Fisetin CMS121 slows down the aging of brain cells and reverses Alzheimer’s disease

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A drug candidate developed by Salk researchers, and previously shown to slow aging in brain cells, successfully reversed memory loss in a mouse model of inherited Alzheimer’s disease.

The new research, published online in July 2020 in the journal Redox Biology, also revealed that the drug, CMS121, works by changing how brain cells metabolize fatty molecules known as lipids.

“This was a more rigorous test of how well this compound would work in a therapeutic setting than our previous studies on it,” says Pamela Maher, a senior staff scientist in the lab of Salk Professor David Schubert and the senior author of the new paper. “Based on the success of this study, we’re now beginning to pursue clinical trials.”

Over the last few decades, Maher has studied how a chemical called fisetin, found in fruits and vegetables, can improve memory and even prevent Alzheimer’s-like disease in mice.

More recently, the team synthesized different variants of fisetin and found that one, called CMS121, was especially effective at, improving the animals’ memory, and slowing the degeneration of brain cells.

In the new study, Maher and colleagues tested the effect of CMS121 on mice that develop the equivalent of Alzheimer’s disease.

Maher’s team gave a subset of the mice daily doses of CMS121 beginning at 9 months old–the equivalent of middle age in people, and after the mice have already begun to show learning and memory problems.

The timing of the lab’s treatment is akin to how a patient who visits the doctor for cognitive problems might be treated, the researchers say.

After three months on CMS121, at 12 months old, the mice–both treated and untreated–were given a battery of memory and behavior tests.

In both types of tests, mice with Alzheimer’s-like disease that had received the drug performed equally well as healthy control animals, while untreated mice with the disease performed more poorly.

To better understand the impact of CMS121, the team compared the levels of different molecules within the brains of the three groups of mice.

They discovered that when it came to levels of lipids–fatty molecules that play key roles in cells throughout the body–mice with the disease had several differences compared to both healthy mice and those treated with CMS121.

In particular, the researchers pinpointed differences in something known as lipid peroxidation–the degradation of lipids that produces free radical molecules that can go on to cause cell damage. Mice with Alzheimer’s-like disease had higher levels of lipid peroxidation than either healthy mice or those treated with CMS121.

“That not only confirmed that lipid peroxidation is altered in Alzheimer’s, but that this drug is actually normalizing those changes,” says Salk postdoctoral fellow Gamze Ates, first author of the new paper.

The researchers went on to show that CMS121 lowered levels of a lipid-producing molecule called fatty acid synthetase (FASN), which, in turn, lowered levels of lipid peroxidation.

When the group analyzed levels of FASN in brain samples from human patients who had died of Alzheimer’s, they found that the patients had higher amounts of the FASN protein than similarly aged controls who were cognitively healthy, which suggests FASN could be a drug target for treating Alzheimer’s disease.

While the group is pursuing clinical trials, they hope other researchers will explore additional compounds that may treat Alzheimer’s by targeting FASN and lipid peroxidation.

“There has been a big struggle in the field right now to find targets to go after,” says Maher. “So, identifying a new target in an unbiased way like this is really exciting and opens lots of doors.”

Other researchers involved in the study were Joshua Goldberg and Antonio Currais of Salk.

Funding: The work was supported by grants from the Shiley Foundation, the National Institutes of Health, the Edward N. & Della Thome Memorial Foundation and the Shiley-Marcos Alzheimer’s Disease Research Center at the University of California San Diego.


Geroprotectors: Compounds that Slow Aging

Perhaps the greatest challenge to modern medicine is to prevent or delay the diseases of aging. The most recalcitrant of these is AD. One approach to AD therapies is to identify drug candidates that slow aging and extend lifespan in model organisms.

These compounds are called geroprotectors and it has been argued that their ability to extend lifespan in model organisms may translate to a healthier lifespan in humans.

The hypothesis is that by delaying biological aspects of aging there will be a simultaneous delay in the chronic diseases associated with aging because they share the same physiological risk factor, the aging process itself.

To date, there are about 200 compounds that extend either median or maximal lifespan in yeast, flies, and/or worms [1]. Analysis of these compounds points to the central importance of several key molecular pathways in life extension and healthy aging, including the AMP-activated kinase (AMP kinase) and mechanistic target of rapamycin (mTOR) pathways [2,3].

Since the AMP kinase and mTOR pathways exist in all cells, but are engaged by a variety of mechanisms, it is currently unclear whether geroprotectors extend lifespan by slowing aging in all tissues equally or whether different geroprotectors protect specific tissues thereby decreasing overall mortality.

In the few cases in which the targets of geroprotectors were identified, they were expressed in a tissue-specific manner [4]. These observations suggest that by using the proper selection criteria, geroprotectors could be identified that pass the blood–brain barrier and protect the brain in addition to affecting aging pathways that are shared with other tissues.

We term these compounds GNPs and describe herein the set of phenotypic screening assays that have been used to identify these compounds.

A Novel Screening Platform Identifies GNPs

If the goal is to promote healthier brain aging and long-term neural function, what are the criteria that can be used to identify functional GNP drug candidates?

First, a GNP should protect from multiple brain toxicities that are known to increase with age. Examples of age-associated brain toxicities include reduced energy metabolism, proteotoxicity, inflammation, and oxidative damage.

Second, GNPs should not be disease specific but should show beneficial effects in multiple neurodegenerative diseases as well as other age-related conditions. Thus, GNPs should have therapeutic efficacy in animals that die from age-related multimorbidity.

Third, it is not sufficient to simply show an effect on lifespan, because a new GNP drug candidate will be required to have a disease-modifying property to obtain FDA approval for clinical trials.

Finally, a GNP must have the ability to reduce, or at least to slow, age-associated physiological and molecular changes even if treatment is initiated in aged animals or at symptomatic stages of the disease.

While most published geroprotector studies have initiated treatment in young adults [1], a GNP should ideally function when given to older individuals.

Here, we outline a plausible drug discovery pipeline for the identification and characterization of GNP drug candidates using cell culture assays that recapitulate multiple age-associated toxicities of the brain.

The pipeline is based on a group of phenotypic screening assays described in [5] and listed in Table 1. Importantly, all of the assays reflect conditions that are more robust in the aged and diseased brain relative to age-matched controls.

Table 1. – Brain Toxicities of Old Age as a Drug Screening Platforma

AssayPathwayMetforminRapamycinCurcumin J147CAD31FisetinCMS121
MC65 cellsAb toxicity1.5 mM>10 µM>20 µM34 nM12 nM2.7 µM90 nM
OxytosisOxidative stress10 mM>10 µM6 µM11 nM20 nM3 µM200 nM
In vitro ischemiaEnergy loss>10 mM>10 µ M100 nM180 nM47 nM3 µM7 nM
Microglial activationInflammation2.5 mM100 nM8 µM>10 µM10 µM5 µM1 µM
Trophic factor withdrawalGrowth factor loss>2 mM>10 µM>20 µM27 nM18 nM2.2 µM70 nM
PC12 neuritesNerve cell differentiation>10 mM>10 µM>10 µM>10 µM>10 µM5 µM2.5 µM
aCurcumin and fisetin along with their synthetic derivatives were screened from the highest nontoxic amount (100 µM) down to 10 nM in the six screening assays described in [5]. The concentration that produces 50% of the maximal effect in each assay (EC50) is given. For comparison, two well-studied geroprotectors, metformin and rapamycin, were included in these assays. The data are from [2,3,5,6,8–12,17,18].

These include assays for amyloid beta proteotoxicity, protection against oxidative stress (oxytosis), protection against a reduction of ATP synthesis (in vitro ischemia), anti-inflammatory activity (microglial activation), protection against loss of the trophic factors that nerve cells depend on for survival (trophic factor withdrawal), and the ability to induce the differentiation of PC12 sympathetic-like neurons (PC12 cells), a property of some trophic factors such as nerve growth factor (NGF). Screening of natural product libraries using these assays yielded fisetin and curcumin as lead compounds [5,6,12].

We and many others have studied the preclinical efficacy and molecular pathways initiated by these natural compounds in multiple cell culture systems and animal models of neurodegeneration, including stroke, AD and dementia, Huntington’s disease, and Parkinson’s disease [6,7].

Subsequent medicinal chemistry based on structure–activity relationship studies and the GNP selection assays listed in Table 1 as phenotypic readouts yielded the synthetic derivatives of curcumin, CAD31, and J147 and the synthetic derivative of fisetin, CMS121 [8–12]. These compounds are several orders of magnitude more potent than their natural precursors, yet they maintain the biological activities of the parent compounds (Table 1).

New GNPs Share Antiaging Pathways with Geroprotectors

As predicted from the GNP selection assays, J147, CAD31, and CMS121 are effective in multiple rodent models of AD. They improve memory, reduce inflammation, maintain synapses, and remove toxic amyloid peptide [5,9–13].

More surprising was the finding that they and their natural product precursors extend life-span and/or delay physiological and molecular aspects of aging [9,11,13,14].

A limited number of molecular pathways associated with reducing the rate of aging in model organisms have been identified, most of which are associated with the experimental manipulation of caloric restriction or treatment with metformin or rapamycin [2,3,15].

If CMS121, J147, and CAD31 are bona fide GNPs, the molecular pathways modified by them should include those that are modified by caloric restriction, metformin, and rapamycin. Figure 1 shows that they indeed engage many of the same pathways.

Their robust effects across many in vitro and in vivo systems at nanomolar concentrations should clearly dispel any notion that polyphenolic compounds make poor lead compounds and cannot be chemically improved while still maintaining their biological activities [8–10,12].

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Figure 1.
Geroneuroprotectors (GNPs) Share Molecular Pathways with Caloric Restriction and Geroprotectors.
Heatmap of molecular pathways activated (shown as red) or inhibited (shown as blue) by the different compounds is shown. White spaces denote that the effects were not determined whereas the hatched boxes depict publications showing both activation and inhibition. Data are compiled from [2,3,5–20].

J147 is the best-studied curcumin derivative. It is effective in over a dozen rodent models of neurodegenerative diseases and memory enhancement [9,10,12,13].

Its mechanistic target is a subunit of mitochondrial ATP synthase that was also found to promote life extension in worms [9]. These data strongly support the designation of J147 as a GNP. CAD31 is a derivative of J147 that has many of the same neuroprotective properties and also stimulates nerve stem cell division [10,11].

Fisetin has been extensively studied in both transgenic mice expressing genes that cause the familial form of AD (APPswe/PSdeltaE9 mice) [16] and old, rapidly aging SAMP8 mice [14].

SAMP8 mice are perhaps the best rodent model to study drug effects on multimorbidity and sporadic dementia [13,14]. CMS121 has the same GNP properties as fisetin but a much higher level of potency (Table 1).

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Source:
Salk Institute

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