Fragment-based drug discovery and biological evaluation of novel cannabinol-based inhibitors of oxytosis/ferroptosis for neurological disorders.

The oxytosis/ferroptosis regulated cell death pathway is an emerging field of research owing to its pathophysiological relevance to a wide range of neurological disorders, including Alzheimer's and Parkinson's diseases and traumatic brain injury. Developing novel neurotherapeutics to inhibit oxytosis/ferroptosis offers exciting opportunities for the treatment of these and other neurological diseases. Previously, we discovered cannabinol (CBN) as a unique, potent inhibitor of oxytosis/ferroptosis by targeting mitochondria and modulating their function in neuronal cells. To further elucidate which key pharmacophores and chemical space are essential to the beneficial effects of CBN, we herein introduce a fragment-based drug discovery strategy in conjunction with cell-based phenotypic screens using oxytosis/ferroptosis to determine the structure-activity relationship of CBN. The resulting information led to the development of four new CBN analogs, CP1-CP4, that not only preserve the sub-micromolar potency of neuroprotection and mitochondria-modulating activities seen with CBN in neuronal cell models but also have better druglike properties. Moreover, compared to CBN, the analog CP1 shows improved in vivo efficacy in the Drosophila model of mild traumatic brain injury. Together these studies identify the key molecular scaffolds of cannabinoids that contribute to neuroprotection against oxytosis/ferroptosis. They also highlight the advantageous approach of combining in vitro cell-based assays and rapid in vivo studies using Drosophila models for evaluating new therapeutic compounds.

CMS121: a novel approach to mitigate aging-related obesity and metabolic dysfunction.

BACKGROUND: Modulated by differences in genetic and environmental factors, laboratory mice often show progressive weight gain, eventually leading to obesity and metabolic dyshomeostasis. Since the geroneuroprotector CMS121 has a positive effect on energy metabolism in a mouse model of type 2 diabetes, we investigated the potential of CMS121 to counteract the metabolic changes observed during the ageing process of wild type mice. METHODS: Control or CMS121-containing diets were supplied ad libitum for 6 months, and mice were sacrificed at the age of 7 months. Blood, adipose tissue, and liver were analyzed for glucose, lipids, and protein markers of energy metabolism. RESULTS: The CMS121 diet induced a 40% decrease in body weight gain and improved both glucose and lipid indexes. Lower levels of hepatic caspase 1, caspase 3, and NOX4 were observed with CMS121 indicating a lower liver inflammatory status. Adipose tissue from CMS121-treated mice showed increased levels of the transcription factors Nrf1 and TFAM, as well as markers of mitochondrial electron transport complexes, levels of GLUT4 and a higher resting metabolic rate. Metabolomic analysis revealed elevated plasma concentrations of short chain acylcarnitines and butyrate metabolites in mice treated with CMS121. CONCLUSIONS: The diminished de novo lipogenesis, which is associated with increased acetyl-CoA, acylcarnitine, and butyrate metabolite levels, could contribute to safeguarding not only the peripheral system but also the aging brain. By mimicking the effects of ketogenic diets, CMS121 holds promise for metabolic diseases such as obesity and diabetes, since these diets are hard to follow over the long term.

Differential Interferon Signaling Regulation and Oxidative Stress Responses in the Cerebral Cortex and Cerebellum Could Account for the Spatiotemporal Pattern of Neurodegeneration in Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative condition caused by genetic mutations of the NPC1 or NPC2 genes that encode the NPC1 and NPC2 proteins, respectively, which are believed to be responsible for cholesterol efflux from late-endosomes/lysosomes. The pathogenic mechanisms that lead to neurodegeneration in NPC are not well understood. There are, however, well-defined spatiotemporal patterns of neurodegeneration that may provide insight into the pathogenic process. For example, the cerebellum is severely affected from early disease stages, compared with cerebral regions, which remain relatively spared until later stages. Using a genome-wide transcriptome analysis, we have recently identified an aberrant pattern of interferon activation in the cerebella of pre-symptomatic Npc1-/- mice. Here, we carried out a comparative transcriptomic analysis of cerebral cortices and cerebella of pre-symptomatic Npc1-/- mice and age-matched controls to identify differences that may help explain the pathological progression within the NPC brain. We report lower cerebral expression of genes within interferon signaling pathways, and significant differences in the regulation of oxidative stress, compared with the cerebellum. Our findings suggest that a delayed onset of interferon signaling, possibly linked to lower oxidative stress, may account for the slower onset of cerebral cortical pathology in the disease.

REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria.

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.

CMS121 Partially Attenuates Disease Progression in Mouse Models of Huntington's Disease.

There are currently no drugs that meaningfully slow down the progression of Huntington's disease (HD). Moreover, drug candidates against a single molecular target have not had significant success. Therefore, a different approach to HD drug discovery is needed. Previously we showed that the flavonol fisetin is efficacious in mouse and fly models of HD (Hum. Mol. Gen. 20:261, 2011). It is also effective in animal models of Alzheimer's disease (AD), ischemic stroke, and the CNS complications of diabetes, all of which share some pathological features with HD. Potent derivatives of fisetin with improved pharmacology were made that maintain its multiple biological activities (J. Med. Chem. 55:378, 2012). From 160 synthetic fisetin derivatives, one, CMS121, was selected for further study in the context of HD based on pharmacological parameters and its efficacy in animal models of AD. Both R6/2 and YAC128 mouse models of HD were used in these studies. We examined motor function using multiple assays as well as survival. In the R6/2 mice, we also looked at the effects of CMS121 on striatal gene expression. In both models, we found a slowing of motor dysfunction and an increase in median life span. Interestingly, in the YAC128 mice, the effects on the slowing in motor function loss became increasingly more pronounced as the mice aged. CMS121 also reduced HD-driven changes in the expression of genes associated with the proteasome and oxidative phosphorylation. Overall, these results suggest that CMS121 could provide some benefits for HD patients, particularly with regard to increasing health span.

A structural comparison of salt forms of dopamine with the structures of other phenylethylamines.

The structures of four salt forms of dopamine are reported. These are dopamine [2-(3,4-dihydroxyphenyl)ethan-1-aminium] benzoate, C8H12NO2+·C7H5O2-, I, dopamine 4-nitrobenzoate, C8H12NO2+·C7H4NO4-, II, dopamine ethanedisulfonate, 2C8H12NO2+·C2H4O6S22-, III, and dopamine 4-hydroxybenzenesulfonate monohydrate, C8H12NO2+·C6H5O4S-·H2O, IV. In all four structures, the dopamine cation adopts an extended conformation. Intermolecular interaction motifs that are common in the salt forms of tyramine can be found in related dopamine structures, but hydrogen bonding in the dopamine structures appear to be more variable and less predictable than for tyramine. Packing analysis discovered three dopamine-containing groups of structures that can be described as isostructural with regards to the cation positions. Two of these groups contain both dopamine and tyramine species, and one of these is also highly variable in other ways too, containing anhydrous and hydrated forms, different anion types and ionized and neutral phenylethylamine species. As such, the group illustrates that packing behaviour can be robust and similar even where intermolecular interactions such as hydrogen bonds are very different.

Attenuation of Age-Related Hearing Impairment in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice Treated with Fatty Acid Synthase Inhibitor CMS121.

In the senescence-accelerated mouse prone 8 (SAMP8) mouse model, oxidative stress leads to premature senescence and age-related hearing impairment (ARHI). CMS121 inhibits oxytosis/ferroptosis by targeting fatty acid synthase. The aim of our study was to determine whether CMS121 is protective against ARHI in SAMP8 mice. Auditory brainstem responses (ABRs) were used to assess baseline hearing in sixteen 4-week-old female SAMP8 mice, which were divided into two cohorts. The control group was fed a vehicle diet, while the experimental group was fed a diet containing CMS121. ABRs were measured until 13 weeks of age. Cochlear immunohistochemistry was performed to analyze the number of paired ribbon-receptor synapses per inner hair cell (IHC). Descriptive statistics are provided with mean ± SEM. Two-sample t-tests were performed to compare hearing thresholds and paired synapse count across the two groups, with alpha = 0.05. Baseline hearing thresholds in the control group were statistically similar to those of the CMS121 group. At 13 weeks of age, the control group had significantly worse hearing thresholds at 12 kHz (56.5 vs. 39.8, p = 0.044) and 16 kHz (64.8 vs. 43.8, p = 0.040) compared to the CMS121 group. Immunohistochemistry showed a significantly lower synapse count per IHC in the control group (15.7) compared to the CMS121 group (18.4), p = 0.014. Our study shows a significant reduction in ABR threshold shifts and increased preservation of IHC ribbon synapses in the mid-range frequencies among mice treated with CMS121 compared to untreated mice.

The Geroprotective Drug Candidate CMS121 Alleviates Diabetes, Liver Inflammation, and Renal Damage in db/db Leptin Receptor Deficient Mice.

db/db mice, which lack leptin receptors and exhibit hyperphagia, show disturbances in energy metabolism and are a model of obesity and type 2 diabetes. The geroneuroprotector drug candidate CMS121 has been shown to be effective in animal models of Alzheimer's disease and aging through the modulation of metabolism. Thus, the hypothesis was that CMS121 could protect db/db mice from metabolic defects and thereby reduce liver inflammation and kidney damage. The mice were treated with CMS121 in their diet for 6 months. No changes were observed in food and oxygen consumption, body mass, or locomotor activity compared to control db/db mice, but a 5% reduction in body weight was noted. Improved glucose tolerance and reduced HbA1c and insulin levels were also seen. Blood and liver triglycerides and free fatty acids decreased. Improved metabolism was supported by lower levels of fatty acid metabolites in the urine. Markers of liver inflammation, including NF-κB, IL-18, caspase 3, and C reactive protein, were lowered by the CMS121 treatment. Urine markers of kidney damage were improved, as evidenced by lower urinary levels of NGAL, clusterin, and albumin. Urine metabolomics studies provided further evidence for kidney protection. Mitochondrial protein markers were elevated in db/db mice, but CMS121 restored the renal levels of NDUFB8, UQCRC2, and VDAC. Overall, long-term CMS121 treatment alleviated metabolic imbalances, liver inflammation, and reduced markers of kidney damage. Thus, this study provides promising evidence for the potential therapeutic use of CMS121 in treating metabolic disorders.

A class of anti-inflammatory lipids decrease with aging in the central nervous system.

Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

Structural Requirements for the Neuroprotective and Anti-Inflammatory Activities of the Flavanone Sterubin.

Alzheimer's disease (AD) is the most frequent age-associated disease with no treatments that can prevent, delay, slow, or stop its progression. Thus, new approaches to drug development are needed. One promising approach is the use of phenotypic screening assays that can identify compounds that have therapeutic efficacy in target pathways relevant to aging and cognition, as well as AD pathology. Using this approach, we identified the flavanone sterubin, from Yerba santa (Eriodictyon californicum), as a potential drug candidate for the treatment of AD. Sterubin is highly protective against multiple initiators of cell death that activate distinct death pathways, potently induces the antioxidant transcription factor Nrf2, and has strong anti-inflammatory activity. Moreover, in a short-term model of AD, it was able to prevent decreases in short- and long-term memory. In order to better understand which key chemical functional groups are essential to the beneficial effects of sterubin, we compared the activity of sterubin to that of seven closely related flavonoids in our phenotypic screening assays. Surprisingly, only sterubin showed both potent neuroprotective activity against multiple insults as well as strong anti-inflammatory activity against several distinct inducers of inflammation. These effects correlated directly with the ability of sterubin to strongly induce Nrf2 in both nerve and microglial cells. Together, these results define the structural requirements underlying the neuroprotective and anti-inflammatory effects of sterubin and they provide the basis for future studies on new compounds based on sterubin.

The Role of AMP-activated Protein Kinase in Oxytosis/Ferroptosis: Protector or Potentiator?

Significance: Evidence for a role for the oxytosis/ferroptosis regulated cell death pathway in aging and neurodegenerative diseases has been growing over the past few years. Because of this, there is an increasing necessity to identify endogenous signaling pathways that can be modulated to protect cells from this form of cell death. Recent Advances: Recently, several studies have identified a protective role for the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase 1 (ACC1) pathway in oxytosis/ferroptosis. However, there are also a number of studies suggesting that this pathway contributes to cell death initiated by various inducers of oxytosis/ferroptosis. Critical Issues: The goals of this review are to provide an overview and analysis of the published studies and highlight specific areas where more research is needed. Future Directions: Much remains to be learned about AMPK signaling in oxytosis/ferroptosis, especially the conditions where it is protective. Furthermore, the role of AMPK signaling in the brain and especially the aging brain needs further investigation.

Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria independently of cannabinoid receptors.

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.

The Alzheimer's disease drug candidate J147 decreases blood plasma fatty acid levels via modulation of AMPK/ACC1 signaling in the liver.

J147 is a novel drug candidate developed to treat neurological dysfunction. Numerous studies have demonstrated the beneficial effects of J147 in cellular and animal models of disease which has led to the transitioning of the compound into human clinical trials. However, no biomarkers for its target engagement have been identified. Here, we determined if specific metabolites in the plasma could be indicative of J147's activity in vivo. Plasma lipidomics data from three independent rodent studies were assessed along with liver lipidomics data from one of the studies. J147 consistently reduced plasma free fatty acid (FFA) levels across the independent studies. Decreased FFA levels were also found in the livers of J147-treated mice that correlated well with those in the plasma. These changes in the liver were associated with activation of the AMP-activated protein kinase/acetyl-CoA carboxylase 1 signaling pathway. A reduction in FFA levels by J147 was confirmed in HepG2 cells, where activation of the AMPK/ACC1 pathway was seen along with increases in acetyl-CoA and ATP levels which correlated with enhanced cellular bioenergetics. Our data show that J147 targets liver cells to activate the AMPK/ACC1 signaling pathway and preserve energy at the expense of inhibiting FFA synthesis.

Profiling the chemical nature of anti-oxytotic/ferroptotic compounds with phenotypic screening.

Because old age is the greatest risk factor for Alzheimer's disease (AD), it is critical to target the pathological events that link aging to AD in order to develop an efficient treatment that acts upon the primary causes of the disease. One such event might be the activation of oxytosis/ferroptosis, a unique cell death mechanism characterized by mitochondrial dysfunction and lethal lipid peroxidation. Here, a comprehensive library of >900 natural compounds was screened for protection against oxytosis/ferroptosis in nerve cells with the goal of better understanding the chemical nature of inhibitors of oxytosis/ferroptosis. Although the compounds tested spanned structurally diverse chemical classes from animal, microbial, plant and synthetic origins, a small set of very potent anti-oxytotic/ferroptotic compounds was identified that was highly enriched in plant quinones. The ability of these compounds to protect against oxytosis/ferroptosis strongly correlated with their ability to protect against in vitro ischemia and intracellular amyloid-beta toxicity in nerve cells, indicating that aspects of oxytosis/ferroptosis also underly other toxicities that are relevant to AD. Importantly, the anti-oxytotic/ferroptotic character of the quinone compounds relied on their capacity to target and directly prevent lipid peroxidation in a manner that required the reducing activity of cellular redox enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and ferroptosis suppressor protein 1 (FSP1). Because some of the compounds increased the production of total reactive oxygen species while decreasing lipid peroxidation, it appears that the pro-oxidant character of a compound can coexist with an inhibitory effect on lipid peroxidation and, consequently, still prevent oxytosis/ferroptosis. These findings have significant implications for the understanding of oxytosis/ferroptosis and open new approaches to the development of future neurotherapies.

The search for anti-oxytotic/ferroptotic compounds in the plant world.

Oxytosis/ferroptosis is a form of non-apoptotic regulated cell death characterized by glutathione (GSH) depletion and dysregulated production of mitochondrial ROS that results in lethal lipid peroxidation. As the significance of oxytosis/ferroptosis to age-associated human diseases is now beginning to be appreciated, the development of innovative approaches to identify novel therapeutics that target the oxytosis/ferroptosis pathway could not be more timely. Due to their sessile nature, plants are exposed to a variety of stresses that trigger physiological changes similar to those found in oxytosis/ferroptosis. As such, they have evolved a rich array of chemical strategies to deal with those challenging conditions. This review details a drug discovery approach for identifying potent inhibitors of oxytosis/ferroptosis from plants for the treatment of Alzheimer's disease and related dementias, thereby highlighting the tremendous potential of plant-based research for developing new medicines while simultaneously being a catalyst for sustainability.

Defining a pharmacological inhibitor fingerprint for oxytosis/ferroptosis.

Ferroptosis was first described in 2012 as an iron- and lipid peroxidation-dependent form of regulated cell death. Since its initial description, these two characteristics have informed numerous cell culture studies where inhibitors of lipid peroxidation and/or iron chelators have been shown to prevent cell death induced by a wide range of insults. However, it is not clear whether these two characteristics are sufficient to distinguish ferroptosis from other forms of regulated cell death. Thus, the primary goal of this study was to determine whether a unique combination of features could be identified that would provide an approach to more clearly separate ferroptosis from other forms of regulated cell death. To this end, multiple pharmacological inhibitors based on a variety of studies were tested. Many of these inhibitors were previously shown to protect cells from oxytosis, a regulated cell death pathway that mechanistically overlaps with ferroptosis and is induced by some of the same chemicals as ferroptosis. These inhibitors were not only tested against both known ferroptosis and oxytosis inducers but also a number of other insults that have been suggested to induce ferroptosis. The results show that a pharmacological fingerprint for ferroptosis can be established and used to categorize toxic insults into those that overlap with oxytosis/ferroptosis and those that do not.