Main Research Findings

1) CMS-121 has the potential to mitigate and treat metabolic disorders such as obesity and diabetes.

1) CMS-121 was shown to protect against inflammation and lipid peroxidation while alleviating cognitive impairments.

 

Selected Data
1) The study performed by Dafre et al investigated whether CMS121 could counteract age-associated weight gain and metabolic dysfunction in otherwise healthy wild-type mice. The study was motivated by previous findings showing that CMS121 improved glucose and lipid metabolism in leptin-receptor-deficient db/db obese mice and that it had beneficial effects on brain metabolism in models of aging and Alzheimer’s disease. The authors were particularly interested in CMS121 because previous work indicated that it inhibits fatty acid synthesis through effects involving fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1), thereby increasing the availability of acetyl-CoA. The present study extended this work to normal C57BL/6 mice to determine whether CMS121 could influence body composition, energy expenditure, glucose regulation, lipid metabolism, mitochondrial function, hepatic metabolism, inflammation, and circulating metabolites. The investigators specifically examined whether the metabolic effects of CMS121 resembled those produced by ketogenic diets, which are known to promote weight loss and alter glucose and lipid metabolism but can be difficult to maintain long term [1].
The principal experiment used male C57BL/6 mice beginning at five weeks of age. Twelve mice were randomly assigned to either a control diet or a standard rodent diet containing CMS121, and animals were housed three per cage. Treatment continued for six months, meaning that the mice were approximately seven months old when the experiment ended. Both diets were supplied ad libitum. Body weight and food consumption were monitored weekly throughout the study. Based on average cage food consumption, CMS121 exposure was estimated at approximately 9.4 mg/kg/day during the first 17 weeks, when the dietary concentration was 200 ppm, and approximately 18.8 mg/kg/day during weeks 18–24, when the concentration was increased to 400 ppm. The investigators therefore examined CMS121 exposure over an extended period rather than evaluating a short-term pharmacological response. Importantly, behavioral testing and data collection were performed by researchers blinded to treatment allocation, reducing the potential for observer bias. Body composition was evaluated at week 13, metabolic status was assessed at week 15, and the animals were sacrificed after six months for collection of blood, liver, and subcutaneous adipose tissue [1].
A variety of metabolic assessments were used to determine how CMS121 influenced body composition and energy expenditure. At week 13, the investigators used an EchoMRI 100 system to quantify body mass composition, including lean and fat mass. At week 15, mice were placed in metabolic cages for five days. Software systems allowed investigators to measure and analyze food and water consumption, locomotor activity, oxygen consumption, carbon dioxide production, respiratory exchange ratio (RER), and energy expenditure using indirect calorimetry. This approach enabled the reseatchers to distinguish changes in body weight from changes in metabolic activity and physical activity. The design was particularly relevant because a reduction in body weight could theoretically result simply from reduced food intake or increased movement; therefore, measuring oxygen consumption, carbon dioxide production, RER, and ambulatory activity provided additional information regarding the metabolic basis of the observed effects.
Glucose metabolism was assessed using several complementary measurements. Blood glucose was measured using enzymatic hexokinase-based assays or Accu-Chek Aviva test strips, depending on the sample. Glycated hemoglobin (HbA1c) was measured enzymatically, while insulin concentrations were determined using an ELISA. A glucose tolerance test (GTT) was conducted after overnight food deprivation. The mice received glucose by gavage at 0.5 g/kg, and blood glucose was subsequently measured at 0, 30, 60, 90, and 120 minutes through tail-vein sampling. Lipid metabolism was evaluated by measuring plasma and liver triglycerides, cholesterol, and free fatty acids. Plasma triglycerides and cholesterol were measured using commercial kits, liver cholesterol was measured fluorometrically, and free fatty acids were assessed using a fluorescent assay. At the end of the experiment, animals were anesthetized and blood was obtained by cardiac puncture [1].
The investigators then examined protein markers associated with mitochondrial function, glucose metabolism, lipid synthesis, and inflammation in adipose tissue and liver. The protein panel included Nrf1 and TFAM as markers of mitochondrial biogenesis; NDUFB8, SDHB, UQCRC2, and ATP5A as markers of mitochondrial respiratory complexes; TOM20 as a mitochondrial membrane marker; phosphorylated ACC1 and FASN as markers of lipid synthesis; GLUT4 as a glucose-transport marker; and several hepatic proteins associated with glycolysis, gluconeogenesis, TCA-cycle metabolism, and inflammation [1].
2) The study by Ates et al investigated the effects of CMS121, a small-molecule derivative of the flavonoid fisetin, on Alzheimer’s disease (AD)-associated cognitive dysfunction and examined the molecular mechanisms underlying its protective effects. CMS121 had originally been developed through a phenotypic drug-discovery approach designed to identify compounds capable of protecting cells against several toxic processes associated with neurodegeneration, including oxidative stress, inflammation, and ischemia. In particular, the compound had demonstrated the ability to protect neuronal cells from oxytosis/ferroptosis, forms of regulated cell death associated with glutathione depletion, reactive oxygen species generation, and excessive lipid peroxidation. The present study was designed to determine whether these cellular effects translated into improved cognition in an established transgenic AD model and to identify a molecular target through which CMS121 altered lipid metabolism and protected against neurodegenerative toxicity. The investigators specifically examined spatial learning and memory, contextual memory, and behavioral disinhibition in APPswe/PS1ΔE9 double-transgenic mice. They also evaluated lipid peroxidation, neuroinflammation, lipid metabolites, and fatty acid synthase (FASN), a central enzyme involved in lipid biosynthesis [2] .
The primary in vivo model consisted of male APPswe/PS1ΔE9 transgenic mice and age-matched C57BL/6J wild-type controls. The transgenic mice express a mouse/human chimeric APPswe gene and mutant human presenilin-1 PS1ΔE9 on a C57BL/6J genetic background. Treatment began when the animals were nine months old, an age at which the investigators had previously demonstrated cognitive deficits and at which AD-like pathology was already substantially established. Twelve animals were randomly assigned to each experimental group. CMS121 was incorporated into the diet at a concentration of 400 ppm, producing an estimated average intake of approximately 34 mg/kg/day. Treatment continued for three months, after which the animals underwent behavioral testing and tissue collection. Behavioral testing and data analysis were conducted by different investigators who were blinded to the treatment group, reducing the possibility of observer bias. Body weight and food consumption were monitored weekly, and no significant differences in these measures were reported among the groups.
The investigators used three behavioral paradigms to evaluate different dimensions of cognition and behavior. Spatial learning and memory were assessed using a two-day Morris Water Maze (MWM). On the first day, animals were trained to locate a visible platform using environmental cues surrounding the pool. Four visible-platform trials were conducted, with each trial lasting up to 180 seconds. On the second day, 24 hours after the final visible-platform trial, the platform was hidden and animals completed three hidden-platform trials. Escape latency, defined as the time required to locate the hidden platform, was automatically recorded using Noldus EthoVision video-tracking software. The elevated plus maze (EPM) was used to assess disinhibition, an abnormal behavioral characteristic associated with AD. Animals were placed on an elevated plus-shaped apparatus containing two open and two enclosed arms for five minutes, and the amount of time spent in the open versus closed arms was quantified using the same automated tracking system. Contextual memory was evaluated with a fear-conditioning paradigm. On the first day, mice explored the chamber before receiving a 30-second, 2-kHz tone followed immediately by a 0.7-mA foot shock lasting two seconds. This tone-shock pairing was repeated, after which animals were allowed to explore the chamber again. Twenty-four hours later, the mice were returned to the same environment without receiving a tone or shock, and freezing behavior was measured as an indicator of contextual memory [2].
The study incorporated several biochemical and molecular approaches to determine how CMS121 affected AD-associated pathology. Western blotting was performed on hippocampal tissue and cultured-cell lysates to quantify proteins associated with lipid peroxidation, inflammation, amyloid pathology, and lipid metabolism. Tissue was homogenized in a RIPA buffer containing protease and phosphatase inhibitors, sonicated, centrifuged, and analyzed for protein concentration. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with antibodies against 4-hydroxynonenal (4HNE), 15-lipoxygenase-2 (15LOX2), amyloid-β, cyclooxygenase-2 (COX2), FASN, glial fibrillary acidic protein (GFAP), and inducible nitric oxide synthase (iNOS). Protein expression was normalized to actin. These measurements allowed the investigators to determine whether CMS121 reduced oxidative lipid damage and inflammatory activation in the brains of treated animals.
The investigators additionally examined lipid metabolism using untargeted metabolomics of mouse cortical tissue. Cortex samples were snap frozen and processed using an automated extraction procedure. Extracted metabolites were analyzed using multiple mass-spectrometry platforms, including UPLC-MS/MS in both positive- and negative-ion modes and GC-MS. Metabolites were identified through comparison with authenticated standards and metabolomic databases, while peak areas were used for quantification. The study also employed several complementary cell-culture models. HT22 mouse hippocampal neuronal cells and HeLa cells were cultured in DMEM, while BV2 microglial cells were maintained in low-glucose DMEM. MC65 cells, an inducible model of intracellular Aβ toxicity, were cultured with tetracycline to suppress intracellular Aβ production. Lipid peroxidation was measured using the fluorescent probe Bodipy 581/591 in HT22 neurons and BV2 microglia. RSL3, a GPX4 inhibitor, was used to induce lipid peroxidation in HT22 cells, while LPS was used to activate inflammatory responses in BV2 microglia. Furthermore, the researchers examined whether FASN suppression protected HT22 cells from glutamate-, erastin-, or RSL3-induced oxytosis/ferroptosis and whether it reproduced CMS121’s effects on lipid peroxidation and inflammatory signaling [2].

 

Discussion
1) The results of the study by Dafre et al demonstrated that long-term CMS121 administration substantially altered body composition and metabolic physiology in wild-type C57BL/6 mice. The investigators first found that food consumption was slightly lower in CMS121-treated mice, with linear regression demonstrating a significant difference between the treatment groups. However, the magnitude of the reduction in body-weight gain was considerably greater than could be explained by the relatively modest difference in food intake. Control mice increased from approximately 29.2 g at five weeks of age to 48.0 g after six months, corresponding to an average weight gain of 18.8 g. CMS121-treated mice increased from 27.61.3 g to 41.1 g, corresponding to a weight gain of only 13.4g. Thus, CMS121 reduced weight gain by approximately 40%. Importantly, the CMS121 group did not simply become smaller overall; these mice also possessed significantly greater lean mass and significantly less fat mass [1].
CMS121 also increased measures of metabolic activity. Indirect calorimetry demonstrated significantly higher oxygen consumption and carbon dioxide production in treated animals, whereas the respiratory exchange ratio was not significantly altered. Overall locomotor activity in the metabolic cages was also not significantly different between groups. Consequently, the increase in energy expenditure could not be definitively attributed to greater physical activity. The authors noted that the metabolic-cage assessment involved relatively few animals and could itself have been stressful, limiting interpretation of the locomotor findings. They also reported that an earlier open-field assessment conducted two weeks after introduction of the diet showed increased locomotor activity in CMS121-treated mice, although no later open-field measurement was available to establish whether that effect persisted. Taken together, the body-composition and calorimetry findings suggested that CMS121 promoted a metabolic state characterized by reduced fat accumulation, greater lean mass, and increased basal metabolic activity [1].

 

Figure 1: Changes in nutrition, body mass, locomotion, and metabolism following treatment with CMS-121.
Glucose measurements generally indicated an improvement in glycemic status, although not every outcome reached statistical significance. During the GTT, CMS121-treated mice tended to have lower glucose concentrations, with the clearest statistically significant difference occurring at 60 minutes. The overall glucose area under the curve was lower in the CMS121 group but did not reach statistical significance. Fasting blood glucose also tended to be lower in treated animals, while glucose measured under fed conditions was not significantly different. HbA1c showed a tendency toward reduction, suggesting an overall improvement in longer-term glycemic exposure. The most pronounced glucose-related finding was the marked reduction in circulating insulin, which reached statistical significance. Thus, CMS121 appeared to improve glucose handling while requiring substantially less insulin. The authors suggested that the increased adipose-tissue GLUT4 expression observed later in the study could contribute to enhanced glucose uptake and insulin sensitivity [1].

 

Figure 2: Changes in blood glucose parameters following administration of CMS-121.
CMS121 also produced substantial changes in lipid metabolism. Plasma free fatty acids were significantly reduced, while plasma triglycerides remained approximately unchanged. Interestingly, plasma cholesterol increased significantly. In the liver, both free fatty acids and triglycerides were significantly reduced, whereas hepatic cholesterol was not significantly altered. Figure 3 provides a direct visual comparison of these lipid outcomes, showing reductions in plasma FFA, hepatic FFA, and hepatic triglycerides. These findings were interpreted as evidence for reduced lipid accumulation and altered fatty-acid handling rather than a uniform reduction in all circulating lipids. The combination of reduced liver triglycerides, lower free fatty acids, increased lean mass, and decreased fat mass suggested that CMS121 substantially remodeled lipid metabolism in peripheral tissues.
The adipose-tissue protein analyses provided evidence for enhanced mitochondrial biogenesis and altered glucose and lipid metabolism. CMS121 significantly increased the mitochondrial biogenesis-associated transcription factors Nrf1 and TFAM. There were also tendencies toward increased levels of respiratory-chain complexes I, II, III, and V, suggesting enhanced mitochondrial capacity. TOM20, a marker associated with the mitochondrial outer membrane, was significantly increased. At the same time, phosphorylation of ACC1 was increased. Because phosphorylation inhibits ACC1 activity, this finding was consistent with reduced conversion of acetyl-CoA into malonyl-CoA and therefore reduced fatty-acid synthesis. GLUT4 levels were also significantly increased in adipose tissue. These findings suggest that CMS121 increased mitochondrial biogenesis while simultaneously suppressing adipose lipogenesis and increasing the machinery available for glucose uptake [1].
The liver also displayed a complementary pattern of metabolic remodeling. CMS121 reduced FASN, supporting inhibition of de novo fatty-acid synthesis. PEPCK, a marker of gluconeogenesis, was also reduced, suggesting diminished hepatic gluconeogenesis. Increased PFKFB3 was interpreted as evidence for increased glycolytic flux, while decreased TXNIP could favor glucose uptake by reducing removal of glucose transporters from the plasma membrane. CMS121 also reduced hepatic MLYCD and protein malonylation, while fumarate hydratase (FH) levels decreased. The authors noted that the combination of decreased FASN, reduced PEPCK, increased PFKFB3, and altered TXNIP suggested a complex metabolic shift toward greater glucose utilization and reduced lipid synthesis. However, mitochondrial respiratory-chain markers in the liver were not significantly changed, despite an increase in TOM20. Therefore, unlike adipose tissue, the liver did not show a broad increase in respiratory-complex proteins.
Overall, the study demonstrated that CMS121 produced broad metabolic effects in aging wild-type mice, including approximately 40% less body-weight gain, greater lean mass, reduced fat mass, increased metabolic activity, improved insulin and glucose profiles, reduced circulating and hepatic free fatty acids, reduced hepatic triglycerides, enhanced adipose mitochondrial markers, increased GLUT4, suppression of lipid synthesis, and reduced hepatic inflammatory markers. The metabolomic findings further suggested a shift toward increased fatty-acid oxidation and ketogenic metabolism. The authors proposed that inhibition of FASN and activation of AMPK-associated ACC1 phosphorylation may increase acetyl-CoA availability and redirect metabolism away from de novo lipogenesis. They concluded that this metabolic remodeling could potentially contribute not only to peripheral metabolic health but also to the previously observed neuroprotective effects of CMS121 [1].
2) The results of the study performed by the research team of Ates et al revealed that three months of CMS121 treatment substantially improved several behavioral abnormalities in APPswe/PS1ΔE9 transgenic mice. In the Morris Water Maze, all groups were able to learn the visible-platform task during the first day, and there were no significant differences in escape latency during the visible-platform trials. However, when the platform was hidden on the second day, untreated AD mice required significantly more time to locate it, demonstrating impaired spatial learning. CMS121-treated AD mice performed comparably to wild-type animals, indicating that treatment prevented the spatial-learning deficit observed in untreated transgenic mice. CMS121 also improved abnormalities in other behavioral domains. Untreated AD mice spent significantly more time in the open arms of the elevated plus maze, consistent with a disinhibition phenotype, whereas CMS121-treated AD mice did not differ significantly from wild-type controls. Similarly, untreated AD mice exhibited impaired contextual memory, demonstrated by reduced freezing when returned to the conditioning chamber 24 hours after training. CMS121 treatment prevented this deficit, restoring freezing behavior to levels comparable with wild-type animals. Thus, the compound improved spatial learning, contextual memory, and behavioral disinhibition simultaneously [2].

 

Figure 1: Changes in parameters of the MWM test across the experimental treatment groups.
The behavioral findings were accompanied by evidence that CMS121 reduced excessive lipid peroxidation. The investigators first tested the compound in cultured neuronal and microglial cells. In HT22 neuronal cells, RSL3 was used to inhibit GPX4 and stimulate lipid peroxide accumulation. CMS121 significantly prevented the RSL3-induced increase in lipid peroxidation. CMS121 also reduced lipid peroxidation in BV2 microglial cells activated with LPS. These findings demonstrated that the compound could suppress lipid oxidative damage in both neuronal and inflammatory cell models. The in vivo findings were consistent with the cell-culture results. Untreated AD mice showed increased hippocampal 4HNE protein adducts, an important marker and toxic product of lipid peroxidation. CMS121 treatment reduced hippocampal 4HNE levels to approximately those observed in untreated wild-type animals. The researchers also detected increased hippocampal 15LOX2 in untreated AD mice, another protein associated with lipid oxidation and inflammatory signaling, whereas CMS121 reduced 15LOX2 levels toward wild-type values. GFAP, a marker of glial activation and inflammatory stress, was also elevated in untreated AD mice and was reduced following CMS121 treatment [2].

 

Figure 3: Changes in lipid peroxidation in A) HT22 neuronal cells and B) BV2 microglia following treatment with CMS-121
The study next examined the relationship between CMS121 and intracellular amyloid-β toxicity using MC65 cells. Withdrawal of tetracycline from these cells induces production of the C-99 APP fragment, which is subsequently processed to intracellular Aβ and produces inflammation, proteotoxicity, and cell death. CMS121 prevented the accumulation of intracellular Aβ and protected the cells from Aβ-induced death. Importantly, CMS121 also prevented the broad increase in eicosanoids associated with intracellular Aβ production. Forty-three eicosanoids were detected in the culture medium, with most derived from arachidonic acid, followed by linoleic acid and docosahexaenoic acid. Intracellular Aβ production increased eicosanoid levels across these different lipid sources and metabolic pathways, including cyclooxygenase, cytochrome P450, lipoxygenase, and nonenzymatic pathways. CMS121 prevented this increase regardless of the specific lipid source or pathway involved.
These observations suggest that CMS121 may exert a broad effect on lipid dysregulation rather than acting exclusively through inhibition of one particular lipoxygenase pathway. Lipid-related metabolites were elevated in untreated AD mice compared with CMS121-treated animals. More specifically, endocannabinoids, fatty acids, and polyunsaturated fatty acids (PUFAs) were significantly higher in untreated AD mice than in CMS121-treated AD mice. The researchers also found that untreated AD mice had increased levels of palmitate and FASN protein. CMS121 normalized palmitate levels to those of control mice, although it did not significantly alter FASN protein levels in AD mice [2].
Because FASN catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA, these findings suggested that abnormal lipid biosynthesis could contribute to AD-related lipid peroxidation and that CMS121’s protective effects could involve suppression of FASN activity rather than simply reducing the amount of FASN protein. That being said, FASN was the only protein appearing among the top three candidate targets in both cell lines. Direct enzymatic analysis subsequently demonstrated that CMS121 inhibited FASN activity in a dose-dependent manner. The investigators then used siRNA to reduce FASN expression in HT22 cells. FASN knockdown protected neurons from oxytosis/ferroptosis induced by glutamate, erastin, and RSL3. In glutamate- and erastin-induced toxicity, CMS121 provided additional protection even after FASN knockdown
The FASN experiments also linked lipid metabolism directly to lipid peroxidation. FASN knockdown significantly reduced baseline 4HNE adduct formation in HT22 cells. RSL3 increased 4HNE formation in control cells, but this increase was not observed following FASN knockdown, and CMS121 did not provide additional protection against 4HNE formation in the FASN-deficient cells. A strong positive correlation was observed between FASN protein expression and 4HNE adduct formation, the investigators extended these findings to inflammation using BV2 microglia. LPS increased iNOS, COX2, and TNFα, whereas both CMS121 treatment and FASN knockdown prevented these inflammatory responses. Furthermore, FASN protein levels were significantly increased in human AD brain tissue, and FASN knockdown protected MC65 cells from intracellular Aβ-induced cell death. Together, these results strengthened the conclusion that FASN is an important biological target through which CMS121 can influence lipid peroxidation, inflammatory signaling, and Aβ-associated toxicity [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Dafre AL, Zahid S, Probst JJ, et al. CMS121: a novel approach to mitigate aging-related obesity and metabolic dysfunction. Aging (Albany NY). 2024;16(6):4980-4999. doi:10.18632/aging.205673

[2] Ates G, Goldberg J, Currais A, Maher P. CMS121, a fatty acid synthase inhibitor, protects against excess lipid peroxidation and inflammation and alleviates cognitive loss in a transgenic mouse model of Alzheimer’s disease. Redox Biol. 2020;36:101648. doi:10.1016/j.redox.2020.101648

 

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