Alzheimer\u27s 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

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\u27s 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\u27s 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\u27s therapeutics

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.

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

Effect of Abeta control peptides in membrane trafficking assay.

<p>Oligomer preparation of Abeta 1–40 is 200 times less potent than Abeta 1–42 in binding assay (<b>A</b>) and in membrane trafficking (<b>B</b>). Scrambled Abeta 1–42 is not active in the membrane trafficking assay and is not detectable by the antibody used for the Abeta binding assay.</p

Correlation of brain concentration of compounds with behavioral efficacy and estimated receptor occupancy at sigma-2/PGRMC1 receptor.

<p>CT0093 and CT0109 were dosed subcutaneously in mice by continuous osmotic minipumps infusions at the doses indicated. CT01344 and CT01346 were dosed by once daily oral gavage. Twenty four hours after the last dose, animals were euthanized and drug concentration in the brain was measured. Ki  =  binding affinity of compound at the sigma-2/PGRMC1 receptor. Measured efficacy: statistically significant improvement (+), or no significant improvement (-) seen in behavioral tests. Estimated % receptor occupancy was calculated according to the formula (concentration/Ki)/[(concentration/Ki) + 1)], where Ki is determine by radioligand competition binding.</p><p>Correlation of brain concentration of compounds with behavioral efficacy and estimated receptor occupancy at sigma-2/PGRMC1 receptor.</p

Characterization of human Abeta 1–42 oligomers isolated from patient frozen, unfixed 1 gram brain samples by non-denaturing Western blot, MALDI-TOF and ELISA.

<p><b>A</b>, Non-denaturing Western blots of immunoprecipitated Alzheimer's patient hippocampal samples demonstrates heterogeneous populations of oligomer assemblies. 6E10 antibody labeling of western blots from four different AD patients (lanes 1–4) detects major bands ≥250 kDa, and multiple discrete bands between 50–75 kDa. In contrast, APP antibody detects a faint band at 125 kDa (lane 5). Significant amounts of monomeric Abeta 1–42 were not observed in any individual. MALDI-TOF analysis of immune-precipitated human brain samples demonstrates heterogeneous populations of oligomer assemblies, both between individual Alzheimer's patients (<b>B, D</b>) and between age-matched histologically normal individuals (<b>C, E</b>). Significant amounts of monomeric Abeta 1–42 were not observed in any individual. Albumin was added to samples as an internal size control (arrow in <b>B–E</b>).</p

Small molecule Abeta binding antagonists prevent Abeta 1–42 oligomer-induced synaptic regression in cultured neurons.

<p><b>A</b>, Abeta oligomers bound to a subset of neurites (red) reduces synaptophysin-immunoreactive synaptic puncta (green). <b>B</b>, Treatment with sigma-2/PGRMC1 antagonists reduces oligomer binding and restores normal immunoreactivity for the synaptic marker. <b>C</b>, Oligomers induce an average 18%±2 s.e.m. loss in the number of immunoreactive puncta per micron length of neurite (red bar) compared to vehicle-treated cultures (blue bar). Treatment of cultures with sigma-2/PGRMC1 antagonists (closed bars) restores synaptophysin immunoreactivity to normal, but has no effect when antagonists are dosed alone (open bars). *p = 0.05, Student's paired t-test.</p

Potency of compounds in membrane trafficking assay.

<p>Prevention: compound added 1 hr prior to Abeta oligomers. Treatment: compound added 1 hr after Abeta oligomers. N =  number of experimental repeats (four replicate wells per experiment). n.d.  =  not determined.</p><p>Potency of compounds in membrane trafficking assay.</p

Small molecule Abeta binding antagonists do not act directly on Abeta oligomers.

<p>An ELISA specific for oligomeric forms of Abeta 1–42 shows that (A) preformed oligomers are dissociated by 8-OH quinoline but not by CT0109 or CT0093, and; (B) assembly of oligomers are inhibited by Tween but not by CT0109 or CT0093 at concentration of up to 20 µM for 24 hr, ruling out a direct effect of these compounds on oligomer assembly or disruption.</p

Characterization of synthetic human Abeta 1–42 oligomers by non-denaturing Western blot, MALDI-TOF.

<p><b>A</b>, Freshly prepared solutions of synthetic human Abeta 1–42 (lane 1) or 1–40 (lane 3) peptide loaded onto non-denaturing western gels immediately after reconstitution contain large amounts of monomer (arrow; fainter lower molecular weight band represents peptide degradation product) and little higher molecular weight material. In contrast, the same solution of Abeta 1–42 peptide that is allowed to oligomerize for 24 hours (lane 2) contains much larger amounts of higher molecular weight material >50 kDa, and less monomeric protein. The full length of gel lanes are shown from loading well to dye front. Note that oligomers run differently on non-denaturing gels than globular molecular weight protein size standards <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111898#pone.0111898-Tseng1" target="_blank">[49]</a>. <b>B</b>. The presence of significant amounts of monomer in oligomer preparations is also confirmed by MALDI-TOF analysis of the same Abeta 1–42 oligomer preparation that shows both a 4.5 kDa monomer peak and multiple lower abundance peaks corresponding to oligomers of various sizes. MALDI-TOF (detection range 3–100 kDa) of vehicle (media without Abeta) is shown below for comparison (<b>C</b>).</p

Binding affinity (Kd) of Abeta preparations to cellular compartments of neurons and glia.

<p>Mean Kd ± S.E.M. Data are results of 13 replicates for oligomer binding and 4 replicates for monomer binding. NS  =  no significant binding above background.</p><p>Binding affinity (Kd) of Abeta preparations to cellular compartments of neurons and glia.</p