Antimicrobial peptides: a promising frontier to combat antibiotic resistant pathogens

Abstract

Antimicrobial peptides (AMPs) are varied naturally occurring compounds that are crucial to the innate immune system among several organisms. These peptides are effective against various bacteria, viruses, fungus, and cancer cells. Alternative therapeutic options are becoming more important as drug-resistant diseases become a global concern nowadays. AMPs unique modes of action and benefits over traditional antibiotics make them potential candidates for improving drug-resistant disease treatment. The capacity to target microbial membranes, alter intracellular processes, and bypass resistance systems distinguishes AMPs, making it challenging to develop resistance. This review examines how AMPs can combat drug-resistant bacteria and also, emphasizes on the broad-spectrum antibacterial properties of AMPs and their many mechanisms like, AMPs can permeabilize bacterial membranes, limit biofilm formation, and alter immune responses, making them promising therapeutics for infections that defy conventional treatments. As antibiotic resistance threatens global health, AMPs offer a possible path for next-generation antimicrobials.

Introduction

A growing number of pathogenic bacteria have developed antibacterial resistance, making conventional antibiotics less effective in fighting infectious diseases^[1]. This crisis has ignited a global quest for innovative solutions, leading researchers to explore an ancient yet underappreciated defense mechanism: antimicrobial peptides (AMPs). AMPs are molecules that by their chemical nature are short peptides (chains of amino acids), usually consisting of 12–50 amino acid residues. These peptides, found in many organisms, give hope for fighting antibiotic-resistant infections^[2]. In this research, we discover AMPs distinctive features, modes of action, and potential to revolutionize infectious diseases management. Antibiotics, formerly considered medical marvels, have revolutionized contemporary medicine by treating once-fatal infections. However, antibiotic misuse and overuse have expedited the emergence of resistant strains, which today threaten human health. Antibiotic-resistant illnesses kill hundreds of thousands of people each year, with the WHO predicting 10 million deaths by 2050 if effective measures are not implemented^[3].

HIGHLIGHTS

• Potential of AMPs against antibiotic-resistant bacteria: AMPs, as naturally occurring molecules, have shown significant promise in addressing the global challenge of antibiotic-resistant bacterial pathogens. Their unique mechanisms, including the ability to permeabilize bacterial membranes and alter intracellular processes, provide a strong alternative to traditional antibiotics.

• Mechanisms of action: AMPs primarily target microbial cell membranes through electrostatic interactions and alter intracellular processes, which result in destabilization and cell death. This mechanism is effective against a broad range of bacteria, including gram-positive and gram-negative bacteria, making it difficult for pathogens to develop resistance.

• Challenges for clinical application: Despite their potential, AMPs face challenges like non-specific binding, poor solubility, susceptibility to proteases, and potential toxicity to host cells. Addressing these issues is critical to harnessing their therapeutic potential.

• Innovative delivery solutions: Advances such as structural modifications and the use of nanoparticle delivery systems have been explored to enhance AMP stability, bioavailability, and targeted delivery. These strategies aim to overcome delivery challenges and improve the clinical efficacy of AMPs.

• Future prospects: As antibiotic resistance continues to threaten global health, AMPs offer a promising direction for developing next-generation antibacterial therapies. Ongoing research and innovation in optimizing their therapeutic properties could position AMPs as vital tools in managing drug-resistant infections.

Different studies had established “AMPs” as one of the defense systems of the most living organism. From insects to plants and invertebrates, AMPs have developed over millions of years to fight bacteria, fungus, viruses, and parasites^[4]. Their abundance and diversity suggest they could yield many new therapeutic medicines. AMPs target several pathogenic components, unlike antibiotics, which target specific cellular processes from which microbial cell membrane disruption is a major process^[5]. AMPs naturally interact with negatively charged microbial membranes, leading to destabilizing, pore-forming, and cell death. Also, some AMPs can infiltrate the cell wall and disrupt protein synthesis and DNA replication. Pathogens are unlikely to acquire mutations to resist all four systems at once, making evolution of resistance more difficult^[6]. Broad spectrum activity of AMPs allows them to target many bacteria, decreasing the need for individual or targeted therapies. Besides this, their rapid activity eliminates pathogen faster, reducing treatment failure as well as AMPs target the same bacteria via different mechanism and it becomes difficult for the bacteria to develop resistance against them^[7]. Antibiotic-resistant bacteria are threatening modern medicine, prompting scientists to find new antibiotics. With their different mode of action, AMPs show great promise as a novel antibacterial resistant infection treatment. But their stability, synthesis, and toxicity remain a challenge which must be addressed before its clinical implication. A new era of infectious disease management may dawn as researchers discover the secrets and possibilities of AMPs, giving renewed hope in the fight against antibiotic-resistant bacteria. Objective of the current review is to explore the distinctive features, mechanisms of action, and therapeutic potential of AMPs in the context of antibiotic resistance. While previous studies have documented the biological functions of AMPs, this article uniquely focuses on their versatility, evolutionary advantages, and challenges in clinical translation. We highlight their ability to overcome the limitations of traditional antibiotics by targeting multiple microbial components simultaneously, making it harder for pathogens to develop resistance. Current review also highlighted the gaps in current knowledge and propose directions for future research, paving the way for their integration into clinical practice. Additionally, we discuss critical barriers to their widespread adoption, such as stability, synthesis, and toxicity, while offering insights into ongoing advancements in these areas.

Emergence of antimicrobial resistance limits treatment options

Antibiotic resistant bacteria have threatened this medical breakthrough, threatening our ability to treat infections. Drug-resistant bacteria have rapidly spread, limiting our therapeutic repertory and challenging global healthcare paradigms. We must rethink infectious disease intervention tactics. Various different survival strategies have been developed by the bacterial pathogens through different adaptive ways to counteract antibacterial substances which makes these bacterial pathogens as antibacterial resistant strains (Fig. 1) ^[8]. Antibacterial resistance has many effects, but the most important is its reduction in therapy alternatives. Food and drug safety authority (FDA) approved several antibiotics which had been commercialized for the treatment of bacterial illnesses but infection caused by multidrug-resistant bacterial strains leave patients with few treatment options which makes treatable infection into a life-threatening infection^[9]. As well as it increases the economic burden. The protracted medication development process and the shrinking pipeline of new antibiotics have caused drug production costs to exceed revenues. Pharmaceutical companies steer away from antibiotic research and development due to this economic calculus, reducing innovation in this vital area^[10]. Furthermore, the elderly, children, and immunocompromised ones are particularly affected by antibiotic resistant bacterial infections. Marginalized groups with limited healthcare access face additional problems in controlling drug-resistant illnesses, worsening global health inequities. Also, the modern medicine faces a unique dilemma as antibiotic resistance rises^[11]. The rise of drug-resistant bacteria puts existing therapies at risk of obsolescence, turning once-manageable diseases into dangerous risks. A coordinated worldwide response requires antibiotic stewardship, infection control, medication research, and international cooperation. These coordinated efforts are the only way to mitigate the impending disaster and preserve the efficacy of our vital medicinal tool “antibiotics.”

Figure 1.

Bacterial resistance mechanism.

Mechanism of action of antimicrobial peptides

One of the fascinating host-defense mechanisms is that AMPs interacts with the microbial surface. A peculiar positive cationic (Positive charge) nature makes AMPs selectively attach to microbial surfaces and specifically targets the peptidoglycan layer of gram-positive bacteria and outer membrane of gram-negative bacteria. When AMPs penetrate the cytoplasmic membrane, they also disrupt cellular processes and translocate across it, targeting microbial survival components^[12]. AMPs complex interactions have established a model of their membranolytic action, helping us to understand how they break bacterial membranes and affect cellular processes (Figs. 2 and 3).

Figure 2.

AMPs mode of action for targeting bacterial cell membrane.

Figure 3.

AMPs mode of action for targeting bacterial intracellular component.

Targeting bacterial cell membrane

AMPs primarily targets the bacterial membranes and penetrate bacterial cytoplasm to interact with internal components^[13]. A key component of bacterial cell walls is peptidoglycan. Peptidoglycan production relies on Lipid II, and a peptidoglycan layer is crucial to bacterial viability. AMPs like bacitracin and vancomycin selectively bind to lipid II which is a cell wall precursor molecule, inhibiting cell wall production^[14]. Some AMPs can also delay cell wall synthesis and damage cell wall structure. Besides negatively charged phospholipids, teichoic acids in gram-positive bacteria and lipopolysaccharides in gram-negative bacteria contribute to the bacterial surface’s electronegativity, attracting positively charged AMPs and facilitating their membrane-binding and disruption^[15]. AMPs are more selective for bacterial targets because bacterial cells have larger negative transmembrane potentials than mammalian cells. These characteristics allow AMPs to target bacteria while sparing mammalian cells from their antibacterial effects^[16].

Targeting bacterial intracellular components

The mechanism of translocation begins when proline-rich AMPs congregate on the membrane surface and interact with lipids. A toroidal gap forms in the membrane due to this contact^[17]. The hydrophobic amino acids enter the lipid bilayer and the cationic component adheres to the phosphate groups. This reaction turns the peptide vertical, creating immediate toroidal pores^[18]. Cateslytin, an AMP with plenty of Arg, binds together on the membrane and generates membrane border pores^[19]. Direct translocation processes, which depend on membrane instability, require high membrane affinity, a pH gradient, and transmembrane potential. Since AMPs include changeable regions, they can alter their functioning sequences to diverse situations. Gram-negative bacteria have robust outer membranes because lipopolysaccharides bind divalent cations like Ca²⁺ and Mg^2+[20]. These interactions briefly breach the outer membrane, allowing AMPs in. When AMPs reach the cytoplasmic membrane, they alter shape to interact with it. Their charged sections engage with phospholipid head groups, and their hydrophobic parts get stuck in the lipid bilayer center. Membrane permeabilization leaks ions and metabolites, lowers transmembrane potential, and stops membrane function. Membrane rupture kills the bacterial cell^[21].

Therapeutic applications of AMPs

AMPs are attractive antibacterial medicinal candidates due to their broad-spectrum, quick bactericidal effect and low resistance risk^[22]. Several Peptides such as Dalbavancin (semisynthetic lipoglycopeptide), Telavancin (lipoglycopeptide), Oritavancin (glycopeptide), Bacitracin (polypeptide), Colistin (Polypeptide), Polymyxin B (Polypeptide), Tyrothricin (Polypeptide), Vancomycin (glycopeptide), Gramicidin S (Polypeptide), and Gramicidin-D (Polypeptide) are FDA-approved antibacterial peptides. For instance, Dalbavancin were approved to treat complicated skin and skin-structure infections (cSSSI) by targeting gram-positive bacteria, primarily Staphylococcus aureus. Dalbavancin has a long half-life for less frequent dosage^[23,24]. Telavancin targets gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). It is widely used in case of cSSSI and hospital-acquired bacterial pneumonia^[25]. Vancomycin, which fights MRSA and other gram-positive bacteria, had been used clinically for decades. It inhibits cell wall formation and is essential for treating severe systemic infections^[26]. Gramicidin S and D, which cannot be used systemically due to hemolytic activity, but are used locally to treat superficial wounds, nasal, ocular, and throat infections^[27]. Many AMPs and peptides are in clinical trials to treat various bacterial illnesses (Table 1), and many have been FDA approved to treat diverse microbial infections (Fig. 4, Table 2).

Table 1.

Antimicrobial peptides under different phase of clinical trail

Peptide name	Therapeutic utility	Target site	Target organism	Synergistic activity reported with FDA approved antibiotics	Toxicity trait	Developmental stage	Ref
HB-50	A synthetic natural peptide mimicking cecropin, HB-50, may prevent wound infections.	Lipid bilayer	Gram-positive and gram-negative bacteria and yeast	Piperacillin, Ceftazidime, Meropenem, Clarithromycin, Chloramphenicol, Doxycycline, Rifampin, Ofloxacin, Netilmicin, Polymyxin E	Nontoxic	Preclinical	[71]
HB-107	HB-107, a 19-amino-acid cecropin B fragment, may improve wound healing.	Lipid bilayer	Parasite (Leishmania)	Not reported	Hemolytic at >100 µg/ml	Preclinical	[72]
Mersacidin	Mersacidin, a type-B lantibiotic with three methyllanthionine and S-(2-aminovinyl)-3-methylcysteine residues and four intra-chain thioether bridges, fights gram-positive infections like MRSA.	Lipid II	MRSA, VRE, Clostridium difficle	Not reported	Not reported	Preclinical	[73]
Ruminococcin C	Ruminococcin C, a Lanthionine-containing peptide bacteriocin, has antibacterial activity against pathogenic bacteria and may be useful in treating gram-positive bacterial infections.	Cell membrane	Gram-positive bacteria	Fosfomycin, Ampicillin, Amoxicillin, Imipenem, Ciprofloxacin, Vancomycin	Non-Hemolytic	Preclinical	[74]
Planosporicin	Planomonospora sp. strain DSM 14920 produces lantibiotic planosporicin. Its elongated structure has one methyl-lanthionine and four lanthionine bridges. It is highly effective against multi-drug-resistant hospital-acquired infections.	Lipid bilayer	Gram-positive and gram-negative bacteria	Not reported	Not reported	Preclinical	[75]
Colicin E1	Escherichia coli’s bacteriocin Colicin E1 targets OMPs TolC and BtuB to enter the cell. Treating gastrointestinal infections is suggested.	Cell membrane	Gram-negative bacteria	Not reported	Not reported	Preclinical	[76]
Pediocin PA-1	Pediococcus acidilactiti produces a 62-amino-acid classes IIa bacteriocin called PA1. It effectively inhibited L. monocytogens and other gram-positive bacteria.	Cytoplasmic membrane	Gram-positive bacteria	Colistin	Nontoxic	Preclinical	[77]
Nisin A	Lactococcus species produce type A (1) lantibiotic nisin A. Its 34 FDA-approved amino acids are GRAS and can be used clinically to target gram-positive and gram-negative bacteria.	Lipid bilayer	Gram-positive and gram-negative bacteria	Colistin, Ceftazidime, Tobramycin, Ciprofloxacin, Doripenem	6.58 ± 1.87% Hemolysis at 4000 µg/ml	Preclinical	[78]
Temporin A	European red frog skin contains hydrophobic AMP Temporin A. It inhibited Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans.	Lipid bilayer	Gram-positive and gram-negative bacteria, yeast virus, parasite, cancer, fungus	Gentamicin	28% Hemolysis at 40 µM	Preclinical	[79]
Plectasin	It fights pneumococcal and streptococcal infections as a fungal defensin.	Lipid II	Gram-positive and gram-negative bacteria and fungus	Vancomycin, Ampicillin, Chloramphenicol, Gentamicin, Rifampicin, Tetracycline	5% hemolysis at 125 µg/ml	Phase I	[80]
Friulimicin B	A natural antibiotic from Actinoplanes friuliensis treated pneumonia and staphylococcal skin infections.	Cell wall	Gram-positive bacteria	Not reported	Not reported	Phase I	[81]
Human lactoferrin 1-11	Bacteremia and fungal diseases in immunocompromised HSC transplant patients	Inhibition of bacterial growth by iron scavenging	Gram-positive and gram-negative bacteria	Gentamicin, tigecycline, rifampicin, clindamycin and clarithromycin	No toxicity up to 10 mg/kg	Phase I	[82]
LL-37 (Cathelicidin)	Developed from human neutrophils and epithelial cells. Effective antibacterial.	Membrane disruption	Gram-positive and gram-negative bacteria	Nafcillin, meropenem, colistin, teicoplanin, Vancomycin, tigecycline, gentamycin, azithromycin	No toxicity up to 100 μg /kg Toxicity observed at high dose (3000 μg/kg) in rats	Phase I	[83]
LTX-109	Topical broad-spectrum, fast-acting bactericidal antibacterial LTX-109 disrupts membranes and lyses cells. It treats simple gram-positive skin infections, impetigo, and S. aureus nasal colonization.	Membrane disruption	Gram-positive bacteria	Not reported	Toxicity not observed in topical application of 5% LTX-109 in rats	Phase I	[84]
Histatin	Saliva contains naturally occurring cationic peptides that treat oral candidiasis.	Targets and inhibits mitochondrion activity	Candida species	Not reported	Non toxic	Phase II	[13]
Pac113	To treat oral candidiasis in HIV-positive individuals, it uses the active portion of histatin 5 protein found in saliva.	Target mitochondria & causes generation of reactive oxygen species	C. Albicans, C. Glabrate, C. Parapsilosis & C. Tropicalis	Not reported	Non toxic	Phase II	[85]
Czen-002 (Melantropin)	This unique, non-azole anti-fungal synthetic octapeptide is produced from alpha-melanocyte-stimulating hormone (a-msh). It effectively fought candidiasis.	Interfere cAMP induction	Candida albicans	Not reported	Non toxic	Phase II	[86]
Nvxt (novexatin, np213)	Cyclic cationic peptide produced from novabiotics arginine peptide treated onychomycosis effectively.	Cell wall	Dermatophytes	Not reported	Non toxic	Phase II	[86]
Pmx-30 063 (brilacidin)	A non-peptide, small molecule/copolymer defensin structural mimic. It effectively treated staphylococcus-related acute bacterial skin infections.	Acts primarily on the bacterial cell membrane by depolarization	Gram-positive and gram-negative bacteria and yeast	Not reported	Dose-dependent ocular toxicity reported	Phase II	[12]
Ghrelin	Chronic respiratory infection, cystic fibrosis, cancer, endogenous host-defense peptide, synthetic airway inflammation	Ghrelin quenches the negative surface charge influenced by charge-dependent binding	Escherichia coli & Pseudomonas aeruginosa	Not reported	Non toxic	Phase II	[87]
Sifuvirtide	It is designed based on the 3d structure of the hiv-1 gp41 fusogenic core conformation	HIV fusion inhibitor	HIV	Not reported	Cytotoxicity reported against mammalian cells at 406 μm concentration	Phase II	[88]
Dpk-060	Kininogen, cationic random-coil peptide, is its precursor. It effectively treated atopic dermatitis and otitis externa.	Membrane disruption	Gram-positive and gram-negative bacteria	Not reported	Non toxic	Phase II	[89]
Reltecimod (AB103)	The CD28 T-cell receptor peptide reltecimod was previously known as AB103 or p2TA. It significantly modulates the host immune response, especially in necrotizing soft tissue infections. Inducing pro-inflammatory cytokines through its interaction with the CD28/B7-2 co-stimulatory pathway is its main mechanism. Reltecimod targets and reduces this route, not inhibits it. Notably, reltecimod is antimicrobial-free. Instead, it reduces infectious pathology’s excessive and damaging inflammatory cytokine response. It improves the host’s ability to fight infections, minimizing tissue damage to essential organs during acute inflammation.	Reltecimod modulates the inflammatory response by targeting and attenuating the critical CD28/B7-2 co-stimulatory pathway without inhibiting it.	Reltecimod does not have antimicrobial activity, and by targeting CD28, it prevents the excessive harmful inflammatory cytokine response underlying infection pathology; it significantly improves the host’s ability to respond to infection efficiently and, consequently, reduces the tissue damage caused to key organs during acute inflammation	Not reported	Not Reported	Phase III	[90]
Ramoplanin (NTI-851)	A macrocyclic glycolipodepsipeptide generated by Actinoplanes spp. VRE/Clostridium difficile oral therapy.	Disrupt cell wall biosynthesis	Staphylococcus aureus, Staphylococcus epidermidis, streptococci, vancomycin-resistant Enterococci, Bacillus spp., Listeria monocytogenes & Clostridium difficile	Not reported	Nontoxic	Phase III	[91]
Demegal (D2A21)	An α-helix peptide with 22 residues. Burn, multidrug-resistant pathogen skin infection	Gram-positive and gram-negative bacteria	Pseudomonas, Chlamydia trachomatis	Not reported	Not reported	Phase III	[92]
Glutoxin (NOV-002)	Stabilized disodium glutathione disulfide and cisplatin create glutoxin. It boosts immunity to halt tumor growth and fights TB.		Mycobacterium tuberculosis & lung cancer	Not reported	Not reported	Phase III	[93]
Murepavadin (POL7080)	Substituting amino acids from protegrin I produces it. The antibacterial effect involves binding the outer membrane protein LptD, which is implicated in lipopolysaccharide synthesis in gram-negative bacteria, causing ventilator-associated bacterial pneumonia (VABP)	Lipopolysaccharide transport protein D	Pseudomonas aeruginosa	Not Reported	Nontoxic up to 766 mg/day	Phase III	[94]
Peceleganan (PL-5)	Drug-resistant microorganisms in skin and wound infections are targeted.		Gram-positive and gram-negative bacteria	Levofloxacin	Low toxic	Phase III	[95]
Surotomycin (MK-4261/CB-183,315)	Surotomycin, a benzenebutanoic acid derivative, may treat diarrhea and Clostridium Difficile infection.	Disrupt cytoplasmic membrane	Clostridium Difficile	Not reported	Cytotoxic and Hemolytic	Phase III	[96]
TD-1792 (Cefilavancin)	The covalently linked heterodimer glycopeptide-cephalosporin (beta-lactam) cefilavancin kills cells by targeting D-Ala-D-Ala-containing peptidoglycan precursors and penicillin-binding protein’s active site (Gram positive)	Peptidoglycan precursors (D-Ala-D-Ala)	Gram-positive	Not reported	No toxicity up to 2 mg/kg	Phase III	[97]

Figure 4.

Clinical potential of AMPs.

Table 2.

Commercialized FDA approved peptides for therapeutic utility

Antimicrobial peptide	Therapeutic utility	Target site	Target organism	Resistance reported (if any and its mechanism)	Ref
Daptomycin	Daptomycin, a cyclic lipopeptide antibiotic, can treat cSSSI and bloodstream infections caused by Staphylococcus aureus.	Cell membrane (Calcium dependent binding with anionic phospholipid phosphatidylglycerol)	Gram-positive cocci	Resistance by MprF protein synthesis, increase of cell-surface charge by overexpression of the dlt opero	[98]
Dalbavancin	Lipoglycopeptide dalbavancin is derived from teicoplanin. It treats ABSSSI, osteomyelitis, and septic arthritis caused by gram-positive bacteria.	Disrupts bacterial cell wall formation by binding to the terminal d-alanyl-d-alanine peptidoglycan	Gram-positive cocci	Resistance is mediated through a plasmid gene VanA or VanB	[99]
Telavancin	Telavancin is a semi-synthetic derivative of vancomycin that is used to treat osteomyelitis and other infections caused by MRSA.	It inhibits peptidoglycan synthesis by binding to late-stage peptidoglycan precursors (acyl-D-alanyl-D-alanine)	Gram-positive cocci	Resistance is mediated through a plasmid gene VanA or VanB	[100]
Oritavancin	Oritavancin is a glycopeptide antibiotic that used to treat ABSSSI.	It binds to the stem peptide of peptidoglycan precursors, inhibiting trans-glycosylation, inhibiting transpeptidation, and cell membrane interaction/disruption.	Gram-positive cocci	Resistance is mediated through a plasmid gene VanA or VanB	[100]
Colistin	Colistin, an eight-amino-acid polypeptide, targets cell membranes. The medicine treats infections caused by multidrug-resistant gram-negative bacteria.	Polymyxin electrostatically bind to the negatively charged lipopolysaccharide and disorganize bacterial cell osmotic equilibrium	Gram-negative bacilli	Resistance is mediated through a plasmid gene mcr 1-9	[101]
Tyrothricin	The polypeptides from Bacillus brevis contain tyrocidines and gramicidin. Due to harmful effects on blood, liver, kidneys, meninges, and olfactory system, it is used as an ointment to treat skin infections caused by gram-positive bacteria and some fungal diseases.	The tyrocidine dimer is able to disrupt the cell membrane producing leakage of cell contents.	Gram-positive cocci	Not reported	[102]
Vancomycin	Vancomycin, a glycopeptide antibiotic, treats MRSA infections such as septicaemia, infective endocarditis, skin, bone, and respiratory tract infections.	Vancomycin binds to the acyl-D-ala-D-ala portion of the growing peptidoglycan cell wall	Gram-positive cocci	Resistance is mediated through vanA gene	[103]
Gramicidin S	This cyclic peptide from Bacillus brevis contains two identical pentapeptides. It is effective against gram-negative and gram-positive bacteria and fungus without harming sperm. Can cure STD-related genital ulcers.	Delocalizes peripheral membrane proteins involved in cell division	Gram-positive and gram-negative	Not reported	[102]
Gramicidin D	Gramicidin D contains Bacillus brevis’s gramicidin A, B, and C. For skin sores, wounds, and eye infections, Polysporin ophthalmic solution and topical treatment destroy most gram-positive and some gram-negative bacteria.	Gramicidin D binds to and inserts itself into bacterial membranes which results in membrane disruption and permeabilization ultimately leads to loss of intracellular solutes, dissipation of the transmembrane potential, inhibition of respiration, a reduction in ATP pools and inhibition of DNA, RNA, and protein synthesis, causing cell death.	Gram-positive and gram-negative bacteria	Not reported	[104]
Enfuvirtide & Fuzeon	Enfuvirtide is a 36-amino acid biomimetic peptide that mimics HIV proteins that adhere to cell membranes and enter cells. HIV-fusion inhibitors like enfuvirtide are novel antiretrovirals.	It binds with CD4 cells and inhibits HIV spreads	Human immunodeficiency virus	Not reported	[105]

Challenges to utilize AMPs as a therapeutic agent

Non-specific binding of AMPs

Collagen-based biomaterials loaded with AMPs present a promising approach for promoting wound healing while providing protection against infections. In our previous work, we modified the AMP LL37 by incorporating a collagen-binding domain (cCBD) as an anchoring unit for collagen-based wound dressings. We demonstrated that cCBD-modified LL37 (cCBD-LL37) exhibited improved retention on collagen after washing with PBS. However, the binding mechanism of cCBD-LL37 to collagen remained to be elucidated. In this study, we found that cCBD-LL37 showed a slightly higher affinity for collagen compared to LL37. Our results indicated that cCBD inhibited cCBD-LL37 binding to collagen but did not fully eliminate the binding. This suggests that cCBD-LL37 binding to collagen may involve more than just one-site-specific binding through the collagen-binding domain, with non-specific interactions also playing a role. Electrostatic studies revealed that both LL37 and cCBD-LL37 interact with collagen via long-range electrostatic forces, initiating low-affinity binding those transitions to close-range or hydrophobic interactions. Circular dichroism analysis showed that cCBD-LL37 exhibited enhanced structural stability compared to LL37 under varying ionic strengths and pH conditions, implying potential improvements in antimicrobial activity. Moreover, we demonstrated that the release of LL37 and cCBD-LL37 into the surrounding medium was influenced by the electrostatic environment, but cCBD could enhance the retention of peptide on collagen scaffolds. Collectively, these results provide important insights into cCBD-modified AMP-binding mechanisms and suggest that the addition of cCBD may enhance peptide structural stability and retention under varying electrostatic conditions^[28]. In addition, AMPs modulate immune cell activity to regulate the immunological response. However, immune cell binding without specificity can have unforeseen consequences. When AMPs randomly connect to immune cells, they may cause harmful immunological responses. Unspecific binding of cathelicidin-related antimicrobial peptide to macrophages can trigger pro-inflammatory pathways^[29]. Additionally, LL-37 binds to mucus layer glycosaminoglycan, limiting its respiratory tract penetration and respiratory infection treatment^[30].

The solubility challenge

Drug’s bioavailability and efficacy depend on solubility, including AMPs. Poor solubility can cause aggregation, bioavailability, and formulation issues, making AMPs based treatments challenging. Many AMPs are poorly soluble in water, limiting medication formulation choices^[31]. For instance, AMP gramicidin S has high antibacterial action but low water solubility. This limits its injectable therapeutic usage and requires other formulations that may not be as easy or practical for clinical use^[32]. As well, poorly soluble AMPs tend to aggregate, which can change their bioactivity and cause toxicity. Aggregated AMPs may lose structure and function, making them less potent against infections. AMPs aggregates may generate an immunological response or cytotoxic consequences in host cells. The solution-bound AMP protegrin-1 can agglomerate, compromising its safety and therapeutic efficacy^[33].

The concept of cross-resistance

Cross-resistance arises when bacteria develop resistance mechanisms against one AMPs and can resist some others with similar modes of action or structures. This problem calls into question the concept that AMPs could replace antibiotics because resistance to one can render many ineffective^[34]. Certain bacteria have efflux pumps that actively remove AMPs or other peptides from their cytoplasmic membranes. For instance, Pseudomonas aeruginosa can withstand polymyxin B and colistin, another clinically utilized peptides^[35]. Many AMPs or peptides interact with bacterial cell membranes electrostatically due to their charge. Changes in membrane features like surface charge or membrane-protective chemical synthesis can help bacteria adapt. This modification may permit cross-resistance to similar-charged AMPs. For example, LL-37 resistance can give resistance to other cationic AMPs^[36]. Cross resistance also occurs when AMPs share structural homology or motifs. For instance, cross-resistance shown by Staphylococcus aureus’ resistance to human AMPs lactoferrin-derived peptides^[37].

Immunogenicity of AMPs

Immunogenicity is a substance’s ability to induce an immunological response in the host. AMPs may cause allergy responses due to their immunogenicity. Some AMPs share structural similarities with allergenic proteins or other antigens, causing vulnerable people to produce particular antibodies^[38]. Due to its immunogenic qualities, AMPs dermcidin can induce allergic contact dermatitis in some people^[39]. Another example is LL-37, an immune system modulator, can cause inflammation at high doses. Immune activation can cause tissue inflammation or fever, limiting LL-37’s therapeutic usage. Immunogenicity may also be affected by AMPs exposure frequency and duration^[40].

The challenge of AMP delivery

AMPs, like any drug, need effective delivery. Their size, charge and hydrophobicity can make them accessing target bacterial cells or affected tissues difficult. Some AMPs are too big to get through biological barriers or tissues^[41]. Cationic AMPs like human beta-defensin 3 are essential for mucosal defense. Large size can hinder its capacity to penetrate the mucus layer in the respiratory or gastrointestinal systems, where bacterial infections arise. This lower penetration may reduce antibacterial efficacy, especially at high dosages to fight resistant pathogens^[42]. AMPs charges can influence their interaction with host proteins, reducing bioavailability and delivery. Crossing membranes is difficult for intracellular bacterial infection-targeting AMPs. Some AMPs may have trouble crossing host cell lipid bilayers and targeting intracellular bacteria, like Mycobacterium tuberculosis resists numerous AMPs due to its unusual cell envelope structure, which prevents AMPs from reaching the bacterial cytoplasm^[43].

Susceptibility to proteases

Multiple potential AMPs are in development, but they often struggle with protease susceptibility. Omiganan, a synthetic cationic peptide, has been developed preclinically and clinically to fight infections, including antibiotic-resistant bacteria. Despite its antibacterial potential, omiganan is susceptible to protease breakdown in vivo. This susceptibility can lower bioavailability and therapeutic efficacy^[44]. Pexiganan, a synthetic magainin analogue from amphibians, began clinical trials to treat diabetic foot ulcers topically. It showed promise in wound conditions, but protease degradation is its downfall. This constraint made clinical use difficult, raising questions regarding its efficacy^[45]. Novexatin, an antifungal AMP, gained attention^[46]. It was protease-sensitive, which could reduce its therapeutic efficacy. To maximize Novexatin’s fungal infection treatment, this challenge must be overcome.

Antimicrobial peptide toxicity

Several AMPs have emerged as promising broad-spectrum agents that could serve as effective alternatives to antibiotics, especially in cases where resistance to conventional treatments has developed. Their intrinsic cytotoxicity to host cells limits their clinical value. Due to their amphipathic properties, AMPs can target microbial and eukaryotic cell membranes, causing cytotoxicity. For example, Insect-derived Cecropin A has high antibacterial action but harms host cells^[47]. Despite their antibacterial properties, AMPs including magainin-2 (from Xenopus frogs), dermicidin (from sweat glands), and buforin II (from Asian toads) struggle to eliminate pathogens and preserve host cells^[48]. Synthetic AMPs like Pexiganan (MSI-78) were promising topical therapies for diabetic foot ulcers, but cytotoxicity issues limited them^[49]. In therapeutic trials, synthetic defensin-mimetic peptide Brilacidin (PMX-30063) has shown promise against acute bacterial skin and skin structure infections (ABSSSIs). However, cytotoxicity concerns remain, highlighting the persistent issue of therapeutic safety optimization^[50]. Omiganan (CLS001), another clinical candidate, faces protease susceptibility and cytotoxicity, requiring novel clinical applicability procedures^[51]. Porcine protegrin-1-derived MX-2401 (Calcitermin) is being tested for chronic wounds and diabetic foot ulcer, however its cytotoxicity requires careful examination^[52]. Finally, Talactoferrin, an immunomodulatory protein with antibacterial characteristics evaluated in clinical trials for respiratory infections, deserves further evaluation for host cell toxicity. Researchers are still trying to reduce AMPs cytotoxicity while keeping their powerful antibacterial properties to maximize their medicinal potential^[53].

Bioavailability challenges

AMPs face bioavailability issues that hinder their clinical development as antibiotic alternatives. Candidates in clinical trials demonstrate the complexity of this issue as AMPs therapeutic potential and target site accessibility interact. Pexiganan (MSI-78), a synthetic AMP, may treat diabetic foot ulcers but has low topical bioavailability^[54]. The sensitivity of Omiganan (CLS001) to protease degradation affects its bioavailability during preclinical and clinical development, affecting its therapeutic efficacy^[55]. Iseganan (IB-367), an oral and topical drug, has low oral bioavailability, reducing its therapeutic efficacy^[56]. Novexatin (NP213/MSI-1436), an antifungal, has bioavailability issues that limit its efficacy^[46]. LTX-109, tested for skin infections, has bioavailability difficulties that limit its systemic potential. MX-2401 (Calcitermin) may treat chronic wounds and diabetic foot ulcers, however bioavailability concerns require new approaches^[57]. Brilacidin (PMX-30063), a synthetic defensin-mimetic peptide in clinical trials, has bioavailability issues that require improvement. These examples demonstrate the delicate balance between AMPs therapeutic potential and target site delivery. Researchers are investigating formulation upgrades, innovative drug delivery systems, and structural alterations to overcome bioavailability issues and release AMPs full therapeutic potential as a new generation of antimicrobial medicines^[50].

Strategies to enhance AMPs utilization in clinical practice

Structural modifications of AMPs

Synthesizing amino acid substitution, stapling, cyclization, or hybrid AMPs improves stability, specificity, and bioavailability, boosting antibacterial action and minimizing drawbacks. Amino acid substitutions improve peptide characteristics by replacing certain amino acids^[58]. D-amino acids have been added to synthetic AMPs such magainin and dermaseptin from frog skin. This change makes them protease-resistant while preserving antibacterial action. For proteolytic enzyme specificity or resistance, amino acid substitutions can include non-natural amino acids. Stapling creates a covalent “staple” between peptide amino acids. The peptide is less degradable and more effective in penetrating bacterial cell membranes after this alteration^[59]. The AMPs protegrin-1-derived stapled peptide SAAP-148 is an example. Stapling makes it more stable and powerful antibacterial by resisting proteolysis. The peptide sequence forms a closed ring during cyclization. This change improves stability and enzymatic resistance. Gramicidin S, a cyclic peptide, has better antibacterial action and stability than its linear equivalent, making it a good template for stronger cyclic AMPs. AMPs sequences are combined with antibiotic or peptide sequences to form hybrid AMPs. This technique combines the strengths of both components to boost antibacterial action and reduce resistance. The hybrid peptide cecropin A-melittin combines sequences from two AMPs, creating a synergistic effect that boosts its antimicrobial action against several bacterial species^[60,61].

Nanoparticle delivery systems for AMPs

Natural compounds called AMPs destroy bacteria, fungi, and viruses. The innate immune system relies on them since they can kill many infections. Their stability, half-life, and delivery to the proper spot have limited their clinic use. Researchers have developed nanoparticle delivery systems like liposomes to place AMPs inside and transport them where they need to go to avoid these issues^[13]. Many free AMPs have a limited blood half-life, making them ineffective drugs. A nanoparticle-delivered AMP has a longer half-life. In nanoparticles or liposomes, AMPs are protected from early immune system destruction. Therapeutic benefits stay longer. This approach makes the medicine more stable, extends its half-life, provides simpler delivery to infection sites, and reduces tissue damage. Nanoparticle delivery methods address biomedical applications’ AMP delivery problems^[62,63]. For instance, liposomal AMP LL-37 has improved stability and antibacterial action. PLGA-based biodegradable polymeric nanoparticles support prolonged release, increasing AMP pharmacokinetics^[64]. AMPs are protected and therapeutically enhanced by AuNPs and AgNPs. Antibacterial activity of AuNPs functionalized with AMP cecropin A is improved^[65]. Iron oxide magnetic nanoparticles can be targeted using magnetic fields. AMPs are released controlled by dendrimers, highly branched macromolecules^[66]. Self-assembling micelles from amphiphilic compounds boost AMP solubility and stability with nano-sized carriers. Other examples include functionalized carbon nanotubes that carry AMPs with high surface area loading and prolonged release^[67]. Encapsulating AMPs in biocompatible chitosan nanoparticles from a natural polysaccharide allow prolonged release. Nanogels, three-dimensional crosslinked polymer networks, control AMP release and prolong therapeutic benefits^[68]. These nanoparticle delivery technologies demonstrate novel ways to boost AMPs antibacterial efficacy.

Limiting toxicity

AMPs are varied molecules with considerable potential as innovative antibacterial medicines. Clinical applications have grown due to their capacity to target bacteria, fungi, and viruses. Their inherent host cell toxicity has hindered their development into useful medications. Thus, it is crucial to find ways to reduce AMPs host cell damage while maintaining their antibacterial activity^[69]. AMPs hydrophobic amino acid residues like phenylalanine and tryptophan cause cytotoxicity. By substituting hydrophobic residues with less water-repellent amino acids or adding basic groups, researchers have reduced their cytotoxicity. Modified AMP versions are less toxic to host cells and do not damage red blood cells. AMP sequences can now contain non-canonical amino acids thanks to solid-phase peptide synthesis, lowering cell toxicity. For instance, adding thiazole and octahydroindole amino acids to Magainin-2 reduced hemolysis and toxicity^[70]. The wider consequences of these alterations on proteolytic cleavage and immunotoxicity need additional study. AMPs detrimental effects can be reduced by modifying their C-termini. Reduced cell membrane damage, increased antibacterial activity, and longer serum half-life were seen in AMPs containing thiolated or amidated C-termini^[59]. Researchers have altered bigger AMPs to reduce cytotoxicity while keeping antibacterial effects. Modified AMPs pharmacological characteristics must be rigorously assessed. Polyethylene glycol and non-hazardous acyl chains can improve therapeutic peptide safety and reduce in vivo toxicity. However, their effect on antibiotic efficacy must be considered. Host cell toxicity must be managed to advance AMP-based treatments. Changing longer peptide segments or removing hydrophobic residues may reduce cytotoxicity without affecting pathogen effectiveness.

Conclusion

AMPs hold significant potential as alternative therapies to combat microbial infections, particularly in the face of rising antibiotic resistance that highlights the limitations of conventional treatments. The broad-spectrum activity of AMPs and their ability to target diverse pathogens make them a versatile tool in fighting infections. However, translating AMPs into effective clinical therapies is a formidable challenge due to issues like nonspecific binding, low solubility, cross-resistance, immunogenicity, susceptibility to proteases, toxicity, and bioavailability. Addressing these challenges requires innovative and interdisciplinary approaches. Structural modifications of AMPs, such as the incorporation of non-natural amino acids or sequence alterations, can enhance their selectivity and stability while reducing toxicity. Advanced delivery systems, such as nanoparticle-based platforms, offer promising solutions for improving solubility, stability, and targeted delivery of AMPs. Additionally, conjugating AMPs with adjuvants or other therapeutic agents could enhance their efficacy and minimize resistance development. Rigorous research is needed to optimize AMP design, focusing on reducing immunogenicity and improving protease resistance while ensuring that these peptides retain their antimicrobial potency. Long-term studies and clinical trials are essential to evaluate their safety, efficacy, and performance in diverse patient populations. Furthermore, exploring synergistic combinations of AMPs with existing antibiotics or other antimicrobial agents may provide new avenues for overcoming resistance. By overcoming these obstacles through creative solutions and a deeper understanding of AMP mechanisms, these peptides could emerge as transformative agents in the fight against infectious diseases. This advancement would not only address the global challenge of antimicrobial resistance but also significantly improve public health outcomes worldwide.

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Author’s contribution

S.S., A.B.: initial draft & pictorial representations; N.K., S.A.: conceptualisation, manuscript review and curation; M.H., S.A.M.: data collection, analysis, and manuscript writing; D.M., S.C.: manuscript review and revised the final manuscript.

Conflicts of interest disclosure

Authors declare no conflict of interest.

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Guarantor

Shahbaz Aman and Ayesha Bibi.

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