Booster for antibiotics

Transition metals promise to reduce the threat of resistance by giving a new lease of life to antibiotics that are either ineffective or have waning effectiveness against resistant bacteria. Jasmin Fox-Skelly reports.

Every year in the US at least 2.8m people become infected with antibiotic-resistant bacteria.¹ Of these, more than 35,000 die. The crisis is such that a growing number of infections, including pneumonia, tuberculosis, gonorrhoea, and salmonellosis, are becoming harder to treat as the antibiotics used to fight them are no longer effective.

If this continues, organ transplantations, chemotherapy and routine surgeries such as Caesarean sections will become dangerous medical interventions. In short, unless something urgent is done, we are heading for a post-antibiotic era, in which common infections and minor injuries can kill once more.

Mark Blaskovich, a self-described ‘antibiotic hunter’ and Director of the Centre for Superbug Solutions at the University of Queensland, Australia, believes that metal complexes could offer a solution.

‘The only metal complexes that are currently extensively used to treat diseases are anticancer compounds, there are almost no metal complexes used for any other diseases’, says Blaskovich.

This is perhaps surprising. The antimicrobial properties of metals have been known about for thousands of years. Vessels made of copper and silver were first used to keep food clean and free of bacteria during the time of the Persian kings, who ruled between 559 BCE to 331 BCE. Silver was used as a germicide and disinfectant until the 1940s.

Nevertheless, metals fell out of favour after 1928, when Fleming discovered penicillin. Since then, hundreds of antibiotics have been discovered and licensed to treat bacterial infections. But most of these were patented between 1940 and 1960. Over the last few decades, the rate at which we have developed new antibiotics has slowed dramatically.

‘There are just over 40 new antibiotics in clinical trials, which sounds encouraging until you compare this with the more than 1000 medicines and vaccines in clinical trials for cancer treatments,’ says Blaskovich. ‘Of these, around 75% are derivatives of existing antibiotics, so are likely to be vulnerable to exactly the same bacterial drug resistance mechanisms.’

So, what are these resistance mechanisms? Bacteria have evolved several ways of evading antibiotics. These include thick impenetrable cell walls, which prevent drugs from entering bacterial cells in the first place; efflux pumps that transport antibiotics back out of the cell; and enzymes that deactivate any antibiotics they come in contact with.

To look for new candidates that bacteria might not have had chance to evolve resistance to, Blaskovich’s research group recently identified over 900 metal-containing compounds within a larger set of over 300,000 compounds tested for antimicrobial activity using the Community for Open Antimicrobial Drug Discovery (CO-ADD).² This screening service allows scientists to send in compounds that don’t fit the mould for common drug design, and which would otherwise be sitting unused on laboratory shelves³ The compounds are tested on known bacterial and fungal pathogens to see if they have any antimicrobial effect.

In the study, metal-based compounds were seen to display a significantly higher hit-rate (9.9%) than purely organic molecules (0.87%).

‘When we started looking at the subset of metal containing compounds it was quickly obvious they had a much higher hit rate than the traditional organic compounds that are generally the mainstay of drug discovery,’ says Blaskovich. ‘The hit rate was 10-24 times higher for the organometallic compounds than the organic compounds, so it wasn’t just a little bit better – it was substantially better.’

The reasons behind this superior hit-rate aren’t known, but it could have something to do with the shape of the molecules, as well as activity of the metal component itself.

‘A huge benefit of metal complexes is that they naturally have a three-dimensional geometry,’ explains Blaskovich. ‘A lot of organic compounds are mostly flat and planar, and so don’t engage well with biological targets like enzyme pockets or G-protein coupled receptor cavities, which are often globular in shape. With organometallic compounds you can quite easily generate complex and large three-dimensional globular shapes, which are ideal for fitting into ion channels, enzymes or receptor pockets.’

Blaskovich and his colleagues have focused on 23 previously unexplored compounds containing metals such as silver, manganese, zinc, ruthenium and iridium that have antibacterial and antifungal activity. The good news is that whilst these metal compounds are great at killing bacteria, including the potentially deadly methicillin resistant Staphylococcus aureus (MRSA), they are not harmful to human cells at the same concentration they kill the microorganisms.

‘That was a really interesting finding because it overcomes this perception that organometallic compounds must be toxic,’ explains Blaskovich. ‘I think a lot of this perception is simply down to the fact that the only metal complexes used in medicine are for treating cancer. Those drugs are toxic because that is how they kill cancer cells.’

When we started looking at the subset of metal-containing compounds it was quickly obvious they had a much higher hit rate [in antimicrobial screening] than the traditional organic compounds that are generally the mainstay of drug discovery.
Mark Blaskovich Director of the Centre for Superbug Solutions, University of Queensland, Australia

In addition to activity against MRSA, some of the metal containing compounds are active against dangerous Gram-negative pathogens such as Escherichia coli and Acinetobacter baumannii, for which there are few effective antibiotic treatments.

So, could metal-based compounds help solve the antimicrobial resistance crisis? There’s good reason to think so. For one, they are very different to current antibiotics, which are modelled on natural compounds. That means that bacteria are less likely to have come into contact with them in the past, so they won’t have evolved defence mechanisms. Also, unlike the majority of licensed antibiotics, which tend to target just one protein, metal compounds appear to attack bacterial cells in several different ways, making them harder for bacteria to evade.

Going for gold

Blaskovich isn’t the only scientist who thinks that metals could play a role in solving the antimicrobial crisis. Dejian Zhou, Professor of nanochemistry at the University of Leeds, UK, has created antibiotics using tiny particles of gold.⁴

For several years researchers have known that gold nanoclusters – each made up of about 25 atoms of gold – are harmful to bacteria. However, until now scientists have struggled to figure out a way of getting the nanoparticles to the site of a bacterial infection. Zhou’s team believe they have come up with an answer. Their research takes advantage of the fact that bacterial cell walls are strongly negatively charged. Using the idea that opposite charges attract, the team wrapped the gold nanoclusters in a pyridinium-based ligand. As the ligand is positively charged, it acts like a carrier pigeon and finds and delivers the nanoclusters straight to the walls of bacteria cells.

One problem, though, is that the pyridinium-based ligand is also toxic to human cells. To protect host cells, the scientists added a second ligand to the envelope around each nanocluster. These zwitterionic groups carry both positive and negative charges. Similar molecules are found in the outer membranes of most mammalian cells and are therefore not toxic. Shielding the nanoclusters inside zwitterionic groups means that they can also pass easily through the kidney and be excreted from the body.  

According to Zhao, the gold nanoclusters can act as antimicrobials in their own right, but they can also boost the efficacy of existing antibiotic treatments. ‘The positively charged nanoclusters essentially neutralise the electrical potential that exists across the bacterial cell wall,’ explains Zhao. ‘This inhibits the bacteria’s efflux pump, which relies on the proton gradient across the membrane to function. This reduces the ability of bacterial cells to remove any antibiotics from their interior, and so can reduce resistance.’

As well as preventing bacteria from expelling antibiotics directly, the ligand-gold complex also disrupts the bacterial cell wall, making it more permeable to antibiotics. Together these two mechanisms suggest the treatment could effectively give a new lease of life to antibiotics that are either ineffective or have waning effectiveness against resistant bacteria.

To test this theory, the scientists investigated whether the gold nanoclusters could boost the effectiveness of antibiotics. They used a bacterial strain called methicillin resistant Staphylococcus epidermidis (MRSE), which is responsible for many hospital-acquired infections. They tested three classes of antibiotics against MRSE – with and without the gold nanoclusters – and found they had a remarkable impact, reducing the amount of antibiotic needed to inhibit growth of MRSE by a factor of 128 times in one case.

Zhou believes combining gold nanoclusters with existing antibiotics will be a faster and cheaper alternative to developing new antibiotics when it comes to combatting bacterial antibiotic resistance. ‘Although new antibiotics can temporarily address bacterial resistance, unfortunately bacterial resistance to such new antibiotics is inevitable due to natural selection,’ he says. ‘In contrast, as our gold nanoclusters can effectively disrupt bacterial cell membranes, which are vital for all bacterial functions, it would be very difficult, if not impossible, for bacteria to develop resistance against this antibacterial mechanism.’

Tricking bacteria

Another scientist looking at combining treatments in this way is Eszter Boros, a medicinal inorganic chemist at Stony Brook University in the US, who recently developed a therapy using the transition metal gallium. The therapy works by exploiting bacteria’s need for iron, which the microbes use to power reactions inside their cells. As humans, we get all the iron we need from our diet, but bacteria must steal it from their hosts. To do this they release complexes called siderophores that bind to iron. The siderophores are then later reabsorbed back into the bacteria.

In a recent study, Boros and her team designed a special version of a siderophore that binds to gallium instead of iron.⁵ The bacteria are tricked into transporting the gallium into their cells, but unlike iron, which is vital to bacteria, gallium stalls the chemical processes inside the cell and ultimately kills the microbes.

But this isn’t all. Other compounds can be linked to siderophores and taken up inside bacteria too. Boros’s group created a gallium-siderophore complex with the antibiotic ciprofloxacin attached for good measure. The idea is that the bacteria are tricked into absorbing the antibiotic like a trojan horse. Once inside the cell, the link between the siderophore and the antibiotic is severed, allowing the deadly chemical to kill the microbe from the inside. In laboratory tests, the molecule, called galbofloxacin, is four to 10 times as effective at killing Staphylococcus aureus bacteria than ciprofloxacin alone.

Wake up call

Taken together, these results should serve as a wakeup call to other researchers, biotech firms and pharmaceutical companies to conduct more research into metal-based complexes. But there are still massive hurdles preventing pharmaceutical companies from investing in this area.

‘I think part of the reason that pharmaceutical companies traditionally haven’t looked at metals is because of their initial use for cancer therapy. There is a perception that metal complexes must be very toxic to cells because that’s how they kill cancer cells,’ says Blaskovich. ‘There is also a perception that metal complexes won’t be biologically stable and will fall apart within the body before they can reach bacteria.’

However, perhaps the biggest factor preventing investment is the fact that the antibiotics field itself is not particularly profitable. In 2019, for example, biopharmaceutical company Achaogen filed for bankruptcy just one year after having a new antibiotic approved.

‘That would normally cause a small biotech company’s stock market to jump sky high and become very successful, but actually their stock price dropped and within a year they were bankrupt,’ says Blaskovich.

The reason that Archaogen failed is because their profits from selling the antibiotic didn’t cover 100th of the cost of introducing it onto the market, let alone the cost of development.

To give an idea of cost, most new antibiotics coming onto the market are priced around $15,000 for a two-week treatment. That is compared with the newest cancer CAR T-cell therapy, which is over $500,000 for one treatment.

In addition, when new drugs reach the clinic, health professionals tend to use them sparingly to slow the spread of resistance. Doctors will almost always prescribe older, and cheaper, antibiotics in the first instance. For this reason, almost all big pharmaceutical companies have exited the field of antimicrobial research, with most research and development now being conducted by either small biotech companies or academic groups.

That needs to change if we have any hope of solving the looming antimicrobial crisis.

‘It’s predicted that by 2050 there will be 10m deaths/year caused by drug resistant infections,’ says Mark Blaskovich. ‘Compare that with Covid which has killed approximately 5m people around the world over the last year and a half, and you get an idea of how serious an issue this is.’

‘We really need to be working on coming up with new solutions and new antibiotics, and metal complexes are an untapped well of novel structures. I can’t think of any other class of compounds that has as much potential, and that has been as little explored.’

References
1 Antibiotic Resistance Threats in the United States, 2019, The Center for Disease Control and Prevention (CDC), https://www.cdc.gov/drugresistance/biggest-threats.html
2 A. Frei et al, Chem. Sci., 2020, 11, 2627.
3 J. Zuegg et al, ACS Infect. Dis. 2020, 6(6), 1302.
4 Z. Pang et al, Chem. Sci., 2021, 12, 14871.
5 A. Pandey et al, Chem. Sci., 2021, 12, 14546.