Analytical Quality by Design approach in the development of a green reversed-phase ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry method for the simultaneous analysis of synthetic antimicrobial and hypotensive peptides

Antimicrobial peptides in malaria and tuberculosis management: a systematic review of emerging evidence.

Antimicrobial Peptides (AMPs) are a diverse class of host-defence molecules that respond to microbial invasion and challenge. Owing to their broad spectrum of antimicrobial activity and the low rates of induced resistance resulting from the co-evolution of pathogens with host AMPs, these peptides play a crucial immunomodulatory role against microbes. This, in turn, contributes to effective defence against oral infections such as dental caries and periodontal diseases. Numerous studies and existing literature suggest that AMPs have a promising future in controlling the formation and growth of biofilms by oral pathogenic microorganisms, thereby helping to reduce the incidence and prevalence of dental caries, which commonly affects children, as well as other oral diseases. Consequently, researchers are actively developing synthetic AMPs with improved stability and biocompatibility that could potentially replace conventional antimicrobial therapies. The scope of the present review is to summarise AMPs, including their origin, structural characteristics, mechanisms of action and recent advances in their application for combating various oral diseases.

Background/Objectives: Antimicrobial peptides (AMPs) represent a promising class of therapeutics with diverse biological functions, including antibacterial, anti-fungal, anti-parasitic and anti-tumoral activities. Previous works demonstrated the successful repurposing of the two synthetic AMPs 19-2.5 and 19-4LF for cutaneous leishmaniasis, when the compounds were administered in solution on skin lesions caused by Leishmania major in a BALB/c mouse model. In this research project, we assessed the activity of 19-4LF, 19-2.5, and their hybrid 19-2.5LF derivative when formulated as a cream for topical administration in the same animal model. Methods: The peptides were formulated in DAC cream and applied to the wound of BALB/C mice for 30 days. Lesion progression was monitored using a digital caliper. Parasite burden was measured by qPCR. Parasite viability was assessed using MTT and microscopy imaging assays. Results: The three peptides in cream formulation succeeded in reducing the skin lesion. Peptide 19-4LF was the most potent, followed by 19-2.5LF and then 19-2.5. In addition, 19-4LF was able to significantly reduce the parasite burden in the skin lesions of infected mice, as measured by quantifying L. major Lm18S ribosomal gene mRNA levels using qPCR. Moreover, when combined, the peptides exhibited synergistic effects on L. major promastigotes and significantly reduced the number of amastigotes in infected macrophages. Conclusions: These studies support the approach of repurposing these AMPs as antileishmanial drugs and identify 19-4LF as a lead candidate for further studies. While historical barriers to peptide therapeutics included high production costs, recent advancements in biological fermentation and synthesis strategies have significantly improved their economic viability. Furthermore, the use of nanotechnology delivery systems can reduce the required dosage, further making peptide therapy a sustainable option for neglected diseases, including leishmaniasis.

Chagas disease is a neglected disease caused by Trypanosoma cruzi, which affects 6-7 million people worldwide. More than one hundred years after its description, the performance of available drugs for treating the T. cruzi infection remains largely unsatisfactory. Antimicrobial peptides (AMPs) are new alternatives that may have potential as trypanocides. Herein, we assessed Cm-p5, a synthetic peptide with previously shown antimicrobial activity, and 10 derivatives. After screening assays using epimastigote forms of the parasite to test their potential as proliferation inhibitors, Cm-p5 was selected. Cm-p5 showed an EC50 against T. cruzi of 16.9 ± 1.2 μM and a cytotoxicity towards CHO-K1 mammalian cells (CC50) of 124.8 ± 0.1 µM. After further investigation, it was evidenced that part of the epimastigote population underwent necrosis-like cell death, while those that remained alive showed a cell-cycle arrest at the phases G2_M and S_G2. When infected cells were treated, the peptide diminished the release of the infective trypomastigote form, with an EC50 of 25.2 ± 1.4 µM. Furthermore, Cm-p5 inhibited the number of intracellular amastigotes as well as the number of infected cells by 64.3 and 75%, respectively. Taken together, these numbers resulted in a reduction of the infection index by 91.1%. Additionally, we showed that Cm-p5 trypanocidal activity against intracellular amastigotes was attributable to cell membrane damage and cell cycle partial arrest, as described for epimastigotes. Our data suggest that Cm-p5 may be a promising template to design new peptides for the treatment of Chagas disease.

As we face the threat from bacterial pathogens that are resistant to many conventional antibiotics, many current research efforts focus on expanding our arsenal of antimicrobial compounds. However, identifying use cases in which such new antimicrobials can effectively target pathogens while minimizing collateral damage in the commensal microbiota remains a challenge. To tackle this challenge, we focused on one new antimicrobial, the synthetic antimicrobial peptide SET-M33, and examined its ability to target porcine respiratory pathogens and a collection of porcine commensal nasal microbiota members in vitro. Our experiments revealed three key results. First, there were large differences in SET-M33 sensitivity across the tested strains. In particular, pathogenic Glaesserella parasuis was highly sensitive to SET-M33 at concentrations that did not affect the growth of most commensal strains. Second, some of the tested commensal strains (Rothia nasimurium and Staphylococcus aureus) were able to inactivate SET-M33 during in vitro cultivation. Third, despite this potential for SET-M33 inactivation by commensal strains, SET-M33 was still able to selectively eliminate pathogenic G. parasuis from in vitro co-cultures that also contained R. nasimurium. Overall, this study highlights the substantial complexity that emerges from the interplay between antimicrobials, pathogens, and commensals, even within a comparatively simple in vitro system, and provides a template for identifying suitable use cases for newly developed antimicrobials.IMPORTANCEAntimicrobial resistance in pathogenic bacteria is a major global challenge. To tackle it, antimicrobial peptides (AMPs) have emerged as particularly promising candidates, and recent years have seen a rapid expansion of the available AMP arsenal. However, identifying good "use cases" for such AMPs-that is, scenarios in which an AMP not only inhibits a pathogen of interest but also avoids disrupting the commensal microbiota-remains challenging. Here, we aimed to develop an experimental framework that enables the rapid detection of such use cases in vitro. As a test case, we focused on the synthetic AMP SET-M33 and examined its effect on several respiratory porcine pathogens and a collection of commensal nasal microbiota strains. We found that even within such a comparatively simple in vitro model system, there is substantial complexity that emerges from the interplay among AMPs, pathogens, and commensals.

The global rise in antimicrobial resistance among the ESKAPE pathogens represents a major challenge to public health. Here, we report the broad-spectrum antibacterial activity of the synthetic antimicrobial and pore-forming peptide C14R against all six ESKAPE species. Using a radial diffusion assay and resazurin-based viability testing, C14R exhibited a potent bactericidal effect with minimum inhibitory concentrations (MICs), defined as the lowest concentration of an antimicrobial agent that completely inhibits visible growth of planktonic microorganisms, ranging from 3.4 µg/mL (Enterococcus faecium, vancomycin-resistant) to 45.2 µg/mL (Klebsiella quasipneumoniae, ESBL). C14R also inhibited biofilm formation by Gram-positive pathogens, with minimum biofilm inhibitory concentrations (MBICs), referring to the minimal concentration required to prevent the development of biofilms, of 15.0 µg/mL (Staphylococcus aureus, MRSA) and 22.0 µg/mL (E. faecium, VRE), whereas Gram-negative biofilms showed higher tolerance. Together, these findings demonstrate that C14R retains high activity against multidrug-resistant ESKAPE strains, highlighting its potential as a lead compound for the development of next-generation antimicrobial drugs to expand the portfolio of available antibiotics and brace health systems against emerging severe infections.

Chronic wounds and implanted medical devices remain highly vulnerable to biofilm-associated infections, which resist conventional antibiotics and immune clearance. Synthetic antimicrobial peptides (AMPs) have emerged as promising alternatives, offering tunable sequences, short lengths for cost-effective synthesis, and functional modifications that enhance stability and antibiofilm potency. Hydrogels provide an optimal delivery matrix by enabling localized AMP release, maintaining a moist wound environment, and supporting stimuli-responsive or sustained therapeutic action. This review highlights recent advances in peptide engineering strategies-including rational sequence design, chemical modifications, and self-assembling nanostructures-alongside hydrogel integration approaches ranging from physical entrapment to covalent tethering and infection-triggered release systems. Mechanistic insights into antibiofilm activity are discussed, supported by in vitro, ex vivo, and in vivo evaluation models. Beyond antimicrobial efficacy, multifunctional AMP-hydrogel systems can deliver complementary benefits such as hemostasis, anti-inflammation, or enzymatic biofilm dispersal, further accelerating tissue repair. Despite significant progress, translational challenges remain, including peptide stability, manufacturing costs, regulatory hurdles, and host safety. Future directions point toward AI-driven peptide design, programmable hydrogels, and point-of-care integration to realize safe, effective, and multifunctional AMP-hydrogel therapies for chronic wound management and biofilm eradication.

ABSTRACTBackground and Aim:Bacterial contamination during liquid storage of boar semen negatively affects sperm quality and fertility outcomes, necessitating the routine use of antibiotics in semen extenders. However, increasing concerns regarding antimicrobial resistance have encouraged the development of alternative antimicrobial strategies. Synthetic antimicrobial peptides (AMPs) have demonstrated broad-spectrum antibacterial activity and may serve as potential substitutes or adjuncts to conventional antibiotics. This study aimed to evaluate the antimicrobial efficacy of the synthetic peptide PA-13 alone and in combination with reduced gentamicin concentrations against Escherichia coli isolated from boar semen, as well as its effects on semen quality during storage at 18°C.Materials and Methods:Two experiments were conducted. In Experiment I, fresh semen samples collected from seven healthy adult boars were diluted in Beltsville Thawing Solution supplemented with gentamicin (200 μg/mL, positive control), without antibiotics (negative control), or PA-13 at concentrations of 62.5, 31.25, and 15.625 μg/mL. Total bacterial counts were measured at 0, 24, 48, and 72 h of storage, while semen quality parameters, including sperm motility, viability, acrosomal integrity, and mitochondrial membrane potential, were evaluated on days 1, 3, and 5. In Experiment II, isolated E. coli was incubated with PA-13 alone or in combination with varying gentamicin concentrations, and bacterial growth was monitored over 24 h using optical density measurements.Results:PA-13 effectively inhibited bacterial proliferation in extended semen during the first 24 h of storage, with lower concentrations (15.625 and 31.25 μg/mL) showing greater antibacterial activity than the higher concentration (62.5 μg/mL). Semen quality parameters were comparable among groups on day 1; however, prolonged storage demonstrated that the highest PA-13 concentration negatively affected sperm motility and viability. Lower PA-13 concentrations preserved semen quality similar to that of the gentamicin-treated control. In Experiment II, combination treatments exhibited synergistic effects, with PA-13 at 3.906 μg/mL combined with gentamicin at 100 μg/mL inhibiting E. coli growth equivalently to gentamicin at 200 μg/mL alone.Conclusion:PA-13 effectively controls bacterial contamination in stored boar semen while maintaining semen quality at appropriate concentrations. Its combination with gentamicin enables a substantial reduction in antibiotic dosage without compromising antibacterial efficacy. These findings support the use of AMPs as alternative or complementary antimicrobial agents in semen extenders to reduce antibiotic use in the swine industry.

Comparative in silico analysis identifies potent natural and synthetic peptide candidates for targeting SARS-CoV-2 viral proteins.

Aflatoxin contamination of corn (Zea mays) caused by Aspergillus flavus is a major concern in the US and globally due to its acute toxicity and carcinogenicity in humans and livestock. Transgenic expression of synthetic peptides, which have been modified for greater antimicrobial activity and stability, is a promising approach to mitigating aflatoxin contamination in crops. In this study, corn that was modified with the synthetic peptide AGM182, derived from tachyplesin-1, was tested in two inoculated greenhouse trials for resistance to Af70-GFP, an A. flavus strain that expresses green fluorescent protein. A 25-50% reduction in conidia on corn kernels was observed, up to 60% reduction of internal kernel fungal colonization (GFP fluorescence), and 50% reduction in aflatoxin contamination for corn expressing AGM182 from wildtype Hi-II corn. Our results reveal that incorporating AGM182 and other synthetic antimicrobial peptides traits can reduce aflatoxin contamination in corn.

Antimicrobial peptides (AMPs), evolutionarily conserved components of innate immunity characterized by their broad-spectrum efficacy and minimal resistance development, are increasingly recognized as promising therapeutic candidates. This review aims to integrate current knowledge concerning natural and synthetic antimicrobial peptides and their therapeutic effectiveness in addressing gastrointestinal infections. A literature review was performed, evaluating recent peer-reviewed studies on AMPs. The research concentrated on their molecular mechanisms of action, antimicrobial spectrum, and their interactions with standard antibiotics. More in detail, the peptide classes examined herein included defensins, cathelicidins, histatins, and various natural peptides such as lactoferricin, protamines, RegIII, and hepcidin, along with synthetic analogs like WR12, D-IK8, MSI-78, and IMX942. Natural AMPs demonstrated significant antimicrobial and immunomodulatory effects against Escherichia coli, Klebsiella pneumoniae, Salmonella spp., and Shigella spp. Beyond direct antimicrobial activity, antimicrobial peptides act as integrated anti-infective agents not only by modulating host-microbiota interactions, but also preserving epithelial barrier integrity, and limiting inflammation, thereby offering a multifaceted strategy to control gastrointestinal infections. On the other hand, synthetic peptides showed improved stability, reduced cytotoxicity, and synergistic interactions with antibiotics, which suggests that they could be used either alone or in combination with other treatments. AMPs constitute a promising category endowed with anti-infective activity, especially for therapy of intestinal diseases, which is attributed to their distinctive anti-infective mechanisms, immune-modulating characteristics, and a relatively low propensity for resistance development compared to conventional antibiotics. However, more clinical trials and improvements to their formulation are needed to translate promising in vitro results into reliable patient outcomes.

ABSTRACT Tuberculosis (TB) remains a global health concern, primarily due to the alarming increase in multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains of Mycobacterium tuberculosis ( Mtb ) in recent years. To effectively overcome Mtb drug resistance mechanisms, innovative therapeutic modalities such as combinatorial therapy, which integrates conventional anti‐tubercular drugs with antimicrobial peptides (AMPs), are being actively investigated. In this review, we have attempted to establish the therapeutic rationale for proposing membrane‐targeting synthetic AMPs by focusing on the unique lipid composition and structural rigidity of the Mtb cell envelope. Subsequently, we evaluated the properties and molecular mechanisms of action of five key synthetic AMPs demonstrating potent antimicrobial activity against MDR and XDR strains of Mtb , which includes LLAP (a 15‐amino‐acid LL‐37 analogue); CP26 (a 26‐amino‐acid cecropin A / mellitin hybrid); D‐LAK120 (a 27‐amino‐acid containing synthetic AMP); hLF1‐11/ hLF1‐1117‐30 (an 11‐amino‐acid human lactoferrin derivative); and MIAP (a 19‐amino‐acid magainin derivative). A detailed comparative analysis on synthetic peptides' physicochemical properties, efficacy, safety profile, pharmacodynamic characteristics and mechanisms of action are addressed. Finally, we have reported the significant translational challenges associated with the clinical application of synthetic AMPs, including systemic delivery and stability. Based on these systematic analysis, we put forth that synthetic AMPs, when utilized in combination with existing drugs, represent a highly promising therapeutic modality for overcoming drug‐resistant tuberculosis infection.

Antimicrobial resistance in Acinetobacter baumannii has underscored the need for treatments beyond single-drug regimens, given high mortality rates associated with carbapenem-resistant strains. This review explores combination therapy as a framework for addressing the diverse resistance mechanisms presented by A. baumannii, including membrane modifications, efflux regulation, biofilm formation, and target-site alteration. This manuscript compares the potential integration of therapeutic pillars, including macrocyclic peptides, AI-driven drug discovery, and oxidative stress, to understand their underlying mechanisms, current applications, and translational limitations as multimodal treatment strategies. Understanding oxidative stress mechanisms is paramount for addressing persistent Antimicrobial resistance (AMR) challenges, revealing why broad Reactive oxygen species (ROS) flooding is ineffective and how localized oxidants can bypass bacterial defenses. Through case studies such as Zosurabalpin, a macrocyclic peptide with selective inhibition, and AI susceptibility models, this review highlights how an interdisciplinary approach can advance AMR therapies. Zosurabalpin’s specific mechanism of action demonstrates the value of narrow-spectrum molecules that disrupt essential structural pathways. At the same time, deep antimicrobial susceptibility phenotyping enables rapid phenotypic classification and virtual screening, significantly shortening discovery timelines. Finally, emerging compounds—including synthetic nanoparticles and antimicrobial peptides—are discussed that enhance membrane disruption and potentiate ROS-based killing. This review highlights that no single modality can overcome A. baumannii’s adaptability. Instead, the most promising and cost-effective approach is combination therapy, strategically pairing existing drugs to reduce the likelihood of resistance and improve clinical outcomes.

There is an urgent need for new antimicrobial agents to address the emerging antimicrobial resistance and the lack of novel antibiotics on the market. Antimicrobial peptides (AMPs) have gained significant interest as potential antibiotics over the past 30 years due to their broad activity against bacteria. So far, the presence, characteristics, and function of AMPs in camel immunity remain to be explored. Therefore, this study aims to identify and functionally characterize AMPs in Camelus Dromedarius using in-silico and experimental approaches. In-silico identification and prediction of cathelicidin peptides properties were conducted using Blastp, Conserved Domain, Signal P-5.0, Peptide Cutter-Expasy, and the Antimicrobial Sequence Scanning System (AMPA) database. Physicochemical and biological properties were characterized using bioinformatics analysis tools. The experimental assays of synthetic AMPs were performed using circular dichroism (CD) spectroscopy, colony-forming assay, sytox green uptake assay, transmission and scanning electron microscopy, and hemolysis assay. Three cathelicidin peptides were identified from Camelus Dromedarius which were designated as CdPMAP-23, Cdprotegrin-3 (CdPG-3), and Cdcathelin-like (CdCATH). CdPG-3 and CdCATH demonstrated significant antibacterial effects against all tested Gram-negative and Gram-positive strains, including Escherichia. coli (Multidrug resistant) and Methicillin-Resistant Staphylococcus aureus (ATCC 700699). These two peptides caused significant membrane leakage and damage to Escherichia. coli (ATCC 25922), with CdPMAP-23 showing a lesser effect. Lower concentrations of CdPMAP-23, CdPG-3 and CdCATH exhibited low to moderate lytic activity against red blood cells in humans, camels, and chickens. This study identified novel AMPs from dromedary camels with potential therapeutic value against multidrug-resistant strains. The results show that AMPs are present in dromedary camels, setting out a foundation for further studies on the unique features of their innate immune system.

Antimicrobial resistance (AMR), particularly in methicillin-resistant Staphylococcus aureus (MRSA), continues to threaten global health due to its multidrug resistance and strong biofilm-forming ability. Antimicrobial peptides (AMPs) have emerged as promising agents against MRSA biofilms because of their diverse origins, structural versatility, and unique modes of action. Natural AMPs derived from animals, plants, fungi, protists, archaea, and bacteria primarily act by disrupting bacterial membranes, interfering with quorum sensing, and downregulating biofilm-related genes such as sarA, icaA, and icaD. Synthetic AMPs, designed through computational modeling and machine learning, demonstrate enhanced stability, reduced toxicity, and improved target specificity. Synergistic AMP-antibiotic combinations, including nisin, indolicidin, and α-MSH analogs with β-lactams, significantly improve antibiofilm efficacy and bacterial clearance. Despite these advances, challenges persist due to peptide instability, enzymatic degradation, cytotoxicity, and limited in vivo validation. Recent developments in nanoparticle, hydrogel, coatings, and nanofiber delivery systems have improved AMP bioavailability and controlled release within biofilms. Continued integration of peptide engineering, nanotechnology, and bioinformatics-driven design offers promising solutions for clinical translation. Overall, AMPs represent a frontier in combating MRSA biofilms and antibiotic resistance, with future research focusing on stability enhancement, resistance prevention, and optimized therapeutic delivery.

BackgroundPseudomonas aeruginosa is a Gram-negative pathogen frequently responsible for nosocomial infections and a significant problem in intensive care units. P. aeruginosa, as an opportunistic pathogen, increases mortality risks for severely wounded and immunocompromised individuals. The inherent drug-resistance of P. aeruginosa now requires novel therapeutics with multiple mechanisms that will offer lasting potency in the post-antibiotic era. Synthetic antimicrobial peptides (AMPs) are ideal, as their multiple modes of action slow resistance development. In this study, we investigated the potential of novel proprietary AMP OB1111 to effectively treat P. aeruginosa under standard antimicrobial susceptibility testing (AST) and host-mimicking conditions, in planktonic and biofilm states, and at sublethal and lethal concentrations.ResultsThe highly virulent PA14 and moderately virulent PAO1 reference strains were used in these studies. OB1111 effectively displayed inhibitory and bactericidal activity against both strains under AST conditions in planktonic and biofilm states. OB1111 demonstrated anti-virulence activity under host-mimicking conditions by reducing pyoverdine production and early biofilm attachment at sublethal concentrations. Under AST conditions, sublethal doses of OB1111 gradually reduced planktonic PA14 and PAO1 growth but showed less efficacy against biofilms. Additionally, PAO1 biofilms showed reduced susceptibility to OB1111 in comparison to PA14 biofilms at sublethal concentrations. Of significance, scanning electron microscopy revealed that OB1111 effectively deformed and disintegrated PA14 and PAO1 membranes in both the planktonic and biofilm states.ConclusionsOB1111 successfully demonstrated the capacity to combat P. aeruginosa as an anti-planktonic, anti-biofilm, and anti-virulence agent. Future studies should further examine specific mechanisms of action against PA14 and PAO1, along with testing against clinical isolates in AST and host-mimicking conditions.

The synthesis of biocidal peptide materials using simple, low-cost, solvent-free methods is a crucial challenge for developing new antimicrobial approaches. In this study, we produced proteinoid nanostructures through simple, inexpensive, and environmentally friendly thermal reactions between glutamic acid (Glu) and tyrosine (Tyr) in various molar ratios. Mechanistically, the thermal cyclization of glutamic acid into pyroglutamic acid (pGlu) facilitated the formation of short peptide chains containing pGlu as the N-terminus moiety and subsequent L-tyrosine or glutamic acid residues, which self-assembled into nanometric spheroidal structures that exhibit blue emission. Spectroscopic (FTIR, UV-Vis, photoluminescence) and mass (LC-MS) analyses confirmed the formation of mixed pGlu-/Tyr/Glu peptides. All products exhibit dose-dependent antimicrobial activity against Methicillin-Resistant Staphylococcus aureus (MRSA), with a minimum inhibitory concentration (MIC) of 25 mg mL−1 for the GluTyr 1:1 and 2:1 proteinoids. The outcomes observed following 24 h exposure of the HEK293 cell line to the materials indicate their suitability for integration into hybrid systems for antimicrobial surfaces. This work is the first to demonstrate a direct antibacterial activity of proteinoids obtained by thermal condensation, opening up the possibility of designing a new class of synthetic antimicrobial peptides.

Synthetic antimicrobial peptides: Combatting antimicrobial resistance for sustainable aquaculture.

Abstract The ability of microorganisms to form biofilm structures is at the root of many diseases of the oral cavity. Biofilm – in particular its abnormal growth in combination with other factors, such as impaired functioning of specific and non-specific defence mechanisms of the human body or disorders in the quantitative and qualitative composition of the oral microbiota – may lead to the development of caries, gingivitis or periodontitis. Treatment of this type of infections is a challenge for modern dentistry, also due to the increasing resistance of microorganisms. The above requires a search for alternative therapeutic methods. This paper presents a general characteristics of antimicrobial peptides, briefly characterizes oral diseases and provides basic information on infection therapy in dentistry. It also discusses the possibilities of therapeutic use of natural and synthetic antimicrobial peptides in dentistry.