Exploration of Diazaspiro Cores as Piperazine Bioisosteres in the Development of σ2 Receptor Ligands.

A series of σ2R compounds containing benzimidazolone and diazacycloalkane cores was synthesized and evaluated in radioligand binding assays. Replacing the piperazine moiety in a lead compound with diazaspiroalkanes and the fused octahydropyrrolo[3,4-b] pyrrole ring system resulted in a loss in affinity for the σ2R. On the other hand, the bridged 2,5-diazabicyclo[2.2.1]heptane, 1,4-diazepine, and a 3-aminoazetidine analog possessed nanomolar affinities for the σ2R. Computational chemistry studies were also conducted with the recently published crystal structure of the σ2R/TMEM97 and revealed that hydrogen bond interactions with ASP29 and π-stacking interactions with TYR150 were largely responsible for the high binding affinity of small molecules to this protein.

Discovery of Investigational Drug CT1812, an Antagonist of the Sigma-2 Receptor Complex for Alzheimer's Disease.

An unbiased phenotypic neuronal assay was developed to measure the synaptotoxic effects of soluble Aß oligomers. A collection of CNS druglike small molecules prepared by conditioned extraction was screened. Compounds that prevented and reversed synaptotoxic effects of Aß oligomers in neurons were discovered to bind to the sigma-2 receptor complex. Select development compounds displaced receptor-bound Aß 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.

Preclinical and clinical biomarker studies of CT1812: A novel approach to Alzheimer's disease modification.

INTRODUCTION: Amyloid beta (Aß) oligomers are one of the most toxic structural forms of the Aß 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 Aß 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 Aß 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). METHODS: Experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. RESULTS: CT1812 significantly and dose-dependently displaced Aß oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aß oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. DISCUSSION: These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aß oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.

Sigma-2 receptor antagonists rescue neuronal dysfunction induced by Parkinson's patient brain-derived α-synuclein.

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.

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.

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

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.

Discovery of ADDL--targeting small molecule drugs for Alzheimer's disease.

Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.

The discovery of biaryl acids and amides exhibiting antibacterial activity against Gram-positive bacteria.

Solid-phase synthetic methods for biaryl-based compounds were developed resulting in the construction of two 1000-member libraries. Numerous compounds were identified by high-throughput screening using whole cell screens to exhibit anti-microbial activity against Gram-positive bacteria. A series of biaryl compounds containing natural and unnatural amino acids were made to explore the SAR of the amino acid functionality.