Name: Production and Testing of Antimicrobial Peptides and Their Mimics (Video) | JoVE Journal
Uploaded: 2026-04-10T13:08:00.000+00:00
Duration: 10 min 35 s
Description: Learn a unified protocol for synthesizing and testing antimicrobial peptides and peptide mimics using Mel4 and RK-758. This method enables reproducible evaluation of antibacterial activity and cytotoxicity for drug discovery.

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5 minutes

The authors would like to acknowledge the Mark Wainwright Analytical Centre and Recombinant Product Facility (RPF) at the University of New South Wales. In particular, the Bioanalytical Mass Spectrometry Facility for HRMS analysis and the NMR facility for access to NMR instruments.

<ol><li>Naghavi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+burden+of+bacterial+antimicrobial+resistance+1990%E2%80%932021%3A+a+systematic+analysis+with+forecasts+to+2050%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050.</a> Lancet. 404 (10459), 1199-1226 (2024).</li>
<li>Antimicrobial resistance. , World Health Organization. Available at: 
 <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance">https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance</a> (2023).</li>
<li>Puri, B., Vaishya, R., Vaish, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+resistance%3A+Current+challenges+and+future+directions%5BTitle%5D)+AND+%22Med.+J.+Armed+Forces+India%22%5BJournal%5D)">Antimicrobial resistance: Current challenges and future directions.</a> Med. J. Armed Forces India. 81 (3), 247-258 (2025).</li>
<li>Zhang, T., Jin, Q., Ji, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+peptides+and+their+mimetics%3A+promising+candidates+of+next-generation+therapeutic+agents+combating+multidrug-resistant+bacteria%5BTitle%5D)+AND+%22Adv.+Biol%22%5BJournal%5D)">Antimicrobial peptides and their mimetics: promising candidates of next-generation therapeutic agents combating multidrug-resistant bacteria.</a> Adv. Biol. 9 (4), 2400461(2025).</li>
<li>Pasupuleti, M., Schmidtchen, A., Malmsten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+peptides%3A+key+components+of+the+innate+immune+system%5BTitle%5D)+AND+%22Crit.+Rev.+Biotechnol%22%5BJournal%5D)">Antimicrobial peptides: key components of the innate immune system.</a> Crit. Rev. Biotechnol. 32 (2), 143-171 (2012).</li>
<li>Oliveira Júnior, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+peptides%3A+structure%2C+functions+and+translational+applications%5BTitle%5D)+AND+%22Nat.+Rev.+Microbiol%22%5BJournal%5D)">Antimicrobial peptides: structure, functions and translational applications.</a> Nat. Rev. Microbiol. , (2025).</li>
<li>Keshri, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LL-37%2C+the+master+antimicrobial+peptide%2C+its+multifaceted+role+from+combating+infections+to+cancer+immunity%5BTitle%5D)+AND+%22Int.+J.+Antimicrob.+Agents%22%5BJournal%5D)">LL-37, the master antimicrobial peptide, its multifaceted role from combating infections to cancer immunity.</a> Int. J. Antimicrob. Agents. 65 (1), 107398(2025).</li>
<li>Alzain, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+peptides%3A+mechanisms%2C+applications%2C+and+therapeutic+potential%5BTitle%5D)+AND+%22Infect.+Drug+Resist%22%5BJournal%5D)">Antimicrobial peptides: mechanisms, applications, and therapeutic potential.</a> Infect. Drug Resist. , 4385-4426 (2025).</li>
<li>Kuppusamy, R., Willcox, M., Black, D. S., Kumar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short+cationic+peptidomimetic+antimicrobials%5BTitle%5D)+AND+%22Antibiotics%22%5BJournal%5D)">Short cationic peptidomimetic antimicrobials.</a> Antibiotics. 8 (2), (2019).</li>
<li>Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptides+chemistry%2C+manufacturing%2C+and+controls%5BTitle%5D)+AND+%22Peptide+Science%3A+Chemical+Ligation%2C+Lead+Generation%2C+and+Therapeutic+Advances%22%5BJournal%5D)">Peptides chemistry, manufacturing, and controls.</a> Peptide Science: Chemical Ligation, Lead Generation, and Therapeutic Advances. , 471-508 (2025).</li>
<li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+and+purification+of+antimicrobial+peptide+buforin+IIb+in+Escherichia+coli%5BTitle%5D)+AND+%22Biotechnol.+Lett%22%5BJournal%5D)">Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli.</a> Biotechnol. Lett. 33 (11), 2121-2126 (2011).</li>
<li>Cai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide+synthesis%3A+a+review+of+classical+and+emerging+methods%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Peptide synthesis: a review of classical and emerging methods.</a> Biofabrication. 18 (1), 012001(2026).</li>
<li>Gentilucci, L., De Marco, R., Cerisoli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+modifications+designed+to+improve+peptide+stability%3A+incorporation+of+non-natural+amino+acids%2C+pseudo-peptide+bonds%2C+and+cyclization.%C2%A0Curr.+Pharm+pseudo-peptide%5BTitle%5D)+AND+%22Des%22%5BJournal%5D)">Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Curr. Pharm pseudo-peptide.</a> Des. 16 (28), 3185-3203 (2010).</li>
<li>Kuppusamy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tuning+the+anthranilamide+peptidomimetic+design+to+selectively+target+planktonic+bacteria+and+biofilm%5BTitle%5D)+AND+%22Antibiotics%22%5BJournal%5D)">Tuning the anthranilamide peptidomimetic design to selectively target planktonic bacteria and biofilm.</a> Antibiotics. 12 (3), 585(2023).</li>
<li>Kuppusamy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guanidine+functionalized+anthranilamides+as+effective+antibacterials+with+biofilm+disruption+activity%5BTitle%5D)+AND+%22Org.+Biomol.+Chem%22%5BJournal%5D)">Guanidine functionalized anthranilamides as effective antibacterials with biofilm disruption activity.</a> Org. Biomol. Chem. 16 (32), 5871-5888 (2018).</li>
<li>Wiegand, I., Hilpert, K., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+%28MIC%29+of+antimicrobial+substances%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat. Protoc. 3 (2), 163-175 (2008).</li>
<li>Ji, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adsorption+and+morphology+analysis+of+bovine+serum+albumin+on+a+micropillar-enhanced+quartz+crystal+microbalance%5BTitle%5D)+AND+%22J.+Phys.+Chem.+B%22%5BJournal%5D)">Adsorption and morphology analysis of bovine serum albumin on a micropillar-enhanced quartz crystal microbalance.</a> J. Phys. Chem. B. 128 (41), 10247-10257 (2024).</li>
<li>Yasir, M., Dutta, D., Willcox, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+antimicrobial+peptides+and+ciprofloxacin+against+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Activity of antimicrobial peptides and ciprofloxacin against Pseudomonas aeruginosa biofilms.</a> Molecules. 25 (17), (2020).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+determination+of+susceptibility+of+bacteria+to+antimicrobial+agents.+terminology%5BTitle%5D)+AND+%22Clin.+Microbiol.+Infect%22%5BJournal%5D)">Methods for the determination of susceptibility of bacteria to antimicrobial agents. terminology.</a> Clin. Microbiol. Infect. 4 (5), 291-296 (1998).</li>
<li>Saetan, T., Sukwattanasinitt, M., Wacharasindhu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mild+photocatalytic+synthesis+of+guanidine+from+thiourea+under+visible+light%5BTitle%5D)+AND+%22Org.+Lett%22%5BJournal%5D)">A mild photocatalytic synthesis of guanidine from thiourea under visible light.</a> Org. Lett. 22 (20), 7864-7869 (2020).</li>
<li>Woodring, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disrupting+the+conserved+salt+bridge+in+the+trimerization+of+influenza+A+nucleoprotein%5BTitle%5D)+AND+%22J.+Med.+Chem%22%5BJournal%5D)">Disrupting the conserved salt bridge in the trimerization of influenza A nucleoprotein.</a> J. Med. Chem. 63 (1), 205-215 (2019).</li>
<li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+bacterial+expression+systems+for+heterologous+protein+production%3A+from+molecular+and+biochemical+fundamentals+to+commercial+systems%5BTitle%5D)+AND+%22Appl.+Microbiol.+Biotechnol%22%5BJournal%5D)">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Appl. Microbiol. Biotechnol. 72 (2), 211-222 (2006).</li>
<li>Berridge, M. V., Herst, P. M., Tan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tetrazolium+dyes+as+tools+in+cell+biology%3A+new+insights+into+their+cellular+reduction%5BTitle%5D)+AND+%22Biotechnol.+Annu.+Rev%22%5BJournal%5D)">Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.</a> Biotechnol. Annu. Rev. 11, 127-152 (2005).</li>
<li>Deni, W. H., Gao, T., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+reconstituting+peptides%2Fpeptidomimetics+from+DMSO+to+aqueous+buffers+for+circular+dichroism+analyses%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for circular dichroism analyses.</a> STAR Protoc. 5 (1), 102850(2024).</li>
<li>Lou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+production+systems+and+optimization+strategies+of+antimicrobial+peptides%3A+a+review%5BTitle%5D)+AND+%22World+J.+Microbiol.+Biotechnol%22%5BJournal%5D)">Microbial production systems and optimization strategies of antimicrobial peptides: a review.</a> World J. Microbiol. Biotechnol. 41 (2), 66(2025).</li>
<li>Hancock, R. E., Sahl, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+and+host-defense+peptides+as+new+anti-infective+therapeutic+strategies%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.</a> Nat. Biotechnol. 24 (12), 1551-1557 (2006).</li>
</ol>

The authors have no conflicts of interest to declare.

The protocol described here provides a comprehensive framework for the production and functional evaluation of the cationic AMP Mel4 (which can be altered for use with other AMPs) and the peptide mimic RK758. This workflow combines multi-step chemical synthesis or recombinant expression in E. coli with antimicrobial and cytotoxicity assays. The integration of these approaches provides a versatile platform for systematic AMP research, enabling direct comparisons between chemically synthesized and biologically produced peptides. Several steps in this protocol are particularly critical for successful peptide production and activity assessment.
RK758 is a synthetic peptide mimic based on an anthranilamide scaffold. During the compound screening phase for the discovery of novel AMP mimics, it is difficult to use in silico screening to plan a compound library. Therefore, it is important to base these mimics on a scaffold that allows for diversity in the library of compounds. The anthranilamide scaffold was chosen for this reason. An added benefit of an anthranilamide-based scaffold is that the RK758 metabolite, 5-Bromoanthranilic acid, has been shown to possess antimicrobial properties.
Previous works optimizing the structure and antibacterial activity of RK758 and analogs used different synthetic procedures. The original structure-optimization study investigated the effect of different cationic groups and substituents on the anthranilic phenyl group. Hence, the synthesis started with the ring-opening of Isatoic anhydride derivatives with L-tryptophan methyl ester. The product could then be modified as shown by Kuppusamy et al.15. A follow-up study investigated the structure and location of the hydrophobic group. The follow-up study showed that the Bromo-substituted scaffold tolerated a variety of hydrophobic groups at the naphthyl position and that intermediate 4 was amenable to Suzuki cross-coupling at the bromo position, as shown in Figure 8.
The current work has addressed some of the problems of the former approaches. In the approach depicted in Figure 4, despite the synthesis of intermediate 11 reporting purification via trituration with diethyl ether, attempts to replicate this result have since yielded gummy impure mixtures that require chromatographic purification. Additionally, intermediate 6 also required chromatographic purification using this approach. Overall, the original work obtained an intermediate 7 in 21% yield in six steps from L-tryptophan methyl ester hydrochloride. The second approach15, although lower yielding (14% from Cbz-L-tryptophan), produced intermediates that were easier to handle in fewer steps.
This study identified that coupling Cbz-L-Trp with N-Boc-ethylenediamine required optimization to ensure no residual DMF remained in the product, as observed during initial studies to scale up this reaction. Using a mixed solvent system with a 3:8 (v/v) ratio of DMF to DCM reduced the amount of DMF, facilitating easier removal during workup; however, substituting chlorinated solvents could raise concerns about environmental impact. This modification also improved the yield to 87% which is likely attributed to the urea byproduct from EDC precipitating in the DCM mixture, acting as a driving force for the reaction. The hydrogenation was also identified as a bottleneck, as these reactions typically yield&#62;90%. This study found that substituting THF for methanol yielded 3 in quantitative yield, compared with 85% in previous work, and reduced the reaction time to 4 h. This could also be attributed to a purer starting material being used, since the original study likely contained residual DMF, which could have poisoned the palladium catalyst. Overall, compared with previous approaches, the yield to intermediate 7 has improved to 48% in 5 steps from Cbz-L-Tryptophan. In earlier experiments (discussed above), the overall yield was 21%, with 14% for follow-up reactions, whereas in this study, an overall yield of 48% was achieved for compound 7, demonstrating the diversity of analogs that can be synthesized from appropriately substituted isatoic anhydride derivatives.
The highly deactivated amine in anthranilamide 4 (see Figure 8) requires three equivalents of acid chloride for efficient reaction. When only stoichiometric amounts are employed, incomplete conversion of 4 to 6 occurs, complicating purification because of the similar chromatographic and solubility characteristics of intermediates 4 and 6. The authors acknowledge this is a limitation of the scaffold and are working to develop a new methodology to reduce the excess acid chloride required.
The penultimate reaction to attach thiourea 8 to amine 7 requires mercury (II) chloride, which is highly toxic and extremely harmful to the environment, potentially precluding this method for large-scale synthesis. This approach was used in the current work, since the mercury (II) mediated attachment of thioureas proceeds at room temperature. An alternative method using thiourea 8 involves alkylating the sulfur atom, followed by nucleophilic substitution with an amine substrate, as depicted in Figure 9. This type of reaction has been reported, however, in many cases, it requires elevated temperatures and basic conditions that may lead to the formation of side products with the substrates20,21. Alternatively, using a similar methylated thiourea substrate, a photocatalytic approach has been reported by Saetan et al.20. They report that this reaction occurs at ambient temperature under irradiation with light from a white LED. It was reported that this reaction occurs with a variety of primary and secondary amines, yielding products in excess of 80%. For the final reaction, trituration with diethyl ether followed by freeze-drying yields a product typically requiring no further purification (&#62;95% pure by 1H NMR in MeOD-d4). The primary impurity is residual, high-boiling-point 1,2-ethanedithiol, which is readily detectable by its distinctive cabbage-like odor. Recombinant expression of Mel4 requires induction at an optimal optical density (OD600 0.5&#8211;0.6) and careful maintenance of induction temperature to maximize inclusion body formation while preserving bacterial viability22.
Antimicrobial and cytotoxicity assays also include steps essential for obtaining accurate, reproducible results. Precise inoculum preparation and serial dilution of peptides are required, as small variations in bacterial density or peptide solubility can significantly alter measured MIC and MBC values16. In MTT cytotoxicity assays, accurate cell counting, uniform seeding, and complete formazan dissolution are crucial to obtain reliable metabolic readouts23.
Several modifications to the protocol can address common challenges. Peptides with limited aqueous solubility can be dissolved in 0.01% acetic acid16 while peptides or mimics can also be solubilized in DMSO24 or supplemented with 0.1&#8211;0.2% BSA17 to prevent adsorption to plastic surfaces during MIC determination. Recombinant inclusion bodies can be solubilized and refolded by adjusting guanidinium chloride or urea concentrations, followed by stepwise dialysis. For MTT assays, gentle shaking and complete DMSO dissolution can reduce variability in absorbance readings.
The significance of this protocol lies in its integration of complementary production and evaluation strategies, offering advantages over standard single-method approaches. Many studies focus on a single method for peptide or mimic production, but no single approach is ideal for all compounds; selecting the appropriate method based on peptide properties ensures optimal yield and functionality. For example, chemical synthesis allows precise incorporation of non-natural amino acids and peptide mimics4, which may be challenging in recombinant systems. Conversely, recombinant expression offers cost-effective production of highly cationic peptides25 that may aggregate during chemical synthesis. By combining both methods with standardized functional assays, the protocol enables rigorous characterization of AMP activity and cytotoxicity, surpassing the scope of standard MIC assays alone26.
Compared with traditional standalone approaches, this protocol is broadly applicable to antimicrobial peptide research and therapeutic development. It can be adapted to explore structure-activity relationships, optimize peptide potency, and assess toxicity. Moreover, the methods can support high-throughput screening of peptide libraries, study of bacterial resistance mechanisms, and evaluation of combination therapies with conventional antibiotics. Beyond antibacterial applications, this workflow may also be applied to antiviral or antifungal peptide testing, surface coatings for infection control, and biomedical applications such as biofilm disruption or immunomodulation.
In conclusion, this protocol provides a versatile and reproducible workflow for the production, purification, and functional evaluation of AMPs and peptide mimics. By addressing critical methodological considerations, offering strategies for troubleshooting, and enabling diverse applications, it establishes a robust foundation for antimicrobial and biomedical research.

Antimicrobial resistance (AMR) has emerged as a major global health threat1. According to the World Health Organization (WHO), AMR was directly responsible for approximately 1.27 million deaths in 2019 and contributed to an estimated 4.95 million deaths worldwide2. The overuse and misuse of existing antibiotics have highlighted the emergence of multidrug-resistant pathogens, leaving limited treatment options against microbes like bacteria, fungi, viruses, and protozoa2,3. This AMR crisis has initiated the search for alternative antimicrobial strategies that are effective, stable, and less prone to resistance development. Among the most promising candidates are AMPs and their synthetic mimics4.
AMPs are naturally occurring molecules that play a vital role in the innate immune systems of humans, animals, and plants5. They typically exhibit broad-spectrum antimicrobial activity through mechanisms such as membrane disruption, interference with microbial metabolism, and modulation of immune responses6. For example, the human cathelicidin peptide LL-37 has demonstrated potent activity against bacteria, fungi, and viruses7. However, despite their promising potential4, the large-scale therapeutic use of natural AMPs is often restricted by their low natural abundance. Additionally, obtaining their active forms from natural sources can be challenging, as many peptides require specific post-translational modifications to become functionally active8.
To address the limitations associated with natural AMPs, researchers have developed synthetic analogs and peptide mimics. The mimics can be small, rationally designed molecules that preserve the antimicrobial efficacy of natural AMPs while offering improved stability, selectivity, and pharmacokinetic performance9.
Recent advances in peptide chemistry and molecular biology have enabled the efficient production of AMPs through methods such as solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS), and recombinant expression systems, providing precise control over key structural parameters, such as sequence composition, charge distribution, and amphipathicity10. Recombinant expression systems are suitable for large-scale production and for longer or more complex polypeptides. Once optimized, recombinant methods-typically employing fermentation or bioreactor systems- offer a cost-effective and scalable approach for industrial production of therapeutic peptides11.
This article demonstrates complementary approaches for the production and evaluation of AMPs and their mimics. While solid-phase chemical synthesis is widely used for peptide generation, it can become inefficient and costly for longer sequences or highly cationic peptides due to aggregation, low yields, and purification challenges12. In contrast, chemical synthesis remains highly advantageous for generating short peptide mimics with precise structural modifications and non-natural residues13. Recombinant expression systems, on the other hand, enable cost-effective and scalable biosynthetic production of longer or structurally complex peptides in microbial hosts, overcoming length and yield limitations associated with purely synthetic approaches12. By integrating chemical and biological production strategies, this combined framework offers greater flexibility, scalability, and translational potential than either method alone, making it particularly suitable for mechanistic research and therapeutic development.
Following production, the biological evaluation of these compounds is performed through two key assays. The antimicrobial activity assay measures the inhibitory potential of each peptide or mimic against representative microorganisms, providing insights into their spectrum and potency. The toxicity assay assesses cytocompatibility using mammalian cell lines to ensure safety and selectivity. By integrating these experimental step-design, synthesis, and functional testing, this protocol provides a comprehensive framework for characterizing the efficacy and safety of AMPs and their synthetic analogs. Together, these methodologies establish a systematic approach to developing and validating novel antimicrobial agents to address the growing global challenge of AMR.

<table><tbody><tr><td>1,2-ethanedithiol</td><td>Oakwood&amp;nbsp;</td><td>M03555</td><td></td></tr><tr><td>100X Antibiotics</td><td>Sigma Aldrich</td><td>A5955-20ML</td><td></td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride</td><td>Combi Blocks</td><td>SS-7536</td><td>Referred to as EDC hereafter</td></tr><tr><td>2-Naphthoyl chloride</td><td>Combi Blocks</td><td>OR-0173</td><td></td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Ajax Finechem</td><td>R18401O</td><td></td></tr><tr><td>Acetonitrile</td><td>VWR&amp;nbsp;</td><td>20060.35</td><td></td></tr><tr><td>Alkaline phosphatase</td><td>New England Biolabs</td><td>M0525S</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A1933</td><td></td></tr><tr><td>Cation-adjusted Mueller-Hinton Broth (CAMHB)</td><td>Becton, Dickinson and Company</td><td>212322</td><td></td></tr><tr><td>Cbz-L-Trp</td><td>Combi Blocks</td><td>QB-6691</td><td></td></tr><tr><td>Colistin&amp;nbsp;</td><td>Sigma Aldrich</td><td>C4461</td><td></td></tr><tr><td>Dey-Engley (D/E) Neutralising Broth</td><td>Oxoid</td><td>R453042</td><td></td></tr><tr><td>Diatomaceous earth (Celite 545)</td><td>Sigma Aldrich</td><td>102693</td><td>0.02-0.1 mm particle size</td></tr><tr><td>Dichloromethane (DCM)</td><td>VWR&amp;nbsp;</td><td>23366.327</td><td></td></tr><tr><td>Diethyl ether</td><td>Chem-Supply</td><td>EA036</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Chem-Supply Pty Ltd&amp;nbsp;</td><td>LCS-CHEM-0098</td><td></td></tr><tr><td>Dithiothreitol(DTT)</td><td>Thermo Scientific</td><td>J15397.03</td><td></td></tr><tr><td>DMEM culture medium</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>UCS-BIO-0058</td><td></td></tr><tr><td>E. coli BL21(DE3) host</td><td>Sigma Aldrich</td><td>70235-M</td><td></td></tr><tr><td>Electroporator</td><td>Thermo Scientific</td><td>NEON1SK</td><td></td></tr><tr><td>Ethyl acetate</td><td>Chem-Supply</td><td>EA011-2.5L-P</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Thermo Scientific</td><td>118432500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>12003C-500ML</td><td></td></tr><tr><td>His-tagged TEV Protease&amp;nbsp;</td><td>Sigma Aldrich</td><td>T4455</td><td></td></tr><tr><td>Hydroxybenzotriazole Hydrate</td><td>Chem-Implex</td><td>24755</td><td></td></tr><tr><td>Imidazole</td><td>Thermo Scientific</td><td>288-32-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758</td><td></td></tr><tr><td>Luria-Bertani Broth (LB),&amp;nbsp;</td><td>Thermo Scientific</td><td>12795027</td><td></td></tr><tr><td>Lysis Buffer&amp;nbsp;</td><td>Thermo Scientific</td><td>89822</td><td></td></tr><tr><td>Mercury (II) Chloride</td><td>Sigma Aldrich</td><td>215465</td><td></td></tr><tr><td>Methanol (HPLC Grage)</td><td>VWR</td><td>A452-4K</td><td></td></tr><tr><td>MTT reagent</td><td>Sigma Aldrich</td><td>M2128-100MG</td><td></td></tr><tr><td>N,N-Diisopropylethylamine (DIPEA)</td><td>Sigma Aldrich</td><td>3440</td><td></td></tr><tr><td>N,N-Dimethyl formamide (DMF)</td><td>Chem-Supply</td><td>RP1051</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-ONE</td><td></td></tr><tr><td>N-Boc-ethylenediamine</td><td>Combi blocks</td><td>AM-1613</td><td></td></tr><tr><td>NTA Affinity Resin Column</td><td>Sigma Aldrich</td><td>70666</td><td></td></tr><tr><td>Palladium on carbon</td><td>Sigma Aldrich</td><td>205699</td><td>10% w/w Pd</td></tr><tr><td>Plasmid Purification kit</td><td>QIAGEN</td><td>27104</td><td></td></tr><tr><td>Polypropylene microtiter tubes&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>72.690.008</td><td></td></tr><tr><td>Silica Gel for flash column chromatography</td><td>Silicycle</td><td>R12030B</td><td>Silica Gel 60, 230-400 mesh&amp;nbsp;</td></tr><tr><td>Silica gel Thin layer chromatography plate</td><td>Merk</td><td>1055540001</td><td>0.2 mm layer thickness on aluminium backing with 254 nm fluorescent indicator</td></tr><tr><td>Sodium phosphate, 0.2M buffer soln</td><td>Thermo Scientific</td><td>J63816.AP</td><td></td></tr><tr><td>Sterile flat-bottomed 96-well polypropylene microtiter plate with lid</td><td>Corning</td><td>3599</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>Triethylamine (Et3N)</td><td>Chem-Supply</td><td>TR02151000</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Sigma Aldrich</td><td>T6508</td><td></td></tr><tr><td>Tris-HCL</td><td>Thermo Scientific</td><td>J62848.AK</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific</td><td>A16046.AE</td><td></td></tr><tr><td>Trypan blue</td><td>MP BIOMEDICALS AUSTRALIA PTY LTD</td><td></td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Sigma Aldrich</td><td>T4049-500ML</td><td></td></tr><tr><td>Trypticase soya agar&amp;nbsp;</td><td>Becton, Dickinson and Company</td><td>211043</td><td></td></tr><tr><td>Urea&amp;nbsp;</td><td>Thermo Scientific</td><td>29700</td><td></td></tr><tr><td>Vector (T7 promoter)</td><td>-</td><td></td><td>high-copy number plasmid of choice</td></tr></tbody></table>

production and testing of antimicrobial peptides and their mimics

Production and Testing of Antimicrobial Peptides and Their Mimics

Antimicrobial peptides (AMPs) and their synthetic mimics are emerging as promising alternatives to conventional antibiotics, particularly in response to the growing threat of antimicrobial resistance. Naturally occurring AMPs are essential components of the innate immune system and display broad-spectrum activity against diverse microbial pathogens, while their synthetic mimics, including peptidomimetics and polymer-based analogs, are designed to enhance stability, reduce toxicity, and improve pharmacokinetics. This study aims to demonstrate the production, characterization, and biological evaluation of AMPs and small-molecule peptide mimics. AMPs are generated using recombinant expression systems or solid-phase peptide synthesis, enabling precise control over amino acid sequence and structural properties. Small-molecule peptide mimics are synthesized using chemical strategies, including an anthranilic acid (2-aminobenzoic acid) scaffold to enhance structural stability and pharmacological performance. Following synthesis, compounds are purified and chemically characterized prior to biological testing. Antimicrobial activity is evaluated using in vitro minimum inhibitory concentration (MIC) assays, and cytotoxicity is assessed against mammalian cell lines to determine selectivity. Representative results demonstrate that both AMPs and their mimics exhibit potent activity against Klebsiella pneumoniae and Escherichia coli, highlighting their broad-spectrum antibacterial potential. Overall, this protocol provides a comprehensive and reproducible framework for the development and systematic evaluation of antimicrobial peptides and their mimics as next-generation anti-infective agents.

Synthesis of RK758 
Analytical data up to intermediate 7 were published previously14. The analytical data for RK758 and intermediate 8–9 are as follows:
tert-butyl (2-(3-phenylthioureido)ethyl)carbamate (8): 
The product 8 was obtained as an off-white solid (1.89 g, 87%); m.p. 167.4–168.3 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.58 (s, 1H), 7.71 (s, 1H), 7.44–7.29 (m, 4H), 7.11 (t, J = 12.0 Hz, 1H), 6.88 (br s, 1H), 3.55-3.53 (m, 2H), 3.13 (q, J = 4.0 Hz, 2H), 1.59 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ 180.9, 156.3, 139.5, 129.1, 124.7, 123.7, 78.2, 44.3, 28.7; HRMS (ESI+): m/z calculated for C14H21N3O2SNa+ [M + Na]+: 318.1247, found: 318.1252.
tert-butyl (S,Z)-(3-((1H-indol-3-yl)methyl)-1-(2-(2-naphthamido)-5-bromophenyl)- 1,4-dioxo-9-(phenylimino)-2,5,8,10-tetraazadodecan-12-yl)carbamate (9): 
The product 9 was obtained as a white solid (0.253 g, 62%); m.p. 173.4–174.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.86 (s, 1H), 9.61 (s, 1H), 9.27 (d, J = 8.0 Hz, 1H), 8.57-8.54 (m, 2H), 8.44 (s, 1H), 8.11 (d, J = 4.0 Hz, 1H), 8.06-8.03 (m, 3H), 7.84 (dd, J = 4.0, 12.0 Hz, 1H), 7.78 (d, J = 12.0 Hz, 2H), 7.69–7.60 (m, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.27-7.23 (m, 5H), 7.01- 6.97 (m, 3H), 4.82-4.78 (m, 1H), 3.14-3.13 (m, 10H), 1.32 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 172.3, 167.8, 165.1, 156.3, 154.4, 138.9, 136.5, 135.4, 134.9, 132.6, 131.7, 129.9, 129.1, 128.7, 128.4, 128.2, 127.6, 127.6, 126.4, 124.8, 124.1, 123.6, 122.8, 121.4, 118.9, 118.7, 115.1, 111.8, 110.9, 78.4, 55.2, 42.3, 41.9, 39.4, 38.3, 29.5, 28.6, 27.9; HRMS (ESI+): m/z calculated for C45H47BrN8O5 [M + H]+: 859.2926, found: 859.2925.
(S,E)-N-(2-((1-((2-(3-(2-aminoethyl)-2-phenylguanidino)ethyl)amino)-3-(1H-indol- 3-yl)-1-oxopropan-2-yl)carbamoyl)-4-bromophenyl)-2-naphthamide (RK758) (10): 
RK758 was obtained as an off-white solid (0.039 g, 75%); m.p. 240.0–240.9 °C; 1H NMR (400 MHz, MeOD-d4): δ 8.39 (s, 1H), 8.27 (d, J = 12.0 Hz, 1H), 7.98–7.95 (m, 3H), 7.88 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 4.0 Hz, 1H), 7.71-7.60 (m, 4H), 7.40 (t, J = 8.0 Hz, 1H), 7.30-7.20 (m, 4H), 7.16 (s, 1H), 7.05-6.97 (m, 2H), 4.88-4.86 (m, 1H), 3.33-3.32 (m, 3H), 3.27-3.24 (m, 5H); 13C NMR (100 MHz, MeOD-d4): δ 174.3, 168.5, 166.4, 154.6, 137.1, 136.7, 135.1, 134.7, 131.4, 132.6, 131.3, 130.8, 129.8, 128.9, 127.97, 127.8, 127.9, 127.6, 127.5, 127.2, 126.8, 125.6, 123.5, 123.24, 123.2, 118.6, 117.8, 116.1, 111.1, 109.6; HRMS (ESI+): m/z calculated for C40H39BrN8O3 [M + H]+: 759.2401, found: 759.2407.
The synthesis of RK758 was conducted with an overall yield of 17% from Cbz-L-Tryptophan in 7 steps (Figure 4). The overall yield of this process was 44% to key intermediate 7 and 19% to RK758. The 1H NMR data indicate high purity of the final compound with no major detectable impurities.
Colony PCR screening confirmed successful insertion of the Mel4 and His-KSI-Mel4 cassettes in selected transformants (Figure 5A). PCR analysis of miniprep-isolated plasmids further verified the expected amplicon size, supporting correct construct assembly prior to transformation into BL21(DE3) for expression (Figure 5B).
Recombinant Production and Purification of Highly Cationic Antimicrobial Peptide Mel4 
Although Mel4 was obtained commercially from AusPep Peptide Company with a minimum purity of 90%, it was synthesized by SPPS, and a recombinant approach was also employed to produce Mel4 biologically. At the end of the protocol, Western blot analysis of the Total cell protein (TCP) from the lysed cell pellet confirmed the expression of the recombinant HIS-KSI-Mel4 fusion protein. The protein samples were separated by SDS-PAGE and visualized using a protein ladder as a molecular weight reference. A distinct band corresponding to the expected molecular weight of the HIS-KSI-Mel4 fusion protein was observed, confirming successful expression of the target protein in the host cells (Figure 5B).
From the protein sequence of the selected motif, the theoretical amino acid composition was calculated and compared with the experimental amino acid analysis results obtained from the purified HIS-KSI-TEV-Mel4 IB peptide. The comparison showed good correlation between the predicted and experimentally determined amino acid ratios, confirming the accuracy of the expressed peptide sequence and its successful isolation in the inclusion body fraction (Table 2).
Asparagine is converted to aspartic acid and glutamine to glutamic acid. Amino acid analysis method is insensitive to cysteine and tryptophan and those are not analyzed by this method. This consideration must be made in analyzing the results.
Antibacterial activity of Mel4 and RK758 
The minimum inhibitory concentrations (MICs) of RK758, Mel4, and colistin were evaluated against two Gram-negative bacterial strains, K. pneumoniae JIE 2709 and E. coli NCTC 13846 (Table 3). RK758 exhibited an MIC of 16 µg/mL (21 µM) against both K. pneumoniae and E. coli. In comparison, Mel4 showed MICs of 62.5 µg/mL (26.6 µM) for K. pneumoniae and 125 µg/mL (53.2 µM) for E. coli, indicating lower potency than RK758. Colistin was used as a comparator antibiotic, demonstrating the lowest MIC of 1 µg/mL (0.87 µM) for K. pneumoniae, while the colistin-resistant E. coli reference strain exhibited an MIC of 8 µg/mL (6.9 µM).
Cytotoxicity effect of Mel4 and RK758 
The cytotoxic potential of Mel4 and RK758 was assessed in L929 mouse fibroblast cells (ATCC CCL-1) using the MTT assay following 24 h exposure to serial concentrations. Both compounds exhibited typical sigmoidal dose-response curves analyzed by nonlinear regression with the “inhibitor vs. normalized response - variable slope” model in GraphPad Prism (version 9.5.1), confirming concentration-dependent inhibition of cell viability. RK758 displayed a CC50 value of 172.6 µM, whereas Mel4 showed slightly higher cytotoxic potency with a CC50 of 147.5 µM, representing approximately 1.17-fold greater cytotoxicity than RK758 (Figure 6 and Figure 7).
<img alt="Gene expression diagram with T7 promoter, KSI fusion protein, and T7 terminator in plasmid map." class="xfigimg" src="/files/ftp_upload/70178/70178fig1.jpg" data-altupdatedat="1779768095000" data-alt="Figure 1"> 
Figure 1: A schematic representation of the expression cassette of p15TV-L-His-KSI-Mel4. Schematic map of the p15TV-L expression construct showing the T7 promoter, ribosome binding site (RBS), 6×His purification tag, ketosteroid isomerase (KSI) fusion partner, TEV protease cleavage site, and the Mel4 peptide sequence followed by the T7 transcription terminator. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE gel electrophoresis; protein separation; ladder, post-filtration, flow-through; kDa analysis." class="xfigimg" src="/files/ftp_upload/70178/70178fig2.jpg" data-altupdatedat="1779768095000" data-alt="Figure 2"> 
Figure 2: SDS-PAGE and western blot analysis of recombinant protein expression. (A) SDS-PAGE of inclusion bodies and total cell proteins after filtration and flow-through of crude cell lysate. (B) Western blot of total cell protein. Green arrow indicates the position of the protein of interest at the expected molecular weight. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="BCA protein quantification bar chart, protein concentration, inclusion body vs. Tev cleavage comparison." class="xfigimg" src="/files/ftp_upload/70178/70178fig3.jpg" data-altupdatedat="1779768095000" data-alt="Figure 3"> 
Figure 3: Quantification of recombinant protein before and after TEV cleavage. Bar graph showing protein concentrations determined by BCA assay for the isolated inclusion body fraction and the cleaved recombinant Mel4 obtained after TEV protease treatment. Error bars represent experimental variation between measurements. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Synthesis pathway diagram for RK758, showcasing chemical reactions, equations, and intermediates." class="xfigimg" src="/files/ftp_upload/70178/70178fig4.jpg" data-altupdatedat="1779768095000" data-alt="Figure 4"> 
Figure 4: Overall synthetic approach to obtain RK758. Reaction scheme outlining the seven-step chemical synthesis of RK758 starting from Cbz-L-tryptophan, including intermediate formation, functional group transformations, and final deprotection to obtain the peptide mimic. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electrophoresis gel result showing DNA ladder and His-KSI-Mel4 bands for size comparison." class="xfigimg" src="/files/ftp_upload/70178/70178fig5.jpg" data-altupdatedat="1779768095000" data-alt="Figure 5"> 
Figure 5: Agarose gel electrophoresis of recombinant plasmids. (A) Colony PCR of transformants for His-Mel4 and His-KSI-Mel4. (B) PCR confirmation of plasmids after miniprep. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability graph vs concentration; CC&lt;sub&gt;50&lt;/sub&gt; 172.6 µM; half-maximal effect on cells." class="xfigimg" src="/files/ftp_upload/70178/70178fig6.jpg" data-altupdatedat="1779768095000" data-alt="Figure 6"> 
Figure 6. Cytotoxicity of RK758 against L929 fibroblast cells. Dose-response curve showing the effect of RK758 on cell viability across increasing concentrations. CC50 values were calculated by nonlinear regression using the “inhibitor vs. normalized response - variable slope” model in GraphPad Prism, confirming concentration-dependent inhibition of cell viability. Data represents the mean ± SD of three independent biological replicates. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability vs. concentration graph, CC50 at 147.5 μM, log scale, cytotoxicity analysis results." class="xfigimg" src="/files/ftp_upload/70178/70178fig7.jpg" data-altupdatedat="1779768095000" data-alt="Figure 7"> 
Figure 7: Cytotoxicity of Mel4 against L929 cells. Dose-response curve showing the concentration-dependent effect of Mel4 on cell viability. CC50 values were calculated by nonlinear regression using the “inhibitor vs. normalized response - variable slope” model in GraphPad Prism, confirming concentration-dependent inhibition of cell viability. Data represents the mean ± SD of three independent biological replicates. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemical synthesis diagram showing Suzuki cross-coupling steps, reactions, compounds, yields." class="xfigimg" src="/files/ftp_upload/70178/70178fig8.jpg" data-altupdatedat="1779768095000" data-alt="Figure 8"> 
Figure 8: Modified synthetic strategy starting from Cbz-L-tryptophan. Alternative synthetic pathway illustrating the preparation of anthranilamide intermediates using Cbz-L-tryptophan and subsequent functionalization steps to enable structural diversification. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemical reaction diagram of imidazole synthesis using CH3I and amine, showing structural changes." class="xfigimg" src="/files/ftp_upload/70178/70178fig9.jpg" data-altupdatedat="1779768095000" data-alt="Figure 9"> 
Figure 9: Alkylation of thiourea followed by nucleophilic substitution with an amine substrate. Proposed reaction pathway involving alkylation of the thiourea sulfur followed by nucleophilic substitution with an amine substrate to generate guanidine-containing products. <a href="https://www.jove.com/files/ftp_upload/70178/70178fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Section</td>
			<td>Name/Feature</td>
			<td colspan="4">Sequence</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>XbaI_KNRPR_Start</td>
			<td colspan="4">5’-TAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATT-3’</td>
		</tr>
		<tr>
			<td></td>
			<td>T7_TERM_REV</td>
			<td colspan="4">5’-CCAAGGGGTTATGCTAGTTATTGCTCA-3’</td>
		</tr>
		<tr>
			<td>Gene cassette</td>
			<td>His-KSI-Mel4 amino acid sequence</td>
			<td colspan="4">MDHHHHHHMIDETQRKATVLEYFERVNAKDLDGVVKLF 
			ATDAVVADPVGAPPVAGEEALRAYFQRVLHEFDTHDVPGV 
			PSGAQDGQSVALPLKATINNPQDPTGGVRLDVNLVSVFTIG 
			EDGLISEMRAYWGLTDIAPAGASSGRENLYFQGKNKRKRRR 
			RRRGGRRRR</td>
		</tr>
		<tr>
			<td></td>
			<td>DNA sequence</td>
			<td colspan="4">5’-TAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA 
			GATATACCATGGATCACCACCACCATCACCACATGATTGACGAGAC 
			GCAACGTAAAGCGACGGTCTTAGAGTATTTTGAACGCGTGAATGCAA 
			AAGACTTGGATGGCGTGGTTAAACTCTTTGCGACGGACGCCGTTGT 
			AGCCGACCCTGTTGGTGCACCACCAGTGGCTGGTGAGGAGGCACTC 
			CGTGCCTACTTCCAGCGCGTCTTGCATGAATTTGACACCCATGATGTCC 
			CGGGCGTTCCATCTGGTGCTCAGGATGGCCAATCAGTCGCACTCCCAC 
			TTAAAGCAACCATCAACAATCCTCAAGATCCAACAGGCGGGGTCCGCC 
			TTGATGTGAACTTAGTGAGCGTATTCACGATTGGCGAAGACGGGTTAAT 
			CTCCGAGATGCGTGCGTACTGGGGGTTGACTGATATTGCTCCGGCAGGT 
			GCAAGCAGTGGTCGCGAGAACTTGTACTTCCAAGGTAAGAACAAGCGTA 
			AGAGACGCCGGCGCCGCCGGGGAGGACGGAGACGTCGCTAATCGAAC 
			TCGAGATCCGGCT-3’</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences used for PCR verification and full gene cassette sequence of the final plasmid construct.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aminoacid</td>
			<td>Calculated</td>
			<td>Tested</td>
		</tr>
		<tr>
			<td>Histidine </td>
			<td>4.70%</td>
			<td>4.70%</td>
		</tr>
		<tr>
			<td>Serine</td>
			<td>3.60%</td>
			<td>3.40%</td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>10.70%</td>
			<td>9.20%</td>
		</tr>
		<tr>
			<td>Glycine </td>
			<td>9.50%</td>
			<td>11%</td>
		</tr>
		<tr>
			<td>Aspartic acid</td>
			<td>11.30%</td>
			<td>11.50%</td>
		</tr>
		<tr>
			<td>Glutamic acid </td>
			<td>8.90%</td>
			<td>8.90%</td>
		</tr>
		<tr>
			<td>Threonine </td>
			<td>4.70%</td>
			<td>4.60%</td>
		</tr>
		<tr>
			<td>Alanine</td>
			<td>9.50%</td>
			<td>9.70%</td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>5.30%</td>
			<td>5.30%</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>4.10%</td>
			<td>4.50%</td>
		</tr>
		<tr>
			<td>Tyrosine</td>
			<td>2.40%</td>
			<td>2.20%</td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>1.80%</td>
			<td>1.90%</td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>3.00%</td>
			<td>3.20%</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>7.10%</td>
			<td>7.40%</td>
		</tr>
		<tr>
			<td>Phenyalanine</td>
			<td>3.60%</td>
			<td>3.60%</td>
		</tr>
		<tr>
			<td>Valine</td>
			<td>9.50%</td>
			<td>8.90%</td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>0.60%</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2: Analysis of amino acid compositions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Bacterial strains</td>
			<td colspan="3">MIC µg/mL (µM)</td>
		</tr>
		<tr>
			<td>RK758</td>
			<td>Mel4</td>
			<td>Colistin</td>
		</tr>
		<tr>
			<td>K. pneumoniae JIE 2709</td>
			<td>16 (21)</td>
			<td>62.5 (26.6)</td>
			<td>1 (0.87)</td>
		</tr>
		<tr>
			<td>E. coli NCTC 13846</td>
			<td>16 (21)</td>
			<td>125 (53.2)</td>
			<td>8 (6.9)</td>
		</tr>
	</tbody>
</table>
Table 3: MIC of RK758, Mel4, and colistin against Gram-negative bacteria.

This protocol describes a method for producing and evaluating the antimicrobial activity and cytotoxicity of the cationic peptide Mel4 and peptide mimic RK758. By integrating chemical synthesis, recombinant expression, and standardized functional assays, it provides a streamlined workflow for the production and testing of peptides and small-molecule mimics.

Antimicrobial peptides (AMPs) and their synthetic mimics are emerging as promising alternatives to conventional antibiotics, particularly in response to ...

This protocol describes a method for synthesizing and testing the antimicrobial activity and cytotoxicity of cationic peptide Mel4 and a peptide mimic RK-758. Previously published work has been fragmented and non-standardized, limiting reproducibility and translation. This study integrates synthesis, biological testing, and mechanistic studies into a unified workflow.To begin, obtain the anthranilamide peptide mimic RK-758 and the highly cationic antimicrobial peptide Mel4. Dissolve the peptides in sterile water at a high concentration of 20 millimolar and filter sterilize the peptide stocks using a 0.22 micrometer syringe filter. Streak the bacterial isolate to be tested onto trypticase soy agar to obtain pure and well isolated colonies.Incubate the plate aerobically at around 36 degrees Celsius for 18 to 24 hours. The next day, select three to five morphologically similar and well isolated colonies using a sterile inoculating loop. Transfer the colonies into a sterile tube containing five to 10 milliliters of cation adjusted Mueller Hinton broth Gently rubbed the loop against the inner wall of the tilted tube to obtain a homogeneous suspension.Incubate the broth culture at around 36 degrees Celsius while shaking at 180 revolutions per minute for 18 to 24 hours. After incubation, centrifuge the overnight culture at 3000 G for 10 minutes at 25 degrees Celsius. Discard the supernatant and wash the bacterial pellet three times with sterile PBS.Dispense two milliliters of PBS into a sterile container. Add 50 to 100 microliters of the washed bacterial suspension and vortex for five to 10 seconds. Add 200 microliters of this bacterial suspension into a 96 well plate and add an equal volume of PBS as a blank control.Using a spectrophotometer, measure the optical density at 600 nanometers. The desired absorbance value is 0.1, corresponding to approximately one to two times 10 to the power of eight colony forming units per milliliter. Dilute the adjusted bacterial suspension one to 100 in the cat ion adjusted Mueller Hinton broth to obtain a final inoculum of about 10 to the power of six colony forming units per milliliter.Label a sterile flat bottomed 96 well plate with the bacterial strain antimicrobial peptide name, date, and the scientist initials. Mark the locations for test, blank and control wells on the plate lid. Dispense 200 microliters of antimicrobial peptide solution at the highest concentration into the first column of the plate, and then add 100 microliters of cation adjusted Mueller Hinton broth to the remaining wells from column two to column 12.Using a multichannel pipette, perform twofold serial dilutions of the antimicrobial peptide from column one through column 10. Next, add 100 microliters of the prepared bacterial inoculum at 10 to the power of six colony forming units per milliliter to each well except the blank wells. Dispense 100 microliters of cation unadjusted Mueller Hinton broth to the blank wells to reach a final volume of 200 microliters.Cover the plate with its lid and seal with parafilm to prevent evaporation. Incubate the plate at 36 degrees Celsius for 18 to 24 hours. After incubation, measure the optical density at 600 nanometers of each well using a microplate reader.Calculate the percentage reduction in growth relative to the untreated growth control and identify the minimum inhibitory concentration. Then determine the minimum bactericidal concentration. Seed L-929 mouse fibroblast cells in a 75 square centimeter flask containing DMEM supplemented with 10%FBS and 1%antibiotics including streptomycin sulfate and penicillin G.Incubate the cells at 37 degrees Celsius with 5%carbon dioxide until 75 to 80%confluency is reached.Once confluent, wash the cells with PBS. Then add 0.25%trypsin EDTA to detach the cells. Mix 50 microliters of cell suspension with 50 microliters of 0.4%trypan blue and load 10 microliters of the mixture into a hemocytometer.Count viable unstained cells and dead blue cells in the four corner squares of the hemocytometer and calculate the cell viability using the formula. Next, adjust the final cell concentration to five times 10 to the power of four cells per milliliter using DMEM. Seed 100 microliters of the cell suspension into each well of a sterile 96 well plate and incubate for 24 hours at 37 degrees Celsius with 5%carbon dioxide to allow cell adherence.Next, prepare working solutions of Mel4 and RK-758 in DMEM without FBS and antibiotics using the previously prepared stock solution. Now, remove the culture medium from each well and add 100 microliters of the corresponding working solution to each well. Then add 100 microliters of DMEM as negative control, DMSO as positive control and PBS as blank control to the corresponding wells.After 24 hours of Mel4 or RK-758 exposure, aspirate the test media carefully without disturbing the L-929 mouse fibroblast cells. Prepare a 0.5 milligram per milliliter MTT working solution by diluting the five milligrams per milliliter stock solution nine times with plain DMEM. Add 100 microliters of the prepared MTT working solution to each well.Cover the plate with aluminum foil and incubate for two hours at 37 degrees Celsius until purple formazan crystals form. After incubation, examine the cells for violet precipitation under the microscope. Remove the MTT solution carefully from each well and add 100 microliters of DMSO to each well to dissolve the formazan crystals.Place the plate on a shaker for five minutes to ensure complete dissolution and measure the absorbance at 570 nanometers using a microplate reader. After subtracting the blank absorbance values, calculate cell viability using the equation and then calculate IC50 or CC50 values. RK-758 exhibited a minimum inhibitory concentration of 16 micrograms per milliliter against both Klebsiella.pneumoniae JIE 2709 and Escherichia. coli NCTC 13846 bacterial strains. Mel4 showed minimum inhibitory concentrations of 62.5 micrograms per milliliter for K.Pneumoniae and 125 micrograms per milliliter for E.Coli.Colistin, which was used as a comparative antibiotic demonstrated the lowest minimum inhibitory concentration of one microgram per milliliter for K.pneumoniae and eight micrograms per milliliter for colistin resistant E.coli reference strain. RK-758 showed concentration dependent inhibition of cell viability in L-929 fibroblast cells with a CC50 value of 172.6 micromolar Mel4 showed slightly higher cytotoxicity than RK-758 in L-929 cells with a CC50 value of 147.5 micromolar. This protocol demonstrates the scalable production of the peptide mimic RK-758 and evaluation of peptides and their mimics for further drug discovery.Follow up analysis could include mechanistic studies, stability testing, formulation optimization, resistance development, and advanced efficacy studies in relevant models. Further studies could seek greener production of peptide mimics, improve cost effectiveness, and evaluate in vivo efficacy, safety, and clinical translation.

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Research

JoVE Journal

Biology

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

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Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

Learn a unified protocol for synthesizing and testing antimicrobial peptides and peptide mimics using Mel4 and RK-758. This method enables reproducible evaluation of antibacterial activity and cytotoxicity for drug discovery.

Production and Testing of Antimicrobial Peptides and Their Mimics

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