Jul 2026· Journal of Medicinal Chemistry· Vol 69, pp. 18580 - 18591· 0 citations· 52 references
Medicine
TL;DR
It is demonstrated how targeted sequence refinement can substantially enhance antimicrobial potency while modulating interactions with bacterial membranes and the transcription/translation machinery.
Abstract
Antimicrobial peptides are promising alternatives to conventional antibiotics, yet systematic strategies to enhance their potency and elucidate their mechanisms of action remain limited. Here, we generated and evaluated a focused library of 20 peptides derived from the lead peptide L3. Across clinically relevant pathogens, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans, several variants showed enhanced antibacterial activity, reducing MIC values to as low as 32 μg/mL (G2-4). Additional candidates (G1-8, G2-1, G2-2, G2-10) achieved MICs of 64 μg/mL against E. coli. Studies in environmental Escherichia isolates revealed species-specific susceptibility patterns. Mechanistic investigations demonstrated minimal membrane-lytic activity at concentrations exceeding their MICs, indicating that membrane disruption is not their primary mode of action. In contrast, in vitro transcription/translation assays demonstrated potent inhibition of protein expression. These results demonstrate how targeted sequence refinement can substantially enhance antimicrobial potency while modulating interactions with bacterial membranes and the transcription/translation machinery.
Antimicrobial peptides (AMPs) are promising alternatives to antibiotics, but discovering potent, low-toxicity candidates and improving their delivery remain challenging. In this study, novel AMPs were identified by constructing and screening a synthetic random peptide library using a bacterial surface display system. Rational design generated derivative peptides, among which WP-4 and WP-6 showed high antimicrobial activity, good biocompatibility, rapid bactericidal effects, and low propensity for resistance development. WP-6 also showed good in vivo therapeutic potency in a murine Escherichia coli systemic infection model. Mechanistic studies indicated that WP-4 and WP-6 target bacterial cell membranes, disrupt the proton motive force, and induce excessive reactive oxygen species accumulation. To further improve their activity and in vivo performance, WP-4 and WP-6 were encapsulated within zeolitic imidazolate framework-8, yielding improved antimicrobial activity and proteolytic resistance. These nanoparticles exhibited superior therapeutic efficacy in a Streptococcus suis-induced arthritis model. Our study identified potent AMPs with promising therapeutic potential.
Shuai-Yang Wang, S.-S. Wang, Xiu-Jian Liu et al.· Journal of Medicinal Chemist...· 0 citations
ABSTRACT Antibiotic-resistant bacteria are a global public health threat that is becoming increasingly difficult to address with conventional therapeutics. Consequently, there is much interest in studying alternative biologics that circumvent antibiotic resistance. The Brevinin-2 family of antimicrobial peptides (AMPs) is a group of naturally occurring molecules that have the potential for high activity and low toxicity. Herein, we investigated the potential of a consensus-sequence-driven approach to Brevinin-2 peptide synthesis and evaluated their action against a panel of multidrug-resistant (MDR) bacteria, including carbapenemase-resistant Escherichia coli. We used the positional frequency of amino acids to generate novel synthetic peptides representative of the Brevinin-2 family. Four templates—G30, G33, S33, and G37—were synthesized by standard fluorenylmethyloxycarbonyl (FMOC) chemistry, purified by reverse-phase fast protein liquid chromatography (RP-FPLC), and tested for antibacterial activity and hemolytic toxicity. The results demonstrated a broadly applicable chemical peptide synthesis pipeline for the Brevinin-2 family with a high degree of purity (>90%). Two peptides—S33 and G33—exhibited activity consistent with potential selectivity toward gram-positive and -negative bacteria, respectively, while G37 displays broad-spectrum activity, with growth of a Class B carbapenemase-resistant E. coli inhibited at 16 µM and a Class A carbapenemase-resistant K. pneumoniae inhibited at 64 µM. G37 acts rapidly, slowing growth within 30 min and fully killing targeted bacteria within 150 min. Although moderate levels of hemolytic toxicity pose a challenge for future development, the consensus-sequence approach toward novel Brevinin-2 AMP discovery via alignment combined with in vitro antimicrobial analysis shows promise to test and initially validate other peptides. IMPORTANCE The escalating threat of antimicrobial resistance (AMR) demands innovative therapeutic strategies beyond traditional antibiotics. This study demonstrates a systematic, consensus-sequence-driven approach to designing antimicrobial peptides (AMPs) from the naturally occurring Brevinin-2 family, offering a replicable framework for accelerated drug discovery. Our novel peptide G37 exhibits bactericidal activity against carbapenemase-resistant Escherichia coli within 150 min while maintaining low hemolytic activity at therapeutic concentrations. The potential preferential activity shown by peptides S33 and G33 for gram-positive versus gram-negative bacteria provides prospective valuable insights into structure-activity relationships that can guide further peptide optimization. By combining computational sequence analysis, standardized solid-phase peptide synthesis, and comprehensive in vitro validation, this work establishes a streamlined pipeline for Brevinin-2 peptide development. This methodology addresses the urgent need for alternative antimicrobials while providing a scalable approach to combat multidrug-resistant (MDR) pathogens. The escalating threat of antimicrobial resistance (AMR) demands innovative therapeutic strategies beyond traditional antibiotics. This study demonstrates a systematic, consensus-sequence-driven approach to designing antimicrobial peptides (AMPs) from the naturally occurring Brevinin-2 family, offering a replicable framework for accelerated drug discovery. Our novel peptide G37 exhibits bactericidal activity against carbapenemase-resistant Escherichia coli within 150 min while maintaining low hemolytic activity at therapeutic concentrations. The potential preferential activity shown by peptides S33 and G33 for gram-positive versus gram-negative bacteria provides prospective valuable insights into structure-activity relationships that can guide further peptide optimization. By combining computational sequence analysis, standardized solid-phase peptide synthesis, and comprehensive in vitro validation, this work establishes a streamlined pipeline for Brevinin-2 peptide development. This methodology addresses the urgent need for alternative antimicrobials while providing a scalable approach to combat multidrug-resistant (MDR) pathogens.
Colin M. McDowell, Jessica D. Carder, J. Brozik et al.· Applied and Environmental Mi...· 0 citations
Antimicrobial peptides (AMPs) are a diverse class of bioactive molecules that exert their effects through multiple mechanisms, including membrane disruption, inhibition of cell wall synthesis, interference with intracellular targets, and binding to nucleic acids or proteins. They exhibit broad-spectrum antimicrobial activity and a low propensity to induce drug resistance, positioning them as promising candidates for combating multidrug-resistant infections. However, their clinical translation faces significant challenges, including protease susceptibility, potential cytotoxicity, high production costs, and suboptimal pharmacokinetic properties. In this study, a strain of Bacillus licheniformis SAM-D318 isolated from Hu sheep intestine was used as the peptide-producing strain; genomic analysis confirmed the absence of classical virulence toxin genes despite intrinsic multidrug resistance. A previously unreported cationic α-helical antimicrobial peptide, SAM-KB40 (4176.92 Da, pI 10.886), was obtained from the fermentation supernatant of B. licheniformis SAM-D318 and purified using cation-exchange chromatography and C18 reversed-phase chromatography, achieving a 70.45-fold increase in specific activity. Structural prediction by PEP-FOLD4 suggested an α-helical propensity in the C-terminal region, while circular dichroism spectroscopy indicated that SAM-KB40 adopts a predominantly random coil conformation in aqueous solution. The peptide exhibited potent antibacterial activity against Clostridium perfringens (MIC = 0.5 µg/mL, MBC = 4 µg/mL) and showed selective inhibition against Staphylococcus aureus and Listeria monocytogenes. SAM-KB40 demonstrated exceptional thermal stability (> 97% activity retained at 100 °C for 60 min), good tolerance to acidic conditions (pH 3-4) and bile salts (up to 0.50%), and remarkably low hemolytic activity (1.96% at 1000 µg/mL). Notably, the peptide neutralized recombinant C. perfringens α-toxin at 16 µg/mL in a Vero cell-based assay and exhibited no cytotoxicity at concentrations up to 128 µg/mL, demonstrating a favorable in vitro safety profile.Sequence homology analysis against UniProt, NCBI, APD, and BACTIBASE databases confirmed that SAM-KB40 has not been previously reported. While the peptide shows promising in vitro dual antibacterial and toxin-neutralizing capacity, its high sensitivity to proteinase K (39.78% residual activity) limits oral application. These findings position SAM-KB40 as a promising lead candidate for further development as a feed additive or therapeutic agent against C. perfringens-associated diseases, pending subsequent in vivo validation.
En Liu, Wei-Ping Huo, Fu-Li Xu et al.· Applied Biochemistry and Bio...· 0 citations
In bacterial-induced mouse models of epidermal and keratitis infections, cHH8-6 effectively reduces the bacterial count at the infection site with negligible in vivo toxicity, and due to its unique mechanism, cHH8-6 is less likely to induce bacterial resistance compared to clinical antibiotics.
Zhe Zong, Guowenlie Gao, Pengqi Wan et al.· Biomaterials Science· 0 citations
A stability-centric computational discovery pipeline to identify and characterize novel antimicrobial peptides with potent and selective bactericidal activity against ESKAPE pathogens and to validate the lead candidates through experimental in vitro assays provided a generally applicable strategy for accelerating the discovery of peptide-based therapeutics.
H. S. Mahrosh, M. Christodoulides, A. Jamil· Probiotics and Antimicrobial...· 0 citations
Antimicrobial resistance has become a serious global health challenge, increasing the need for new therapeutic strategies beyond conventional antibiotics. In this study, FL18 and its chimeric derivatives, FL18–TAT8 and FL18–Ahx–TAT8, were designed to combine antimicrobial activity with the cell-associated advantages of a cell-penetrating peptide motif and were synthesized by Fmoc-based solid-phase peptide synthesis. Their molecular masses were confirmed by MALDI-MS analysis. The antibacterial activities of the peptides were evaluated against representative Gram-negative and Gram-positive bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecium. The tested peptides exhibited minimum inhibitory concentration values in the range of 4–64 μM depending on the peptide sequence and bacterial strain, while TAT8 alone showed no significant antibacterial activity under the tested conditions. Among the constructs, FL18–Ahx–TAT8 displayed the most favorable overall antibacterial profile, particularly against P. aeruginosa and E. faecium. Biocompatibility studies further showed that FL18 and its chimeric derivatives maintained approximately 75–90% HaCaT cell viability across the tested concentration range and exhibited generally low hemolytic activity. In addition, confocal microscopy and flow cytometry revealed peptide-associated fluorescence in HaCaT cells, with the TAT8-containing chimeras showing a broader and more pronounced fluorescence distribution than FL18 alone. Overall, these findings demonstrate that FL18-based chimerization provides a modular and effective strategy for tuning antibacterial activity, biocompatibility, and peptide–cell interaction behavior, underscoring their promise as building blocks for the design of next-generation antimicrobial peptide platforms.
N. Zencirci, Busra Kilic, Öznur Akbal Vural et al.· ACS Omega· 0 citations
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