An unbiased phenotypic neuronal assay was developed to measure the synaptotoxic effects of soluble Aβ oligomers. A collection of CNS druglike small molecules prepared by conditioned extraction was screened. Compounds that prevented and reversed synaptotoxic effects of Aβ oligomers in neurons were discovered to bind to the sigma-2 receptor complex. Select development compounds displaced receptor-bound Aβ oligomers, rescued synapses, and restored cognitive function in transgenic hAPP Swe/Ldn mice. Our first-in-class orally administered small molecule investigational drug 7 (CT1812) has been advanced to Phase II clinical studies for Alzheimer's disease.

Amyloid beta (Aβ) oligomers are one of the most toxic structural forms of the Aβ protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients’ brain tissue. We previously demonstrated that antagonists of the sigma‐2 receptor complex effectively block Aβ oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma‐2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aβ oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567).

α‐Synuclein oligomers are thought to have a pivotal role in sporadic and familial Parkinson's disease (PD) and related α‐synucleinopathies, causing dysregulation of protein trafficking, autophagy/lysosomal function, and protein clearance, as well as synaptic function impairment underlying motor and cognitive symptoms of PD. Moreover, trans‐synaptic spread of α‐synuclein oligomers is hypothesized to mediate disease progression. Therapeutic approaches that effectively block α‐synuclein oligomer‐induced pathogenesis are urgently needed. Here, we show for the first time that α‐synuclein species isolated from human PD patient brain and recombinant α‐synuclein oligomers caused similar deficits in lipid vesicle trafficking rates in cultured rat neurons and glia, while α‐synuclein species isolated from non‐PD human control brain samples did not. Recombinant α‐synuclein oligomers also increased neuronal expression of lysosomal‐associated membrane protein‐2A (LAMP‐2A), the lysosomal receptor that has a critical role in chaperone‐mediated autophagy. Unbiased screening of several small molecule libraries (including the NIH Clinical Collection) identified sigma‐2 receptor antagonists as the most effective at blocking α‐synuclein oligomer‐induced trafficking deficits and LAMP‐2A upregulation in a dose‐dependent manner. These results indicate that antagonists of the sigma‐2 receptor complex may alleviate α‐synuclein oligomer‐induced neurotoxicity and are a novel therapeutic approach for disease modification in PD and related α‐synucleinopathies.

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.

Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.

OBJECTIVE: We sought to determine whether CT01344, an antagonist of Abeta oligomer binding to specific neuronal receptors or antibodies directed against epitopes in the same receptor were capable of displacing native human Abeta oligomers in the immediate vicinity of plaques in frozen unfixed post-mortem tissue sections from Alzheimer's patient brains. BACKGROUND: Synaptic loss is known to occur in a region surrounding Abeta plaques in human brain.¹ We have previously shown that CT01344, an experimental therapeutic candidate for Alzheimer's disease, and antibodies directed against specific epitopes in these receptors can competitively displace synthetic Abeta oligomer binding to CNS receptors on rat primary hippocampal and cortical cultures.² It has not yet been shown that native human Abeta oligomers can be displaced from human brain tissue. DESIGN/METHODS: We measured the intensity of oligomer binding in a 2.6 micron halo around Thioflavin-S positive, dense core plaques on tissue sections incubated for 1 hour in the presence of excess concentration of CT01344, antibodies or vehicle via immunohistochemistry with AW7 antibody and automated image processing. RESULTS: CT01344 and antibodies significantly reduced the intensity of Abeta oligomers in plaque halos in a dose dependent manner (one way ANOVA p<0.0001). CT1344 caused a concentration-dependent decrease in oligomer intensity (15% +/-1.6% S.D at 5 µM, 28% +/- 4% at 15 uM (Spearman's Rho =0.038). Antibodies directed against specific target receptor epitopes reduced oligomer intensity by 48% +/-10% at 1 ng/mL (p=0.001). CONCLUSIONS: This study suggests that native Abeta oligomer binding to human brain tissue can be displaced by both antibodies and small molecule receptor antagonists directed against a specific neuronal receptor. Compounds such as CT01344 represent promising disease modifying drug candidates and merit further study in a clinical setting. Study Supported by: Cognition Therapeutics Inc., 2403 Sidney Street, Suite 261,Pittsburgh, PA 15203 1. Koffie RM, Hashimoto T, Tai HC et al. Brain. 2012;135:2155-682. 2. Rehak et al. 2013. 43rd Annual meeting of the Society for Neuroscience

The memory loss that begins in Mild Cognitive Impairment (MCI) and progresses as Alzheimer's disease continues is likely caused by accumulation of Abeta 1-42 oligomers in the patient's brain (Georganopolou et al.,’05, Shankar et al.,’08, Tomic et al.,’09, Fukumoto et al.,’10). Oligomers bind to neuronal surface proteins at the synapse and inhibit synaptic plasticity phenomenon such as LTP via effects on membrane trafficking and induction of reversible spine loss in hippocampal neurons. This results in reversible impairment of memory that culminates in anterograde amnesia in the early stages of Alzheimer's disease. Therapeutics directed against these oligomers should rapidly block their effects on memory and stop disease progress. We have screened a proprietary CNS drug-like library and discovered three structurally distinct lead series that block the synaptic toxicity of Abeta oligomers on primary hippocampal neurons with low micromolar potency. These molecules appear to act via partial antagonism of Abeta oligomer binding to the surface of the neuron, or disruption of the Abeta oligomer ligand itself. These molecules are plasma-stable (plasma t1/2 = 3hr), non-toxic and highly brain penetrant (brain/plasma ratio = 8). Representative members of these compound series were tested in the fear conditioning behavioral task for their ability to preserve normal associative memory. Compounds were injected bilaterally via intrahippocampal cannula (2 pmol) one hour prior to the injection of Abeta 1-42 oligomers (200nM total Abeta) in wild-type C57Bl/6mice. After an additional 20 minutes, animals received a mild electric foot shock. Animals were tested for context-dependent learning 24 hours later. Animals receiving Abeta oligomer injections exhibited significant memory deficits as measured by decreased freezing behavior vs. vehicle (13 +/- 2% vs. 27 +/- 1% respectively). Compounds administered prior to Abeta oligomer completely blocked the effects of Abeta oligomers on memory (CT0109 =30 +/- 2%, CT0093 = 25 +/- 1%), and had no effect on memory when administered without Abeta oligomers (CT0109 =28 +/- 1%, CT0093 = 31 +/- 1%). Systemic administration of these compounds does not induce motor deficits or abnormal behavior. These compounds show promise as a disease-modifying Alzheimer's therapeutics.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

The highly sensitive and artificial biochemical assays that enable high-throughput screening are vulnerable to artifact-generating compounds that occur in drug screening collections. An investigation of known aggregator compounds and amyloid fibrillization inhibitors suggests that such inhibitors operate via a nonspecific mechanism in a well-used assay of amyloid fibrillization.

Small molecule modulators of neural stem cell (NSC) differentiation might potentially be developed into orally administered neurogenic drugs to treat neurodegenerative diseases including Alzheimers disease. New technologies developed for the study of NSC culture, proliferation and differentiation have enabled the establishment of screening platforms to identify small molecules with neurogenic activity. Recent patents claim novel small molecules identified from screening collections that stimulate or otherwise regulate stem cell differentiation and neurogenesis. Several patents claim newly discovered NSC differentiation modulating activity of previously marketed drugs suggesting perhaps a previously unknown mechanism of action of these drugs and/or implicating the target enzyme and receptor pathways as key players in neurogenesis. This relatively new area of research into small molecule modulators of neurogenesis is reviewed and recent patents claiming small molecule neurogenic compounds, potentially orally administered CNS regenerative therapies are summarized.

The history of drug development has its foundation firmly set in the study of natural remedies used to treat human disease over centuries. Analysis of medicinal plants, bioactive cultures, and increased understanding of micronutrients in the food chain opened the door to the development of purified and defined chemical compounds as dose-controlled medicines. Thus, with the early discovery of cardiotonics in foxglove, salicylic acid in willow bark, morphine in poppies, and penicillin in mold, the pharmaceutical, industry was launched. Such natural small molecules served as treatments for disease and ultimately, as pharmacologic tools to enable the understanding of the biochemical pathways and mechanisms of disease. In contrast, modern drug discovery technologies coupled with the powerful tools of biotechnology have prompted drug discovery organizations to focus on target-driven drug discovery at the molecular level by launching high-throughput screening programs using artificial biochemical assays. At a time when the pharmaceutical industry has come under scrutiny for high rates of drug development failure, it is interesting to see that natural products drug discovery has been marginalized in favor of this high-throughput biochemical screening paradigm. If modern drug development is once again to benefit from natural products as a source, then the limitations of artificial biochemical assays as applied to the screening of natural extracts must be realized in order to capitalize on the vast natural molecular diversity and rich ethnobotanic data that has emerged worldwide. Natural compounds can again become central players in the treatment of disease and in the understanding of disease mechanisms. (C) 2008 Elsevier Inc. All rights reserved.

Molecular diversity is of vital importance in drug screening in general and for the discovery and development of new pharmacophores in particular. Biochemical screening is a powerful tool for pharmacophore development given understanding of the properties of a good lead compound operating in the biochemical environment. The properties of leadlikeness have evolved to accommodate the artificial conditions of a biochemical assay. Accordingly, the properties of leadlikeness that are suited for screening at protein targets biochemically are different and complementary to the properties of druglikeness used to guide the selection of good compounds studied biologically in cellular studies and animal models. The benefits of leadlikeness in the biochemical screening arena (including fragment-based screening and co-crystallization studies) are described here and recommendations are forwarded for the generation of leadlike molecular diversity. Chemically stable low molecular weight ‘minimalist’ compounds (or fragments) with dense heteroatom substitution and variable conformational constraint are promoted as conceptually superior compounds for biochemical screening.