Lawren VandeVrede

Furoxans (1,2,5-Oxadiazole-N-Oxides) as Novel NO Mimetic Neuroprotective and Procognitive Agents

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO(2)(-), but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatment with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-β peptide (Aβ) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.

Design and Synthesis of Neuroprotective Methylthiazoles and Modification as NO-Chimeras for Neurodegenerative Therapy

Learning and memory deficits in Alzheimers disease (AD) result from synaptic failure and neuronal loss, the latter caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a lead neuroprotective pharmacophore acting in part by GABA(A) receptor potentiation. MZ derivatives were assayed for protection of primary neurons against oxygen-glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class, selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of cognitive deficits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders, such as AD.

A multifunctional therapeutic approach to disease modification in multiple familial mouse models and a novel sporadic model of Alzheimer’s disease

BackgroundClinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer’s disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD.ResultsNMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aβ in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function.ConclusionsThe multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aβ pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2−/−) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aβ and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.

Novel analogues of chlormethiazole are neuroprotective in four cellular models of neurodegeneration by a mechanism with variable dependence on GABA A receptor potentiation

Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABAA receptor dependence were studied.

An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimers transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.

NO/cGMP/pCREB re-activation reverses cognition deficits and attenuates amyloid-β neuropathology in transgenic models of Alzheimer’s disease

Results and Conclusion The hypothesis that activation of CREB through NO/ cGMP signaling might modify the amyloid-b (Ab) neuropathology, linked to AD pathogenesis, was demonstrated in both APP/PS1 and 3xTg transgenic mouse models of AD using small molecules, termed nomethiazoles, also designed to provide neuroprotection and attenuate pro-inflammatory cytokine release. Functional restoration of long-term potentiation was shown in hippocampal slices from AD transgenic mice in accord with observation of restoration of cognitive function in vivo, and was dependent upon soluble guanylyl cyclase (sGC) activation. Levels of pCREB and BDNF were significantly elevated, whereas TNFa, Ab, oligomeric Ab142, and also tau protein were significantly lowered after drug treatment. In the absence of neuronal loss in animal models of AD, neuroprotection was demonstrated in rat primary neurons after oxygen-glucose deprivation or application of oligomeric Ab. The lead nomethiazole was also studied in a novel transgenic mouse model, E4FAD, which incorporates familial AD mutations, but also has targeted replacement of mouse apolipoproteinE with human ApoE4, the major genetic risk factor for sporadic and age-related AD.

Erratum: A multifunctional therapeutic approach to disease modification in multiple familial mouse models and a novel sporadic model of Alzheimer's disease (Molecular Neurodegeneration (2016) 11 (35) DOI: 10.1186/s13024-016-0103-6)

Author details Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA. Department of Psychiatry, Neuropsychiatric Institute, University of Illinois at Chicago, Chicago, IL, USA. Department of Pathology, The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, NY, USA. Department of Biomedical & Molecular Sciences, Faculty of Health Sciences, Queen’s University, Kingston, ON, Canada. Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA.

Discovery of neuroprotective soluble guanylate cyclase modulators (GCMs) aciting via NO/GC/cGMP/CREB pathway

NO/cGMP signaling is essential for normal brain function, including learning and memory, and mediation of long-term potentiation (LTP). NO/cGMP signaling is coupled to cholinergic, glutamatergic, and dopaminergic systems and plays key roles in motor function associated with Parkinson’s disease pathogenesis and L-DOPA therapy. In Alzheimer’s Disease (AD), early synaptic failure, has been linked to dysfunction of gene expression programs mediated via the transcription factor cAMP-response element binding protein (CREB), activation of which is tightly regulated by NO/cGMP. Since several neurodegenerative disorders continue to be dogmatically linked to the chemical toxicity of NO, activation of soluble guanylyl cyclase (sGC) represents a potential therapeutic approach in both AD and PD; whereas negative modulation of sGC may be of use in some stages of PD. Such small molecule sGC modulators (GCMs) can be assayed in cell cultures by measuring levels of phosphorylated CREB (pCREB). GCMs that allosterically potentiate NO activation of sGC have proven successful in clinical trials for peripheral indications, however, there are no reports directed at therapeutic activity in the CNS. The aim of this study is to develop GCMs for use in CNS disorders, including AD and PD. A library of GCMs was synthesized, including positive allosteric modulators (PAMs) and negative modulators, as assessed by increasing levels of pCREB in SH-SY5Y human neuroblastoma cell cultures. Molecules were designed by classical bioisosteric replacement, aiming for desirable physiochemical properties and chemical diversity. GCM PAMs were assayed for cGMP elevation and reversal of neurotoxicity induced by 6-hydroxydopamine in dopaminergic neuronal cells. One GCM PAM, active in cell culture, was further tested for the reversal of memory deficits in mice treated with scopolamine, and drug levels in brain and plasma measured. The ligand binding site for sGC stimulators and GCM PAMs on the sGC protein remains to be definitively defined. Photoaffinity probes were designed that retained the activity of the parent GCM PAM in cell cultures in order to elucidate the binding mode to sGC and to indicate other protein partners for such compounds.