Protonophores: From Artillery Shells To Anti Ageing Drugs?

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During the First World War, munitions factory workers in multiple countries began to suffer from a mysterious illness. They experienced rapid weight loss and fatigue, and in some cases suffered from dangerously high body temperature and even death. The chemical responsible was dinitrophenol (DNP), a yellow powder used in explosive manufacturing. Little did anyone suspect at the time that, over a century later, the mechanism behind this compound’s toxicity – mitochondrial uncoupling – would be of interest for slowing the ageing process.

What Is Mitochondrial Uncoupling?

Most people know that mitochondria are the ‘power plants’ of our cells, responsible for using nutrients to generate ATP, the cell’s universal fuel. To make ATP, a mitochondrion first strips electrons from nutrients and uses them to pump protons (also known as hydrogen ions, H+) into a space between the double membranes that enclose the mitochondrion (pictured below). These protons then flood back into the inner section of mitochondrion (cristae) through a special turbine-like protein called ATP synthase, which generates ATP as it spins.

Mitochodrial uncoupling occurs when mitochondria allow some protons to leak out of the double membrane without generating ATP. This is primarily useful for generating large amounts of heat to keep the body warm. You can think of it like revving a car’s engine without engaging the clutch – fuel is expended and the engine heats up, but the car doesn’t move. The potential for activating mitochondrial uncoupling for the purpose of weight loss is obvious, as it essentially increases total calorie expenditure without the need for exercise. There is also some reason to think that mitochondrial uncoupling could have metabolic benefits, protect the brain and even slow ageing. This is because mitochondrial uncoupling has been shown to reduce the production of reactive oxygen species (harmful byproducts of mitochondrial metabolism) and preserve mitochondrial function, which is through to play an important role in the ageing process. So, if mitochondrial uncoupling is something our mitochondria do anyway, and we have mitochondrial uncoupling drugs, why aren’t we taking them?

The Problem

Most well-studied drugs that can induce mitochondrial uncoupling are known as protonophores, so-named because they carry protons across the mitochondrial membrane (protonophore translates to ‘proton carrier’). DNP is one such protonophore. Unfortunately, as the factory workers at the start of this story discovered, too much mitochondrial uncoupling is toxic, as it prevents mitochondria from producing the ATP necessary for the cell to fulfil basic functions and can cause fatal hyperthermia. In the United States, DNP was actually sold as a weight loss drug in the 1930s, but the difference between a weight loss-inducing dose and a fatal dose proved to be very small, and so it was soon banned.

Over time, more protonophores have emerged, but all of them either fully uncouple ATP production or are poorly understood. Currently, the only reliable and ‘effortless’ way to induce mitochondrial uncoupling is to get cold, as this activates mitochondrial uncoupling in order to generate heat in the short term. Studies also suggest that repeated exposure to cold temperatures can lead to the long-term expansion of brown fat – a type of fat tissue specialised towards generating heat through mitochondrial uncoupling.

But what if we could develop a drug that only caused mild uncoupling – that is to say, one that causes sufficient leakage of protons to increase energy demands, but not enough to negatively impact ATP production? Such a drug could be a safe and effective way to induce weight loss, and could also have some benefits for mitochondrial health in general. That’s why studies like this one, in which researchers demonstrate that is possible to synthesise ‘milder’ protonophores from existing chemicals, are quite interesting.

Creating Safer Protonophores:

In this study, which was published in Chemical Science, researchers designed and tested a new family of molecules called arylamide-substituted fatty acids (arylamides). Starting with an existing class of protonophores called arylurea protonophores, they synthesized a small library of structurally similar compounds. Using chemical tests and cell culture experiments, they compared mitochondrial effects and membrane proton-transporting properties of these modified protonophores to existing compounds. They found that a specific type of arylamide – 3,4‑disubstituted arylamides – was able to partially uncouple mitochondria, increasing cellular oxygen consumption and proton leakage without significantly lowering ATP or killing cells. Mechanistically, these mild uncouplers formed less stable dimers (when two similar subunits join together) and transported protons across the mitochondrial membranes more slowly.

Let’s break down the above graphs in more detail:

• Graph B shows the rate of oxygen consumption in breast cancer cells treated with the full uncouplers DNP and CCCP, or with 5 different arylamides. Higher oxygen consumption indicates higher mitochondrial activity, as oxygen is consumed in order to move protons into the inter-membrane space, and mitochondria must work harder to maintain proton concentrations if there is a leak. Even though cells were exposed to arylamides at their maximum soluble concentrations in this experiment, they did not increase oxygen consumption as much as DNP or CCCP.
• Graph C shows how mitochondrial polarisation (the charge generated by pumping protons into the intermembrane space) changes according to protonophore concentration, based on a fluorescent dye called JC-1. A loss of polarisation indicates the leakage of protons resulting in a loss of electric charge. The graph shows that most compounds achieved similar depolarisation at the maximum concentrations tested, though some had more gradual effects than others as concentration increased.
• Graph D shows ATP levels within treated cells over time relative to control cells. Statistically significant decreases are marked by *. While there is a trend towards lower ATP levels, arylamides 5b and 6b did not significantly reduce intracellular ATP at any moment in time compared to controls, despite increasing oxygen consumption and depolarising mitochondria in other experiments, suggesting that they act as ‘mild’ protonophores.

While all this doesn’t necessarily mean that the synthesised molecules will work the same way in humans or be safe, it does prove that we can alter existing protonophores to dampen their effects, and also improves our understanding of what makes a protonophore ‘mild’.

Increasing Interest:

Outside of scientific research, there is increasing interest in leveraging mitochondrial uncoupling for the purposes of weight loss and general health, even if mitochondrial uncoupling itself is not explicitly mentioned. Cold showers, cold plunges and cryotherapy have become more popular, and while the effects of these practices are still very much a subject of ongoing study, mitochondrial uncoupling would be one of the underlying mechanisms for their proposed health benefits.

Unfortunately, there has also been a rise in the purchase and consumption of dangerous protonophores, including DNP. That’s right – the chemical that started all this by killing factory workers is still being sold online as a constituent of weight loss pills, and can be bought with varying levels of ease in different countries. It should be re-iterated that while there is a fatal dose of DNP, that dose varies from one person to another, and doses that are not fatal are still unsafe.

Actions You Can Take:

Though not yet scientifically proven, modulation of mitochondrial function through lifestyle practices appears to be a credible strategy to promote healthy ageing. Mitochondrial uncoupling is just one aspect of this – lifestyle interventions like aerobic exercise and caloric restriction can also benefit mitochondrial health in other ways. For more information about the importance of mitochondria in ageing and age-related diseases, check out this article from our hallmarks of ageing series.