N). Compounds did not reduce the intensity of prebound Abeta oligomers as completely as they did when added to cultures before the oligomers. These displacement studies were performed after 1 hour treatment and it is possible that longer treatment with compounds can achieve a greater level of displacement. However, incubation with Abeta oligomers for longer times results in internalization of a portion of the Abeta oligomer labeling (27±8% internalization at 2 hr, Fig. S2), complicating the quantification of the compound treatment effects. In separate biochemical assays using an oligomer-specific ELISA, these small molecules do not appear to directly interact with oligomers and do not disrupt preformed oligomers or block formation of oligomers (Fig. 11). Since glial cell body binding at the oligomer concentrations used in these compound binding prevention and displacement experiments is so low (<10% of puncta binding intensity), we did not analyze the compound's ability to displace Abeta binding.

Bottom Line: We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers.The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking.These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect.

View Article: PubMed Central - PubMed

Affiliation: Cognition Therapeutics Inc., Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Show MeSH
Related in: MedlinePlus