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

角膜-位于眼睛前部的透明窗口-作为正常视力和眼表防御的关键结构。角膜损伤可能导致永久性疤痕，随后视力受损或失明。角膜盲是全球第五大致盲原因，影响世界约200万人口。细菌性角膜感染是最常见的罪魁祸首，隐形透镜佩戴和外伤是主要的危险因素。受影响的患者通常因疼痛和视力受损而虚弱，他们经常需要长期住院接受强化抗生素治疗。然而，由于在影响眼睛和身体其他部位的感染中出现的生物体对抗生素的耐药性（即抗微生物耐药性），抗生素疗效下降的问题越来越受到关注。此外，研究表明，在细菌性角膜感染过程中会形成生物膜（在表面生长的粘糊糊的微生物集合），这增强了它们的毒力和对抗生素的抗性。抗菌肽（Antimicrobial Peptides，AMP）具有独特的生物杀灭（即抗微生物）能力，能有效地杀灭细菌、病毒、真菌和寄生虫等多种感染性生物，因此近年来抗菌肽被认为是一种有潜力的治疗药物。它们是先天免疫系统的重要组成部分，可以在人体的各个部位（包括眼表）中找到。它们由氨基酸（蛋白质的基本结构单元）组成，并且带高度正电荷。它们通过破坏微生物的带负电荷的膜（外表面涂层）来发挥其抗菌作用，最终杀死这些病原体。多年来，我们已经分析了眼表面的AMP谱。值得注意的是，这些人AMP中的一些，包括人β-防御素（HBD）-2，HBD-3和LL-37，在角膜感染期间显示出更高的活性，突出了它们在眼表防御中的关键作用。AMP还表现出抗生物膜、抗癌和伤口愈合特性。然而，AMP的潜在临床应用目前受到几个问题的限制，例如对人细胞的毒性、在某些身体环境中的不稳定性以及对人/细菌酶分解的敏感性。这些导致了新一代AMP的创新，其涉及使用人和非人AMP序列取代AMP内的氨基酸或组合两种不同类型的AMP（杂合肽）。这些新一代抗菌肽已经证明了增强的抗菌效果，更好的稳定性和对人体组织的毒性更低。我们的主要目的是创造和开发有效和安全的人源性杂合AMP用于角膜感染。最近，我们开发了几种对各种细菌具有潜在功效的杂合AMP，我们现在的目标是使用一系列实验来研究和优化我们的杂合AMP的功效和安全性，包括计算机模拟，使用实验室生长和捐赠的角膜组织的实验室实验，以及使用健康志愿者的泪液和血液样本。然后，我们将使用合理和道德设计的动物研究来验证我们设计的AMP的有效性和安全性，这是我们在人体临床试验中测试我们的AMP之前的重要步骤。这些拟议的实验将在英国和新加坡合作进行。这些AMP的成功开发可以使我们能够弥合AMP的基础实验室研究和临床应用之间的差距，最终使眼部和潜在的非眼部感染患者受益。这也可能为目前正在成为全球健康威胁的抗菌素耐药性提供潜在的解决方案。

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.