σ2R/TMEM97 in retinal ganglion cell degeneration.

The sigma 2 receptor (σ2R) was recently identified as an endoplasmic reticulum (ER) membrane protein known as transmembrane protein 97 (TMEM97). Studies have shown that σ2R/TMEM97 binding compounds are neuroprotective, suggesting a role of σ2R/TMEM97 in neurodegenerative processes. To understand the function of σ2R/TMEM97 in neurodegeneration pathways, we characterized ischemia-induced retinal ganglion cell (RGC) degeneration in TMEM97-/- mice and found that RGCs in TMEM97-/- mice are resistant to degeneration. In addition, intravitreal injection of a selective σ2R/TMEM97 ligand DKR-1677 significantly protects RGCs from ischemia-induced degeneration in wildtype mice. Our results provide conclusive evidence that σ2R/TMEM97 plays a role to facilitate RGC death following ischemic injury and that inhibiting the function of σ2R/TMEM97 is neuroprotective. This work is a breakthrough toward elucidating the biology and function of σ2R/TMEM97 in RGCs and likely in other σ2R/TMEM97 expressing neurons. Moreover, these findings support future studies to develop new neuroprotective approaches for RGC degenerative diseases by inhibiting σ2R/TMEM97.

Neuroprotective Effects of σ2R/TMEM97 Receptor Modulators in the Neuronal Model of Huntington's Disease.

Huntington's disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that encodes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration, for which there is no cure or treatment that modifies disease progression. In previous studies, we have shown that small molecules that bind selectively to σ2R/TMEM97 can have significant neuroprotective effects in models of Alzheimer's disease, traumatic brain injury, and several other neurodegenerative diseases. In the present work, we extend these investigations and show that certain σ2R/TMEM97-selective ligands decrease mHTT-induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ2R/TMEM97 and determined their binding profiles (Ki values) for σ2R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ2R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for 7 days and then co-transfected with either a normal HTT construct (Htt N-586-22Q/GFP) or the mHTT construct Htt-N586-82Q/GFP. Transfected neurons were treated with either σ2R/TMEM97 or σ1R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ2R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ1R antagonist known to block neuroprotection by σ1R ligands. These results indicate for the first time that σ2R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ2R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.

Asymmetric synthesis of pharmaceutically relevant 1-aryl-2-heteroaryl- and 1,2-diheteroarylcyclopropane-1-carboxylates.

This study describes general methods for the enantioselective syntheses of pharmaceutically relevant 1-aryl-2-heteroaryl- and 1,2-diheteroarylcyclopropane-1-carboxylates through dirhodium tetracarboxylate-catalysed asymmetric cyclopropanation of vinyl heterocycles with aryl- or heteroaryldiazoacetates. The reactions are highly diastereoselective and high asymmetric induction could be achieved using either (R)-pantolactone as a chiral auxiliary or chiral dirhodium tetracarboxylate catalysts. For meta- or para-substituted aryl- or heteroaryldiazoacetates the optimum catalyst was Rh2(R-p-Ph-TPCP)4. In the case of ortho-substituted aryl- or heteroaryldiazoacetates, the optimum catalyst was Rh2(R-TPPTTL)4. For a highly enantioselective reaction with the ortho-substituted substrates, 2-chloropyridine was required as an additive in the presence of either 4 Å molecular sieves or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Under the optimized conditions, the cyclopropanation could be conducted in the presence of a variety of heterocycles, such as pyridines, pyrazines, quinolines, indoles, oxadiazoles, thiophenes and pyrazoles.

Neuroprotective Efficacy of a Sigma 2 Receptor/TMEM97 Modulator (DKR-1677) after Traumatic Brain Injury.

Compounds targeting the sigma 2 receptor, which we recently cloned and showed to be identical with transmembrane protein 97 (σ2R/TMEM97), are broadly applicable therapeutic agents currently in clinical trials for imaging in breast cancer and for treatment of Alzheimer's disease and schizophrenia. These promising applications coupled with our previous observation that the σ2R/TMEM97 modulator SAS-0132 has neuroprotective attributes and improves cognition in wild-type mice suggests that modulating σ2R/TMEM97 may also have therapeutic benefits in other neurodegenerative conditions such as traumatic brain injury (TBI). Herein, we report that DKR-1677, a novel derivative of SAS-0132 with increased affinity and selectivity for σ2R/Tmem97 ( Ki = 5.1 nM), is neuroprotective after blast-induced and controlled cortical impact (CCI) TBI in mice. Specifically, we discovered that treatment with DKR-1677 decreases axonal degeneration after blast-induced TBI and enhances survival of cortical neurons and oligodendrocytes after CCI injury. Furthermore, treatment with DKR-1677 preserves cognition in the Morris water maze after blast TBI. Our results support an increasingly broad role for σ2R/Tmem97 modulation in neuroprotection and suggest a new approach for treating patients suffering from TBI.

Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of SOS1-Mediated Nucleotide Exchange on RAS.

Son of sevenless homologue 1 (SOS1) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. In its active form, GTP-bound RAS is responsible for numerous critical cellular processes. Aberrant RAS activity is involved in ∼30% of all human cancers; hence, SOS1 is an attractive therapeutic target for its role in modulating RAS activation. Here, we describe a new series of benzimidazole-derived SOS1 agonists. Using structure-guided design, we discovered small molecules that increase nucleotide exchange on RAS in vitro at submicromolar concentrations, bind to SOS1 with low double-digit nanomolar affinity, rapidly enhance cellular RAS-GTP levels, and invoke biphasic signaling changes in phosphorylation of ERK 1/2. These compounds represent the most potent series of SOS1 agonists reported to date.

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at submicromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase-extracellular regulated kinase (MAPK-ERK) pathway, resulting in a decrease in pERK1/2T202/Y204 protein levels at higher compound concentrations.

Small molecule modulators of σ2R/Tmem97 reduce alcohol withdrawal-induced behaviors.

Repeated cycles of intoxication and withdrawal enhance the negative reinforcing properties of alcohol and lead to neuroadaptations that underlie withdrawal symptoms driving alcohol dependence. Pharmacotherapies that target these neuroadaptations may help break the cycle of dependence. The sigma-1 receptor (σ1R) subtype has attracted interest as a possible modulator of the rewarding and reinforcing effects of alcohol. However, whether the sigma-2 receptor, recently cloned and identified as transmembrane protein 97 (σ2R/TMEM97), plays a role in alcohol-related behaviors is currently unknown. Using a Caenorhabditis elegans model, we identified two novel, selective σ2R/Tmem97 modulators that reduce alcohol withdrawal behavior via an ortholog of σ2R/TMEM97. We then show that one of these compounds blunted withdrawal-induced excessive alcohol drinking in a well-established rodent model of alcohol dependence. These discoveries provide the first evidence that σ2R/TMEM97 is involved in alcohol withdrawal behaviors and that this receptor is a potential new target for treating alcohol use disorder.

High-Content Microfluidic Screening Platform Used To Identify σ2R/Tmem97 Binding Ligands that Reduce Age-Dependent Neurodegeneration in C. elegans SC_APP Model.

The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently, we developed a single-copy model of amyloid precursor protein (SC_APP) induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (σ2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our σ2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that σ2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.

Norbenzomorphan Scaffold: Chemical Tool for Modulating Sigma Receptor-Subtype Selectivity.

Some norbenzomorphans exhibit high affinity for sigma 1 and sigma 2 receptors, and varying the position of substituents on the aromatic ring of this scaffold has a significant effect on subtype selectivity. In particular, compounds bearing several different substituents at C7 of the norbenzomorphan ring system exhibit a general preference for the sigma 1 receptor, whereas the corresponding C8-substituted analogues preferentially bind at the sigma 2 receptor. These findings suggest that the norbenzomorphan scaffold may be a unique chemical template that can be easily tuned to prepare small molecules for use as tool compounds to study the specific biological effects arising from preferential binding at either sigma receptor subtype. In the absence of structural characterization data for the sigma 2 receptor, such compounds will be useful toward refining the pharmacophore model of its binding site.

Syntheses of Gliocladin C and Related Alkaloids.

A unique approach to gliocladin C and related alkaloids was developed that features an unprecedented nucleophilic addition of a diketopiperazine to an isatin derivative followed by a Friedel-Crafts alkylation of the resultant tertiary alcohol with indole to set the key quaternary center. Chemoselective oxindole reduction and cyclization delivered a pivotal hexahydropyrrolo[2,3-b]indole diketopiperazine intermediate that was readily converted into (±)-gliocladin C, (±)-T988C, and (±)-gliocladine C, culminating in the shortest approach to these alkaloids reported to date.

Norbenzomorphan Framework as a Novel Scaffold for Generating Sigma†2 Receptor/PGRMC1 Subtype-Selective Ligands.

A novel structural class with high affinity and subtype selectivity for the sigma 2 receptor has been discovered. Preliminary structure-affinity relationship data are presented showing that 8-substituted 1,3,4,5-tetrahydro-1,5-methanobenzazepine (norbenzomorphan) derivatives elicit modest to high selectivity for the sigma 2 over the sigma 1 receptor subtype. Indeed, piperazine analogue 8-(4-(3-ethoxy-3-oxopropyl)piperazin-1-yl)-1,3,4,5-tetrahydro-1,5-methanobenzazepine-2-carboxylate (SAS-1121) is 574-fold selective for the sigma 2 over the sigma 1 receptor, thereby establishing it as one of the more subtype-selective sigma 2 binding ligands reported to date. Emerging evidence has implicated the sigma 2 receptor in multiple health disorders, so the drug-like characteristics of many of the selective sigma 2 receptor ligands disclosed herein, coupled with their structural similarity to frameworks found in known drugs, suggest that norbenzomorphan analogues may be promising candidates for further development into drug leads.