New way to fight multidrug-resistant bacteria spares the good and kills the bad and the ugly

Disease and treatment 9. feb 2021 5 min Associate Professor Benjamin Anderschou Holbech Jensen Written by Morten Busch

If the world fails to act, by 2050, antibiotic-resistant infections will kill 10 million additional people every year across the globe – more people than currently die from cancer. However, no major breakthroughs have been made in developing antibiotics for many years. Now researchers have discovered how to increase the potency of some peptides in the body so that they kill pathogenic bacteria but not the bacteria that promote health. Researchers do not know why this happens, but apparently the pathogenic bacteria do not develop resistance to these peptides.

Defensins had been written off as useless in combating bacteria. They were extraordinarily expensive to manufacture, had limited effectiveness against infections and mostly disintegrated easily. But a group of researchers discovered a little more than a decade ago that human beta-defensin 1 (hBD-1) changes function and becomes extremely active in fighting various bacteria under the acidic and oxygen-poor conditions in the human intestines. A new study shows surprising potential.

“Previous studies showed that the peptides that arise when hBD-1 is cleaved effectively fight bacteria but are also very unstable. We tried to stabilize them, and we produced a stable compound that not only kills pathogenic bacteria in the gut but also protects the gut’s natural bacterial flora. We are therefore taking this further and developing and testing a treatment for bacteria both inside the body and on the skin,” says a researcher behind the study, Benjamin Anderschou Holbech Jensen from the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen.

Greatly underestimated

The spread of antibiotic-resistant bacteria is a growing threat to public health. Specifically, six pathogens collectively called ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) account for most of the more than 700,000 deaths annually from hospital-acquired multidrug-resistant infections worldwide. This figure is estimated to increase 15-fold over the next 30 years if no new antibiotics are developed.

“No new antibiotics have been developed in the past 30 years, and the existing antibiotics have limited effectiveness and adversely affect people’s natural gut flora. The antimicrobial peptides found naturally in all multicellular organisms are therefore an interesting alternative with the potential to become a new generation of antibiotics to fight multidrug-resistant pathogens,” explains Benjamin Anderschou Holbech Jensen.

Most existing antibiotics are derived from bacteria, fungi, plants and animals, but researchers have recently begun to search the human body for new ways to fight pathogens, including the defensins, which are peptides that protect the body against various microbes, such as on mucosal surfaces. Researchers did not see much potential in hBD-1 until it was tested under the reduced conditions, an environmental characteristic of the intestines.

“Ten years ago, our collaborators found that hBD-1 changes function and becomes active in killing bacteria when it enters the intestines. Unfortunately, this activation led to a breakdown of the protein structure and thereby enzymatic degradation, which was previously thought to inactivate this otherwise potent molecule,” says Benjamin Anderschou Holbech Jensen.

Fatty acids ensure stability

Since then, however, the same group of researchers, including Danish participation, found that both hBD-1 and the related defensins, HD-5 and HNP4, either maintained or increased their antimicrobial repertoire when the linearized peptides were fragmented. Now defensin fragments are one of the most promising candidates to save people worldwide from the growing threat posed by multidrug-resistant bacteria.

“Fragmented hBD-1 has strong antimicrobial activity but unfortunately also low stability. We therefore tried to stabilize the hBD-1-derived fragments with palmitic acid and with various spacers, such as sugars or amino acids,” explains Benjamin Anderschou Holbech Jensen.

This enabled the researchers to produce numerous lipopeptides (Pams) with increased stability and bactericidal activity. The fatty acids ensured stability, a well-known biotechnological method for stabilizing linear peptides.

“Unfortunately, in some cases this process also affects effectiveness, such as for hBD-1. Conversely, the various spacers help to control the activity,” says Benjamin Anderschou Holbech Jensen.

Bacteria did not develop resistance

After being produced, the most promising peptides were tested against many multidrug-resistant pathogens and biofilms. One of them, Pam-3, proved extremely effective. In addition to increased stability, it proved more effective against the multidrug-resistant bacteria than both the unmodified peptide and the last-resort antibiotics used today.

“We do not yet know why the modified peptide is more active against the pathogenic bacteria than the original one, but we assume that the small spacers are important for folding and thus activity. Pam-3 even eradicated established S. aureus and P. aeruginosa biofilms, which are otherwise notoriously resistant to antibiotics,” explains Benjamin Anderschou Holbech Jensen.

In addition, the researchers observed that the pathogenic bacteria did not develop resistance, even after culturing them for 25 passages with subinhibitory concentrations of either Pam-3 or traditional antibiotics. Conversely, the same bacteria treated with traditional last-resort antibiotics developed resistance after only five passages.

“One reason resistance did not develop may be that several simultaneous effects create the overall bactericidal effect: the peptides destroy the bacterial membranes by creating small pores in them and disrupt other mechanisms and physiological functions in the bacteria,” says Benjamin Anderschou Holbech Jensen.

From laboratory to people

Pam-3 affects several mechanisms simultaneously, making bacterial survival incredibly difficult. The researchers were therefore very surprised that the peptides do not affect the health-promoting bacteria in our intestinal flora.

“We do not yet understand why, but whereas Pam-3 eradicates the bad bacteria, it spares the good ones. One problem with traditional antibiotics is that they destroy the body’s own health-promoting bacteria, which increases the risk of developing resistance and creates many subsequent health problems such as an increased risk of secondary infections, a reduced ability to digest nutrients and diarrhoea,” explains Benjamin Anderschou Holbech Jensen.

Although the researchers performed most of the experiments in the laboratory, they also tested the efficacy of Pam-3 in two in vivo models that replicated physiological conditions as closely as possible. Although these results were also convincing, the researchers are aware that the hardest work may be ahead of them.

“The leap from the laboratory and testing in animals to humans is always challenging, but we are already discussing with a company that wants to test specific candidates for non-pharmaceutical use. We strongly suspect that our modified fragments may be effective against, for example, acne, which often results from an imbalance in the composition of skin bacteria,” says Benjamin Anderschou Holbech Jensen.

In addition, the researchers are seeking partners and investors to develop targeted antibiotics, which have enormous potential to improve public health in the coming years.

“We have indications that our now thoroughly developed panel of both naturally occurring and modified fragments has both antibacterial and antiviral potential. We therefore have a very well-organized plan for which parts can be developed non-pharmaceutically, with a partner close to being in place, and which should be developed under a solely pharmaceutical framework, such as for atopic dermatitis, which is also characterized by a marked imbalance in the bacterial composition of the skin,” explains Benjamin Anderschou Holbech Jensen.

Promising business model

Atopic dermatitis is also known as asthmatic or childhood eczema. In addition to severe itching, dry skin and blisters, it can also increase the risk of developing both asthma and hay fever, and the researchers also hope that the antimicrobial peptides will be effective in fighting these disorders.

“We clearly also hope to eventually move from treating the outer surfaces of the body to treating the internal ones such as the lungs and intestines. The vast knowledge we have accumulated about full-length defensins, their fragments and especially our modified fragments in recent years suggests that we will be able to develop several promising treatments. This is being tested in a newly established biotech company, of which I am currently the CEO and Research Director,” says Benjamin Anderschou Holbech Jensen.

There is great potential, but focus is also absolutely essential for success. The researchers are therefore creating a new company to integrate the production and defensin expertise of three leading biotech companies that will focus exclusively on non-pharmaceutical indications.

“The road to the market and thus a promising business model is somewhat shorter. We are then seeking investors and development partners to follow up on pharmaceutical indications with closely related products. Initially, we are obtaining experience from the tests for non-pharmaceutical use. The road to pharmaceutical use will be much shorter once we better understand the effects and possibly the challenges of dosage, formulations and potential side-effects,” concludes Benjamin Anderschou Holbech Jensen.

Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota” has been published in Communications Biology. In 2017, the Novo Nordisk Foundation awarded a grant to co-author Benjamin Anderschou Holbech Jensen for the project A Novel Oral Combination Therapy Targeting the Gut Microbiome to Alleviate Insulin Resistance and Type 2 Diabetes–linked Aortic Valve Stenosis. Benjamin Anderschou Holbech Jensen is employed at the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen.

Benjamin A. H. Jensen is a tenured Associate Professor in Nutritional Immunology, consultant, and biotech entrepreneur. He leads the Jensen Group at D...

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