Universidade Catolica De Brasilia

In recent decades, the methylotrophic yeast Komagataella phaffii has emerged as a powerful host for the heterologous production of antimicrobial peptides (AMPs) as an economical and scalable platform. K. phaffii combines several important advantages for a recombinant expression system, such as rapid growth, high-density cell culture, efficient protein secretion, and the ability to perform essential post-translational modifications. The methanol-inducible AOX1 promoter (P_AOX1), generally employed through the pPICZalpha vector, allows strong and tightly regulated heterologous expression and is a central factor in the expression system of this yeast. However, several strategies have been employed to produce recombinant AMPs in K. phaffii, among them codon optimization, engineered methanol-resistant strains, alternative promoters, and the use of different secretory peptides. This review highlights the latest advances and practical considerations in the use of K. phaffii for AMP production, discussing challenges such as peptide stability, proteolytic degradation, and yield optimization. The insights provided contribute to the expansion of biotechnological applications of K. phaffii, reinforcing its potential as an efficient and safe platform for large-scale production of therapeutic AMPs.

Novel solutions to combat the rapid evolution of antimicrobial resistance in human and animal pathogens are urgently required. Antimicrobial peptides (AMPs) represent promising therapeutic molecules, as they exhibit structural nuances and distinct molecular targets against pathogenic microorganisms. In this Review, we explore the multifaceted structural nature of AMPs and advanced structural conformations, discuss the distinct mechanisms of action and explore novel targets. Additionally, we discuss resistance mechanisms, cross-resistance and innovative strategies for AMP design and optimization. We argue that gaining insight into novel AMP structural arrangements, targets and design optimization is crucial for the development of innovative therapies that can be translated into clinical as well as broader applications.

Cutaneous melanoma is an aggressive skin cancer with high metastatic potential and frequent resistance to apoptosis-inducing therapies. Ibrutinib (IBR), a Bruton's tyrosine kinase inhibitor, has demonstrated antitumor potential, yet its topical application is not well established. This study evaluated the cytotoxic, antiproliferative, and skin permeation effects of free IBR and IBR-loaded nanostructured lipid carriers (IBR + NLC) using human melanoma cells (SK-MEL-28) and ex vivo human skin models. NLC + IBR presented enhanced toxicity towards melanoma cells by promoting necroptosis, as evidenced by the absence of caspase-3/7 activation, increased expression of RIPK1 and RIPK3, elevated LDH release, and plasma membrane disruption. In addition, mitochondrial depolarization was observed, likely as a consequence of necroptotic signaling and associated cellular stress. NLC + IBR significantly enhanced reactive oxygen species production, induced cell cycle arrest in the sub-G1 phase, and reduced CDK1 and CDK2 gene expression. Clonogenic and migration assays confirmed complete suppression of tumor cell colony formation and reduced migratory capacity following NLC + IBR treatment. Skin permeation studies revealed that NLC + IBR enabled deeper drug skin penetration, with reduced retention in the stratum corneum and higher accumulation in the viable skin layers, the primary therapeutic target in cutaneous melanoma. Finally, using an ex vivo human skin model of melanoma, NLC + IBR promoted melanoma cell necrosis, while maintaining a favorable topical safety profile with no significant irritation. These findings suggest that NLC + IBR topical application is a promising strategy for localized treatment of cutaneous melanoma, particularly in early-stage or adjuvant contexts.