Supermetals versus superbugs

Liquid suspensions of heavy metal complexes are generally not meant to be swallowed. But nestled between lead and polonium at position 83 on the periodic table is an elemental exception. Bismuth is a heavy metal with long history of medical use. Bismuth subsalicylate has been used to treat gastrointestinal troubles since the 18th century, and in the US has been marketed as over-the-counter medicine Pepto-Bismol since 1919. Today it is widely prescribed as part of a combination therapy to treat stomach ulcers caused by the drug-resistant Gram-negative bacterium, Helicobacter pylori.

‘Bismuth has this strange double character of being a heavy metal with this powerful antimicrobial activity, but at the same time seemingly very low toxicity within humans,’ says Philip Andrews, an organometallic chemist at Monash University in Melbourne, Australia.

With bacterial infections from drug-resistant pathogens now leading to tens of thousands of deaths per year across the US and EU, bismuth’s antimicrobial properties are no longer just a curiosity. Andrews’ group is one of a growing number around the world actively investigating metal complexes as potential antibiotics.

If we are going to look at future antimicrobial compounds, metals really have to be part of the game

As well as bismuth, complexes based on metals such as silver or gallium, ruthenium or zinc are increasingly being investigated – either alone, in combination with other metals, or paired with conventional organic antibiotics – as a way to treat drug-resistant pathogens. Across the field there’s a cautious optimism that metal complexes may represent a new way of fighting bacteria that is far harder for them to evolve resistance against.

These unconventional antimicrobial compounds could be the perfect tonic to our dwindling range of options against superbugs. ‘If we are going to look at future antimicrobial compounds, metals really have to be part of the game,’ Andrews says. ‘And it is going to take chemists who have the real understanding and knowledge of how to design, develop and apply those compounds, to bring them through in the medicinal chemistry sphere,’ he adds.

A multitude of metals

Metal complexes and metalloids have been used in medicine since ancient times. Antimony, for example, was used by the ancient Egyptians to treat fevers and skin irritation. More recently, since the start of the 20th century, antimony complexes have been a frontline treatment for leishmaniasis caused by infection with Leishmania parasites. Around the same time, arsenic-containing arsephenamine (known as Salvarsan) was discovered as the first effective treatment for syphilis.

With Alexander Fleming’s 1928 discovery of penicillin, however, metal complexes rapidly fell out of favour as potential medicines. Medicinal chemistry evolved into an almost exclusively all-organic pursuit, often inspired by natural products. But the wellspring of new antimicrobials based on natural products seems to have run dry, notes medicinal chemist Mark Blaskovich, who directs the Centre for Superbug Solutions at the University of Queensland, Australia. Of all the antibiotics currently in clinical trials, around 75% are simply derivatives of existing antibiotics, so are likely to be vulnerable to existing bacterial drug resistance mechanisms.

‘Similarly, pharma companies have large libraires of compounds but have been very unsuccessful at discovering antibiotics within those collections,’ Blaskovich adds. One reason for this failure, he suggests, could be that these collections are curated to include compounds that are ‘drug like’ in physical chemical properties such as size, shape and charge. ‘But if you look at the majority of approved antibiotics, they break most of the rules those companies are using to make their collections.’

Since 2015, Blaskovich and his colleagues have been gathering a broader set of structures, including as many rule-breakers as they can find, from which to hopefully regenerate the pipeline of novel structures to test as potential antibiotics. With funding from the Wellcome Trust and the University of Queensland, the team set up an initiative called the Community for Open Antimicrobial Drug Discovery (Co-Add).

‘Over the last 100 years chemists around the world have been making weird and wonderful molecules for a variety of reasons – some to develop new methodology, some to develop bioactive molecules against other disease target, some just usual shapes or sizes,’ Blaskovich says. ‘Our idea was to tap into this diversity by making it very easy for chemists around the world to test their compounds for antimicrobial activity,’ Blaskovich says. Co-Add offers a free screening service for any submitted compound. It even pays for shipping.

Each compound is tested against five different types of bacteria and two different types of fungi. ‘Anything active, we confirm the activity in a concentration-dependent test, and counter-screen for toxicity against mammalian cells.’ The results are sent to the submitter, who retains all rights to the compound.

Since Co-Add was established five years ago, over 300 academic groups from around the globe have submitted over 300,000 compounds to the database. Around 2500 have proven to be active antimicrobial compounds with selectivity over mammalian cell toxicity. ‘We now have this very large accessible database that other scientists can use, ideally to develop predictive models for what type of properties give compounds antibacterial activity,’ Blaskovich says.

Co-Add was not set up with metal complexes particularly in mind, but in 2018 Swiss postdoc Angelo Frei joined the team. Frei’s background was in medicinal metal complex research. ‘He looked through the database and found we had close to 1000 different metal complexes in our library,’ Blaskovich says. These complexes proved to be a particularly promising subset in the Co-Add collection. Among purely organic molecules in the database, the hit rate for compounds that are active against at least one bacterial strain, and are non-toxic to mammalian cells, is 0.87%. But for the metals, the hit rate was 9.9%. Compared to organic compounds, metal compounds were more likely to show antibacterial activity, but no more likely to be toxic to mammalian cells.

This lack of toxicity may be surprising to many. The one significant exception to the lack of metals in medicinal chemistry is the platinum-based chemotherapy drug cisplatin, approved in 1978 and still used in most cancer treatments today – despite its side effects. ‘We know cisplatin is very toxic, even though it is very effective,’ Andrews says. Due to the toxicity of this one high-profile metal-based drug, metal complexes in general have become associated with toxicity. ‘The worry has been that, if you ingest metal complexes, there is a real problem with poisoning.’

Blaskovich agrees. ‘One of the reasons metal complexes have not been used much at all in medicinal chemistry, other than as anticancer compounds, is probably that their initial association as an anticancer therapy has given the perception that these compounds are generally quite toxic and not very useful as general drugs,’ he says. The Co-Add analysis suggests that perception is unjustified. ‘Among the compounds we had assembled, the metal complexes did not have any more toxicity than organic compounds against human cells. It opens up the possibility we should be looking at metals as a potentially very rich source of new antimicrobial agents.’

A silver lining

Katharina Fromm was working on the coordination chemistry of silver compounds when, in the early 2000s, she was approached by a team of material scientists interested in developing antibacterial coatings for medical implants. Fromm, now at the University of Fribourg in Switzerland, has been looking at the antibacterial properties of metal compounds ever since.

Silver is one of the most well-known antimicrobial metals and was used at least as far back as the 1920s in certain wound treatments. Yet despite silver’s long history of use and recent revival, precisely how silver works as an antimicrobial is still being established. ‘I was very surprised when I got into the silver story – there are a lot of studies still to be done for this basic understanding,’ Fromm says.

If you know the molecular story you can act at the different pressure points to block the bacteria’s defence systems

Compared to the more than half a century of research into organic antibiotics’ mode of action, metals have received much less attention. But we do know that, compared to organic antibiotics, the activity of metal complexes is – well, complex. Organic antibiotics mainly hit one specific target, whereas metals often appear to be more scattershot. This complexity has added to the challenge of understanding silver’s antimicrobial mode of action, but from a functional point of view potentially adds a key advantage, Fromm notes. ‘If you think about vancomycin, this drug interacts with two amino acids in a protein in the outer membrane of the bacteria,’ she says. ‘If the bacteria mutate just one of those amino acids in the protein, they become immune. Whereas silver ions start at the membrane but also do damage inside, generate ROS [reactive oxygen species], change the function of proteins and so on, so can have multiple places of attack.’

Understanding what silver complexes are doing in the bacteria, to find ways to make better therapeutics, has been a focus of Fromm’s research. ‘If you know the molecular story you can act at the different pressure points to block the bacteria’s defence systems,’ Fromm says. The slow release of silver ions seems to be the key to silver complexes’ bioactivity. But bacteria possess a protein called SilE that seems to act as a sponge for silver ions, detoxifying them. ‘If you block the expression of this protein, you could clearly render the bacteria much weaker,’ she says. Fromm and her team are increasingly focussed on exploring the synergistic effects that can be gained by combining silver with other metal complexes, which may block SilE’s function, for example. ‘If we can learn to hit with two or three weapons at the same time, that will be much more important in the future,’ she says.

Getting down to bismuth

Andrews wasn’t looking for a new antimicrobial when he started researching bismuth. ‘Back in 2001 we got funded to set up the Centre for Green Chemistry within Monash University, and we started looking at benign metals for doing sustainable chemistry,’ Andrews recalls. ‘Bismuth was one of the metals we were very interested in.’

Many bismuth compounds were difficult to make, insoluble and difficult to characterise

But in 2005, researchers Barry Marshall and Robin Warren were awarded the Nobel prize in physiology or medicine for their discovery that H. pylori infection was the cause of stomach ulcers. They also showed Helicobacter infection could be treated with bismuth. Andrews became intrigued by bismuth’s selective toxicity to bacterial cells. ‘We discovered, looking around the literature, that there had been very little done to explore why that might be the case,’ he says. ‘I think the big problem was really a chemistry problem. Many bismuth compounds were difficult to make, insoluble and difficult to characterise.’

Andrews set out to improve these aspects of bismuth chemistry, developing reproducible syntheses and good characterisation, ‘to the extent we knew what we were making, so we could confidently study the biological activity and get a handle on their mode of action’.

Another aspect of organometallic chemistry that could be considered an advantage or disadvantage over organic drugs, depending on your viewpoint, is the potential structural lability of biochemistry of metal complexes. Whereas most drug molecules arrive at their site of action with original structure intact, metal complexes can often switch ligands, depending on what is around them. ‘And the biological environment is just full of potential ligands – proteins, siderophores, sugars, peptides,’ Andrews says. But that susceptibility can be exploited, his group’s work suggests. ‘We find we can tune the kinetic and thermodynamic stability of the complex, based on particular ligands.’ One trend Andrews has observed is that the more ionic the compounds are, the more labile they are – and the more bioactive. Moving from carboxylic to sulfonic acid ligands gives two orders of magnitude higher antimicrobial activity.

Part of the appeal of metal complex chemistry is the ease with which analogues of active compounds can be created, simply by switching ligands. ‘We are really interested in what happens when you put two or three different types of ligand around the metal,’ Andrews adds. ‘The studies we’ve done seem to show that as you move away from one type of ligand, you improve the bioactivity.’

The gold standard

Hongzhe Sun from the University of Hong Kong was also intrigued by bismuth’s capability to treat drug-resistant stomach ulcer infections. ‘Bismuth-containing antibiotic triple therapy has become the standard treatment for resistant Helicobacter infection, and we wondered whether we could extend the concept to other persistent bacteria, to use bismuth to restore the activity of other antibiotics,’ he says.

For the broad β-lactam family of antibiotics, metallo-β-lactamase enzymes such as the relatively recently emerged New Delhi metallo-β-lactamases (NDMs) are the drugs’ nemesis. These zinc(ii)-containing enzymes can cleave the antibiotic’s β-lactam ring, conferring bacterial resistance to all bicyclic β-lactams including carbapenems, the antibiotics of last resort against multidrug resistant infections. To inhibit NDMs, medicinal chemists had been trying to develop substrate mimics to block the NDM active site, or to develop a zinc chelator to remove the zinc. ‘We wondered if the bismuth would have high affinity for the sulfur in the enzyme,’ Sun recalls. The team showed that bismuth simply kicks the zinc out of the NDM active site, displacing the metal and inhibiting the enzyme’s activity. In mice, the bismuth(iii) compound restored the activity of the β-lactam meropenem against NDM-positive bacteria.

The team is continuing to extend the concept to resistance enzymes that bacteria may carry in addition to NDM, such as mobilised colistin resistance enzyme MCR-I. ‘We would like to use one inhibitor to inhibit more than one resistant gene,’ Sun says. The team recently showed that the gold(i)-containing antirheumatic drug, auranofin, was a dual inhibitor of NDM-I and MCR-I, displacing zinc(ii) from their active sites.

The team has also pioneered new approaches to map the impact across the bacterial cell when it is treated with a metal complex. ‘We don’t have sufficient approaches or methodologies to understand metal–protein interaction in cells and tissues – some metal–protein interactions can be labile, so you can’t isolate them,’ Sun says. So the team developed metalloprotemics, in which a fluorescent agent combined with an azide photoaffinity group could be used to capture the interaction between metal and protein. ‘This is a very useful approach – we can dig out lots of metal–protein interactions. We also combine it with metabolomics, to see if that corresponds with certain protein function disruption.’

With bismuth, for example, inhibition of urease activity was long suspected as a mode of action against H. pylori. Rather than inhibit the urease directly, Sun showed bismuth inhibits a urease chaperone protein – and then identified new drug leads that also hit this target. The team has also used this suite of techniques to examine the mode of action of several other metals. They showed that gallium(iii) antimicrobials target the essential transcription enzyme RNA polymerase in Pseudomonas aeruginosa, thereby suppressing RNA synthesis and impairing bacterial metabolism and energy utilization. They also identified 34 proteins directly bound by silver ions in Escherichia coli, impairing their function to the extent that key aspects of the bacterium’s central metabolism are stalled.

Even after 100 years of people swallowing Pepto-Bismol, Helicobacter still hasn’t developed a way to become resistant

In the age of antibacterial resistance, one of the most compelling aspects of bismuth(iii) compounds’ particular biochemistry is that bacteria do not seem to have gained any resistance to it despite its long-term use. One factor may be that because metals can hit multiple targets in the cell, it is harder for bacteria evolve resistance. One additional advantage for ions such as gallium(iii) and bismuth(iii) is that bacteria have to import iron for their survival, and similar ions can ride the same pathway. ‘We can use that trojan horse effect to get bismuth transported in,’ Andrews says. ‘If a cell decides to shut down its iron uptake because it wants to stop a metal that mimics iron from coming in, it is not going to survive anyway.’

Another aspect at play for bismuth, Andrews suspects, comes down to hydrolytic chemistry, which he and his team have explored. ‘When a bismuth complex reacts with water, it starts to form chunks of bismuth oxide. You build up these clusters, maybe nine to 38 bismuth atoms in size.’ These clusters are essentially inactive, shutting down bismuth’s antimicrobial bioactivity. Metals such as silver, in contrast, persist in the body or the environment as silver ions. ‘The problem with these materials is that if the concentration is below that need to completely kill the bug, you create an environment where resistance can develop,’ Andrews says. ‘We hope that by forming these bismuth oxide chunks, you take it away from being an active antimicrobial and then hopefully the bugs don’t develop resistance. Even after 100 years of people swallowing Pepto-Bismol, Helicobacter still hasn’t developed a way to become resistant, which gives us hope,’ he adds.

Precious metals

While bismuth has been one area of recent progress, and there is long-standing interest in silver, plenty of other metals are also showing promise as antimicrobials. Fromm and Blaskovich particularly note the ruthenium complexes being developed, which operate by generating reactive oxygen species upon light irradiation, for use as antibacterial photodynamic therapy agents. ‘We need alternatives to traditional antibiotics, and photosensitisation is one pathway by which metal complexes might give you additional modalities,’ Blaskovich says.

Meanwhile, the Co-Add analysis highlighted that complexes of gallium, palladium, silver, cadmium, iridium and platinum showed the most promising antibacterial activity. The sheer diversity of metals, ligands and geometries of the most active complexes was a notable outcome of the study. ‘We have been following up in collaboration with a couple of the groups, doing some preliminary drug-like assessments, putting them into some early in vivo types of efficacy model – and getting some potentially promising results from those,’ Blaskovich says. The team has also seen an uptick in the number of metal complexes being added to the database since their analysis was published – though that is in part because they are now actively soliciting compounds from groups doing metal complex work.

It needs a concerted drive to show these compounds really do have promise

‘Interest in the area is absolutely growing,’ says Fromm. ‘You can look at the numbers of papers of metal-containing drugs, this is going up.’ But although academia can generate a lot of leads, financially it cannot complete the full clinical trials required to bring a new antimicrobial to market. ‘My personal opinion is probably countries, states, have to see that clinical trials can be financed somehow,’ Fromm says. ‘Big pharma is becoming more interested because they know the pipeline is quite empty against these superbugs – but the first step to be taken is a risk, and it is expensive.’

For now, academia’s role should be to produce enough preliminary studies to prove that metal complexes are a compelling option, she adds. ‘We see it now with vaccines,’ Fromm says. ‘If there hadn’t been the 20 years of basic research on mRNA, we would not have been able to develop these Covid vaccines so quickly.’

Metal-based antibiotics are at a nascent stage of development, Blaskovich says, but are now definitely on the radar. ‘It needs a concerted drive by somebody, and we are trying to help do that, to show these compounds really do have promise. I think that the one key piece of evidence still lacking is to have at least one quite thoroughly characterised candidate show efficacy against resistant bacteria in an in vivo mouse model,’ he says. ‘We are at the stage now where it could blossom very quickly.’

James Mitchell Crow is a science writer based in Melbourne, Australia