Tweaking molecules

It’s a sobering thought to realise that scientists have been looking all over the world for new kinds of antibiotics for decades. But we need them to look, because an increasing number of bacteria are becoming resistant to penicillin, azithromycin, clindamycin, ciprofloxacin, doxycyclin and a host of other antibacterial drugs. And should you catch an infection form the multi-resistant hospital bacterium MRSA, or the just as tough VRE bacterium, you’re in serious trouble.

But RUG chemist Dowine de Bruijn suggests we need to take a closer look at what’s happening around us. Because while researchers are working on developing new drugs in their laboratories, all those new antibiotics we need can be found all around us. What’s more: they’re being made by bacteria. ‘They need to protect themselves from those harmful bacteria as well’, says De Bruijn. ‘It might sound counter-intuitive, but it’s true.’

E-numbers

Take the protein nisin, for example. Nisin is produced by lactic acid bacteria and kills competitors by latching on and making a little hole in the cell wall, killing the bacterium dead. It’s an elegant and effective strategy. This substance and its antibacterial properties had already been described back when Alexander Fleming was alive. You know, the man who discovered penicillin in 1928? The food industry is also a big fan of nisin, using it as E number E234.

‘These proteins are marvellously made’, says De Bruijn. ‘They’re very strange, complex structures consisting of many rings. They don’t even look like normal peptides.’ Unfortunately, that means it’s almost impossible to replicate in a lab. ‘It has been done, but it takes six months to make half a milligram’, says De Bruijn. ‘It’s fairly pointless.’

They’re very strange, complex structures

But it’s still a very potent form of antibiotic, almost as powerful as vancomycin, which is often used as a ‘last resort’ in infections. On top of that, there is almost no resistance against it. ‘In most drugs, all it takes is a small mutation for bacteria to become resistant’, says De Bruijn. ‘But nisin is so complex and versatile that it would take a whole set of specific mutations before bacteria could become resistant to it.’

Tweaking

There’s really only one reason we haven’t already used the substance to combat MRSA or VRE infections; it’s not very stable in a pH neutral environment and is not very soluble in water. ‘In fact, it prefers acid. And unfortunately our blood isn’t acidic’, says De Bruijn. ‘So that’s kind of an issue.’

And so over the past few years, De Bruijn has tried to figure out how to change the characteristics of nisin. But instead of creating entirely new molecules like some of her colleagues at the Stratingh Institute are doing, she has let the lactic acid bacteria do their thing, only to tweak the organically grown molecules in such a way that they could be used as medication.

‘One thing we wanted to do was improve its solubility in water’, says De Bruin, who was awarded a PhD for her research last week. ‘Another thing was to add a group of molecules to see if that would make it easier for the body to absorb the substance.’

Starting point

This did mean she could only make a single adjustment in one location. Too many changes are risky: before you know it, you’ve messed with the antibacterial properties and that’s not something you want. ‘Our first goal was to show that it was possible in the first place.’

Fortunately, nisin turned out to have one very special characteristic. Contrary to most proteins, it has a carbon carbon double bond. ‘This was a unique starting point for us’, says De Bruijn. ‘The molecule uses these bonds themselves to create its rings. But there are a few left that aren’t used, so we decided t make use of them.’

Our first goal was to show that it was possible in the first place

The trick was to do it in water, and in a pH neutral environment. Before De Bruijn, no one had ever pulled this off.

She tried it by using metals such as palladium, rhodium, and iridium as catalysers, with each catalyser enabling a different reaction. ‘Palladium allows you to connect a benzene ring using boric acid, another element. Rodium got rid of the double bond altogether by using formic acid and iridium allowed us to connect a molecule under the influence of light.’

Off limits

But it wasn’t easy. The normal methods of creating bonds by boiling or using aggressive solvents were off limits, as they would damage the protein too much. It was also very difficult to ascertain whether the experiments had actually yielded result.

‘I once compared it to looking for something in the pitch black’, says De Bruijn. ‘You can’t see molecules. All you can do is measure them using a mass spectrometer.’

This led to a few nasty problems. She had calculated that her new molecules consisting of tweaked nisin would have an increased mass of 106. That was the weight of the ring she wanted to attach. ‘And that was the weight I was measuring. Exactly that weight. So I was ecstatic.’

Unfortunately, she soon discovered that the weight of her nisin molecule increased by 106 after every single experiment, no matter how much boric acid she added to it. ‘It turned out that 106 was the weight of the palladium atom. All that time I’d been measuring the catalyser attached to the molecule. Nothing had even happened.’

Frustration

It took her months to figure out a method to detach the palladium from the nisin. ‘But once I did and started measuring the right things, it was an amazing achievement.’

I suddenly couldn’t see the effect anymore

New frustrations arose when the molecule that had given her perfect, reliable measurements suddenly stopped doing its job. ‘15 March, 2016’, she remembers. ‘I suddenly couldn’t see it anymore. We cleaned the machine, tried to figure what had happened to the mass spectrometer, which substances had been in there, who’d used it… But we couldn’t measure it anymore! So we had to find yet another method to make it visible to us.’

What exactly happened is still a mystery. After that came a period of months of experimenting with new molecules and new methods until she finally and definitively figured out how to remove the palladium from her molecules.

So what’s next?

Collaboration

De Bruijn has proven that it’s possible: Scientists can tweak a nisin molecule to such a degree that they’re able to attach other bonds to it without damaging its antibiotic properties. The next step will not be hers, however. It will probably be taken by microbiologists.

‘Chemists can come up with all sorts of things, but it’s the microbiologists who decide what a molecule should be able to do and what the next logical step in the process is’, she says. She thinks the various fields should collaborate more. ‘Chemists should really be trained in microbiology, and microbiologists in chemistry.’

Once that happens, it should be possible. New antibiotics made out of milk.

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