The research internship supported by the European Peptide Society (EPS) Mobility Fellowship was conducted at the Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal (UKZN), Durban, South Africa, under the supervision of Professor Fernando Albericio and Professor Beatriz G. de la Torre. The fellowship spanned a six-month period, from March to September 2025, concluding on September 16th. The objective of this stay was to design, synthesize, and scale up new analogues of the antimicrobial peptide B1CTcu5, a lead compound previously identified for its potent activity against Mycobacterium tuberculosis. This internship represented a critical experimental stage within my doctoral research, aimed at generating optimized analogues with improved pharmacological properties and producing sufficient material for in vivo infection and treatment studies scheduled to take place in Brazil in the following months.

The work developed during the fellowship integrated chemical synthesis, analytical characterization, and process optimization for scale-up production. Initially, rational peptide design strategies were applied, guided by structure–activity relationship data obtained from previous computational simulations. The modifications were intended to improve amphipathicity, hydrophobic balance, and net positive charge distribution, factors known to influence peptide–membrane interactions and stability in biological media. Several analogues were designed, including CRDK22- s18G, CRDK22-s16, and new truncated variants of B1CTcu5, with systematic residue substitutions aimed at enhancing selectivity and stability while maintaining antimycobacterial potency.

Peptides were synthesized through Fmoc/tBu solid-phase peptide synthesis using optimized coupling cycles to ensure high yields in sequences containing hydrophobic and bulky residues. Special attention was given to the coupling of leucine, isoleucine, and lysine, which often determine theamphipathic structure critical for biological performance. The crude peptides were cleaved using TFA-based cocktails containing scavengers tailored for each sequence to minimize oxidation and side reactions. Analytical and preparative high-performance liquid chromatography (HPLC) were performed to assess purity and isolate the target compounds at scales ranging from 100 mg to several grams. Mass verification was achieved through MALDI-TOF and ESI-MS, confirming the molecular weights and integrity of each product. A significant achievement of the internship was the successful development of a reproducible and scalable synthesis workflow that can be implemented in future production batches for preclinical use. This workflow now forms the foundation for the manufacturing of peptide material to be used in murine infection models to evaluate both therapeutic efficacy and host immune response modulation.

During this period, truncated peptides were also generated to identify the minimal sequence motifs responsible for antimycobacterial activity. These fragments allowed the assessment of hydrophobic cluster contribution, charge density, and overall secondary structure stability in aqueous and membrane-mimicking environments. The insights gained from these studies are being integrated into the design of future analogues with improved pharmacokinetic properties and reduced synthetic cost. The optimized analogues synthesized at UKZN will be transported to Brazil for forthcoming in vivo infection studies in M. tuberculosis-infected mice at the UNESP BSL-3 facility, where the evaluation will include bacterial load quantification, granuloma reduction, and immune cytokine profiling. I also had the opportunity to attend The European Peptide Synthesis Conference of 2025 (EPSC 2025) held in Porto, Portugal, together with Professors Fernando Albericio and Beatriz G. de la Torre. This participation strengthened our collaborative links with the European peptide community and provided valuable opportunities to present and discuss the ongoing results derived from this fellowship.

In addition to the experimental synthesis component, the fellowship facilitated an interdisciplinary exchange between chemical synthesis, molecular modeling, and microbiological validation. This collaborative approach was instrumental in strengthening ongoing research links between UKZN, UNESP, and iMed.ULisboa, contributing to a consolidated international network for peptide-based drug discovery. The interaction with the Albericio research group provided valuable experience in large-scale synthesis, advanced purification, and peptide stability studies, complementing the microbiological expertise at UNESP and the structural–computational insight at the University of Lisbon. The fellowship thus bridged the three pillars of my research—design, synthesis, and biological evaluation—under a unified translational perspective.

The internship yielded substantial scientific outcomes. Among them, a key publication derived directly from this collaboration: Integrating Docking, Dynamics, and Assays to Predict AntimicrobialPeptide Interactions with Mycolic Acid Membranes in Mycobacterium tuberculosis, published in ACS Measurement Science Au (DOI: 10.1021/acsmeasuresciau.5c00126). This article integrates computational and experimental methodologies to predict peptide–membrane interactions and was finalized during the EPS fellowship period. Additionally, two other major works that emerged from the extended research collaboration were published in Advanced Science: “Repositioning Antimicrobial Peptides Against WHO-Priority Fungi” and “Antimicrobial Peptides: A Promising Alternative to Conventional Antimicrobials for Combating Polymicrobial Biofilms”. These publications collectively reinforce the international recognition of the project and demonstrate the continuity of collaborative outputs initiated during the EPS-funded stay.

The scientific impact of this fellowship extended beyond peptide synthesis. The training period enhanced my expertise in peptide design and analytical validation, while also deepening my understanding of peptide–membrane dynamics, structural stability, and degradation pathways—key aspects for the rational development of bioactive peptides with clinical potential. The skills acquired will be fundamental for the translational phase of the project, particularly for ensuring reproducibility and consistency in preclinical assays. Furthermore, this internship served as a platform for knowledge transfer, as the protocols optimized in South Africa will now be implemented in Brazil for future synthesis, stability testing, and infection trials.

The EPS fellowship also contributed to the expansion of international collaboration and scientific visibility. Working within an established peptide chemistry hub like the UKZN Peptide Science Laboratory provided access to state-of-the-art facilities, expert mentorship, and an interdisciplinary environment that fostered innovation. The fellowship reinforced ongoing cooperation with leading peptide scientists, including Prof. Albericio, Prof. de la Torre, and Prof. Pavan, as well as collaborative links with European institutions such as the University of Copenhagen and the University of Lisbon. These connections are now facilitating new initiatives, including the design of targeted peptide conjugates and the development of peptide-based bacPROTACs against Mycobacterium tuberculosis.

In summary, the EPS-funded internship at UKZN represented a pivotal step in advancing my doctoral research and consolidating international collaboration in peptide drug discovery. The technical achievements—ranging from the synthesis of new analogues and truncated peptides to the establishment of preparative-scale production—provide the experimental foundation for the ongoing in vivo phase in Brazil. The scientific outputs generated, including the publication in ACS Measurement Science Au, demonstrate the productivity and relevance of this fellowship. Most importantly, the experience strengthened my capacity as a young researcher, expanding both my technical and conceptual understanding of peptide chemistry applied to infectious diseases.

Final Remarks

The European Peptide Society Mobility Fellowship has been instrumental in my professional development, providing an exceptional platform to connect experimental synthesis with translational biomedical research. I express my deepest gratitude to the EPS for their support, to Professors Fernando Albericio and Beatriz G. de la Torre for their mentorship and guidance, and to Professors Fernando Rogério Pavan and João Perdigão for their ongoing collaboration and supervision. The outcomes of this internship will directly contribute to the next stage of preclinical peptide development against multidrug-resistant Mycobacterium tuberculosis, reinforcing the mission of the EPS to promote excellence and collaboration in peptide science across continents. The publications derived from this work are included as annexes to this report.

Advanced Science – 2024 – Roque‐Borda – Antimicrobial Peptides A Promising Alternative to Conventional Antimicrobials for

Advanced Science – 2025 – Roque‐Borda – Repositioning Antimicrobial Peptides Against WHO‐Priority Fungi

ACS meass Au R2 clean version