A weight loss drug targeting the mRNA demethylase, FTO?

Now maybe it’s the fact I ate too many hot cross buns and crème eggs this Easter, or because it vaguely relates to some exam topics and so can count as revision, but I found myself reading about a drug that reduced the body mass of mice. Yep, this new study by Peng et al. 1 identified entacapone, an FDA approved drug used to treat Parkinson’s disease, also functions to inhibit FTO, a protein with genetic mutations associated with obesity. When entacapone was applied to mice, body weight decreased ~10% compared to controls. The team found this weight reduction to be due to increased energy expenditure in part due to downregulation of the protein FOXO1.

The physics of weight loss

Simply put, if we gain weight, we eat more than we burn and conversely to lose weight, we eat less than we burn; Energy in = energy out, then you remain the same (Figure 1). Okay, but if energy in equates with what we eat, what accounts for energy out? Well, it can be split into three major processes; metabolism, physical activity and adaptive thermogenesis.

scalesFigure 1: The physics of weight loss

Simply put, obesity arises therefore when energy in >> energy out. Obesity and diabetes are becoming ever more prevalent in the population, but is there anything that can be done? A number of genome wide association studies (GWAS) (I explain what these are in my last blog – https://asheekeyscienceblog.com/2019/04/10/do-your-genes-dictate-whether-youre-a-morning-person/ ) have identified a number of single nucleotide substitutions (SNPs)  that correlate with obesity – one gene that arises from these studies is FTO – fat mass and obesity-associated gene (that is a pure guess of letter boldening..), a gene that has demethylase activity of m6A (N6-adenosine modified sites) in mRNA. GWAS are great for identifying correlations between genetic variations and phenotypes, but they don’t guarantee causation. However, evidence supports the links between FTO genetic variants and obesity; mouse models over-expressing FTO are obese whilst FTO knock-out reduces body weight. Reducing FTO activity pharmaceutically may therefore be a way to reduce the rate of weight gain and other metabolic disorders such as diabetes.

Identifying entacapone

The team performed a structure-based hierarchical virtual screen followed by target validation to see which compounds could actually inhibit FTO demethylation activity. Entacapone, an FDA-approved compound previously characterised to inhibit catechol-O-methyltransferase (an enzyme which degrades dopamine and used to treat Parkinson’s) met the screen criteria for docking into the substrate binding site of FTO and increased the amount of m6A on mRNA compared to conditions without the drug. The team determined the crystal structure for Entacapone docked in FTO and found it closely matched their prediction from the screen.

In vitro, [√], but what impact will Entacapone have in vivo?

To test this the group applied Entacapone for 5 weeks to the food of high-fat diet-induced obese mice. Compared to the obese mice without Entacapone, the addition of the drug decreased body weight by ~10% in just 3 weeks (Figure 2). Now this isn’t because the mice were eating less, in fact, both test and control mice ate similar amounts. So on our energy scale, if food intake is the same, then the weight reduction must be caused by increased energy expenditure.

graphsFigure 2: Entacapone results in weight loss in mice

Liver gluconeogenesis and fat thermogenesis

In addition to the weight loss, the team noticed that the entacapone treated mice had increased skin temperature surrounding a region of white adipose tissue. This hinted at increased fat thermogenesis in the tissue. Moreover, the mice had reduced fasting blood glucose levels – this hints at reduced flux through gluconeogenesis in the liver (a metabolic pathway that generates glucose). Both increased fat thermogenesis and reduced liver gluconeogenesis could then explain the increased energy expenditure seen in entacapone-treated mice.

To get a molecular handle on these observations, the transcriptome of FTO-knockdown cells was analysed. Key genes involved in gluconeogenesis (glucose 6-phosphate and phosphoenolpyruvate carboxykinase) were found to be down-regulated – these two genes are regulated by a common transcription factor – FOXO1.

Outfoxing the FOXO1-FTO axis

FTO is classified as a member of the iron and alpha-ketoglutarate-dependent oxygenase family with m6A/m6Am demethylating activity. The methylation of mRNA adenosine residues can have a variety of impacts on mRNA from stability, translation efficiency, localisation and alternative splicing – the role in which FTO plays in any of these processes to implicate obesity propensity was of interest in this study. When I briefly name-dropped FTO before (https://asheekeyscienceblog.com/2017/12/31/translating-into-the-new-year-a-new-role-for-eif4a-and-more-methyl-modifications/ ) that was in the demethylation of m6A at the first adenine of the mRNA, but is that the same site being targeted here?

Well, four m6A sites commonly methylated in FOXO1 mRNA had already been identified. Whilst within the body of the transcript instead of at the start, the team found FTO to bind more in regions 1 and 2 (Figure 3) and further work showed these sites to be demethylated by FTO. When FTO was deficient in cells (either by a knock-down or entacapone treatment), increased methylation of FOXO1 mRNA was seen and reduced expression of FOXO1 protein.

foxo1Figure 3: Regions of FOXO1 mRNA methylated at m6A

So, the evidence suggests increased FTO activity results in greater protein levels of FOXO1 which increases levels of gluconeogenesis. And inhibiting FTO activity reduces gluconeogenesis promoting weight loss.

Have we found the cure to the obesity epidemic?

This paper is undeniably pretty cool and shows how many different experimental methods can be used to uncover a molecular pathway from drug screening, testing to translational therapeutics. But, although entacapone is FDA-approved the dose used in mice was high and the side effects of long-term use would need to be evaluated. Moreover, given that the entacapone-FTO crystal structure has been determined, further chemical optimisation of the drug can be designed to develop better inhibitors of FTO – indeed, through a structure-activity relationship exploration, Peng´s team already found that rigidifying the flexible chemical tail of entacapone improved inhibitory activity of FTO. Another consideration to further improve the weight-loss results would be to examine the chronopharmacokinetics – whether the drug would be better to take at the start of the day or the evening – I mention this only as gluconeogenesis is known to oscillate in activity throughout the day to fit in with our feeding/fasting cycles.

Further Reading

  1. Peng, S. et al. Identification of entacapone as a chemical inhibitor of FTO mediating metabolic regulation through FOXO1. Sci. Transl. Med. 11, eaau7116 (2019).

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