Human Hybrid Antimicrobial Peptides for Corneal Infection: From Molecular Dynamic Simulation to In Vivo Animal Studies

Cornea - the transparent window located at the front part of the eye - serves as a critical structure to normal vision and ocular surface defence. Damage to the cornea can lead to permanent scarring with subsequent visual impairment or blindness.

Corneal blindness represents the 5th leading cause of blindness globally, affecting approximately 2 million of the world population. Bacterial corneal infection is the most common culprit, with contact lens wear and trauma being the main risk factors. Affected patients are usually debilitated by pain and visual impairment and they often require long-term hospital admissions for intensive antibiotic treatment. However there has been a growing concern on the declining antibiotic efficacy due to emerging resistance of organisms to antibiotic (i.e. antimicrobial resistance) in infections affecting the eyes and other parts of the body. In addition studies have shown the formation of biofilm (a slimy collection of microorganisms that grow on the surface) during bacterial corneal infection, which enhances their virulence and resistance to antibiotic. These issues highlight the urgent need for alternative effective antimicrobial treatment, ideally with anti-biofilm efficacy.

Antimicrobial peptides (AMPs) have recently shown promise as potential therapeutic agents due to their unique organisms-killing (i.e. antimicrobial) ability against a wide range of infective organisms such as bacteria, viruses, fungi and parasites. They are important components of the innate immune system that can be found in various parts of the human body, including the ocular surface. They are made up of amino acids (the basic structural units of protein) and are highly positive-charged. They exert their antimicrobial effect by disrupting the negatively charged membrane (outer surface coating) of the microorganisms, culminating in killing of these pathogens.

Over the years, we have profiled a spectrum of AMPs from the ocular surface. Notably, some of these human AMPs, including human beta-defensin (HBD)-2, HBD-3 and LL-37 were shown to have higher activity during corneal infection, highlighting their crucial roles in ocular surface defence. The AMPs also exhibit anti-biofilm, anti-cancer, and wound healing properties. However, the potential clinical utility of AMPs is currently limited by several issues such as toxicity to human cells, instability in certain body environment and the susceptibility to breakdown by human / bacterial enzymes. These have led to the innovation of newer generation AMPs, which involves substituting the amino acids within the AMPs or combining two different types of AMPs (hybrid peptide), using human and non-human AMPs sequences. These newer generation AMPs have demonstrated enhanced antimicrobial effect, better stability and lower toxicity to human tissues.

Our primary aim is to create and develop efficacious and safe human-derived hybrid AMPs for corneal infection. Recently we have developed several hybrid AMPs with potential efficacy against a variety of bacteria and we now aim to investigate and optimise the efficacy and safety of our hybrid AMPs using a series of experiments, including computer simulation, laboratory experiments with laboratory grown and donated corneal tissues, as well as using tear and blood samples from healthy volunteers. We will then validate the efficacy and safety of our designed AMPs using rationally and ethically designed animal studies, which is an essential step before we could test our AMPs in human clinical trials. These proposed experiments will be conducted collaboratively in UK and Singapore. Successful development of these AMPs could enable us to bridge the gap between fundamental laboratory research and clinical application of AMPs, ultimately benefitting patients with ocular and potentially non-ocular infections. This may also offer a potential solution to the antimicrobial resistance, which is currently emerging as a global health threat.

Corneal blindness represents the 5th leading cause of blindness globally, with bacterial keratitis (BK) being one of the leading culprits. Broad-spectrum antibiotics remain the mainstay of treatment for BK but they are affected by the emerging antimicrobial resistance (AMR), which are observed in ocular and systemic infections. Biofilm formation may also occur during BK, increasing the bacterial virulence and resistance to antibiotics. These issues highlight the urgent need for alternative effective antimicrobial treatment.

Antimicrobial peptides (AMPs) are emerging as a potential novel class of antimicrobial agent due to their broad-spectrum activity against a wide array of infection. They also exhibit anti-biofilm, anti-tumour, immunomodulatory, and wound healing properties. Newer generation AMPs such as hybrid AMPs have helped to address some of the translational issues of AMPs, including host tissue toxicity, and intolerance to physiological conditions.

We have previously profiled the spectrum of AMPs at the ocular surface. In our recent preliminary work, we have developed a few human-derived hybrid AMP with antimicrobial efficacy against a range of bacteria. We now aim to further optimise these novel hybrid AMPs for a range of ocular surface infection, including methicillin-resistant Staphylococcus aureus (MRSA), using a series of experiments. We will first perform in silico optimisation of the hybrid AMPs using established molecular dynamic simulation. We then aim to examine the in vitro efficacy and safety of the hybrid AMPs using a range of microbiological assays. This is followed by assessment of the antimicrobial and anti-biofilm activity of the AMPs using established ex vivo bacterial keratitis models. Finally we will validate the in vivo efficacy and safety of our AMPs using animal studies.

The successful development of these AMPs will offer exciting new therapeutic avenues for a range of ocular and possibly non-ocular infection in the future.

Who will benefit from this research?
Successful development of these novel hybrid peptides will benefit the population who are at risk of corneal infection, including contact lens wearers and those with increased susceptibility to trauma, amongst others. These factors are commonly associated with young people and working adults, which will have negative impact on the public and private workforce when affected.

In addition to the research scientists in the field of ocular surface infection, researchers in other disciplines - in short- to medium- term - will benefit by utilising our research findings and reproducible experimental models in other types of non-ocular infections. These include researchers in microbiology, dermatology, respiratory, urology and oral medicine, amongst others. The design and optimisation strategies employed for our AMPs could also benefit the computer scientists, chemists, and pharmacologists who are actively involved in the development of AMPs.

Our aim of developing a new class of antimicrobial agent is in line with the World Health Organisation (WHO) 'Global action plan on antimicrobial resistance (AMR)' initiative and the Joint Programming Initiative on AMR (JPI-AMR) set up by the European Countries, including the UK. This exciting innovation could potentially reinvigorate the interest of investing in newer classes of antimicrobial agents by the pharmaceutical companies in the face of emerging AMR. The use of synthetic human-derived AMPs sequence instead of non-human AMPs sequences may help overcome some regulatory hurdles. As one of the research pioneers in the field of AMPs at ocular surface, we continue to promote UK's research competitiveness in this area through our local and international collaboration with the research groups in the UK, Singapore, USA, and India.

How will they benefit from this research?
The primary aim of our research is to design and develop a novel class of antimicrobial agent - human-derived hybrid AMPs - to treat corneal infection. Corneal infection is the most frequent cause for corneal blindness worldwide. It is a serious ophthalmic emergency that often requires hospital admission for intensive antibiotic treatment in order to rapidly eradicate the infective microbes and to preserve good vision. Development of a new class of efficacious antimicrobial agent could potentially expedite patients' recovery, optimise the visual outcome and quality of life, reduce the impact on the limited hospital bed spaces / resources and minimise the impact on private and public workforces. Although uncommon, refractory corneal infection can also lead to corneal perforation, necessitating emergency corneal transplant. This places further strain on the existing shortage of the donated corneal tissues, which is currently a global issue.

More importantly, AMR is an emerging global threat to human health and a novel class of antimicrobial agent is urgently required. A recent report highlighted that AMR accounts for approximately 700,000 death every year and the death toll could potentially increase to 10 million people each year by 2050, which is more than cancer and diabetes, amongst others. It was estimated that AMR is costing US health system 20 billion USD in excess costs each year. The successful development of these novel hybrid peptides will not only foster the global economic performance, but also boost the economic competitiveness in the UK through licensing of intellectual property, drug sales, international research grants / collaboration, etc.

One of the main features of AMPs is their unique, broad-spectrum antimicrobial activity with low risk of AMR. Experimental models could be replicated in other types of ocular and non-ocular infections, including viral, fungal and parasitic, which are all notoriously difficult to treat clinically. These transferrable skills and technologies will be beneficial to all researchers in the field of AMPs and AMR.