Peptides have positively impacted the pharmaceutical industry as drugs, biomarkers, or diagnostic tools of high therapeutic value. However, only a handful have progressed to the market. Toxicity is one of the main obstacles to translating peptides into clinics. Hemolysis or hemotoxicity, the principal source of toxicity, is a natural or disease-induced event leading to the death of vital red blood cells. Initial screenings for toxicity have been widely evaluated using erythrocytes as the gold standard. More recently, many online databases filled with peptide sequences and their biological meta-data have paved the way toward hemolysis prediction using user-friendly, fast-access machine learning-driven programs. This review details the growing contributions of in silico approaches developed in the last decade for the large-scale prediction of erythrocyte lysis induced by peptides. After an overview of the pharmaceutical landscape of peptide therapeutics, we highlighted the relevance of early hemolysis studies in drug development. We emphasized the computational models and algorithms used to this end in light of historical and recent findings in this promising field. We benchmarked seven predictors using peptides from different data sets, having 7–35 amino acids in length. According to our predictions, the models have scored an accuracy over 50.42% and a minimal Matthew’s correlation coefficient over 0.11. The maximum values for these statistical parameters achieved 100.0% and 1.00, respectively. Finally, strategies for optimizing peptide selectivity were described, as well as prospects for future investigations. The development of in silico predictive approaches to peptide toxicity has just started, but their important contributions clearly demonstrate their potential for peptide science and computer-aided drug design. Methodology refinement and increasing use will motivate the timely and accurate in silico identification of selective, non-toxic peptide therapeutics.

Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20-32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD- and the sequence motifs -VALGAVSK- or -GKAAL(N/G/S) (V/A)V- in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3-fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11μM). CZS-1 reached 100% haemolysis at 120.87μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application.

Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. In addition, eight novel peptides were identified, including four dermaseptins, DRS-SP2 to DRS-SP5, one phylloseptin-SP1, and three orphan peptides. Phylloseptin-SP1 and dermaseptins-SP2 were identified in HPLC fractions based on their molecular masses determined by MALDI-TOF MS. Among the antimicrobial peptides, dermaseptin-SP2 was the most potent, inhibiting Escherichia coli, Staphylococcus aureus, and ORSA with a minimum inhibitory concentration (MIC) of 2.68 μM, and Candida albicans with an MIC of 10.71 μM, without haemolytic effects. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. spurrelli.

Antimicrobial peptides have been developed based on plant-derived molecular scaffolds for the treatment of infectious diseases. Chenopodin is an abundant seed storage protein in quinoa, an Andean plant with high nutritional and therapeutic properties. Here, we used computer- and physicochemical-based strategies and designed four peptides derived from the primary structure of Chenopodin. Two peptides reproduce natural fragments of 14 amino acids from Chenopodin, named Chen1 and Chen2, and two engineered peptides of the same length were designed based on the Chen1 sequence. The two amino acids of Chen1 containing amide side chains were replaced by arginine (ChenR) or tryptophan (ChenW) to generate engineered cationic and hydrophobic peptides. The evaluation of these 14-mer peptides on Staphylococcus aureus and Escherichia coli showed that Chen1 does not have antibacterial activity up to 512 µM against these strains, while other peptides exhibited antibacterial effects at lower concentrations. The chemical substitutions of glutamine and asparagine by amino acids with cationic or aromatic side chains significantly favoured their antibacterial effects. These peptides did not show significant hemolytic activity. The fluorescence microscopy analysis highlighted the membranolytic nature of Chenopodin-derived peptides. Using molecular dynamic simulations, we found that a pore is formed when multiple peptides are assembled in the membrane. Whereas, some of them form secondary structures when interacting with the membrane, allowing water translocations during the simulations. Finally, Chen2 and ChenR significantly reduced SARS-CoV-2 infection. These findings demonstrate that Chenopodin is a highly useful template for the design, engineering, and manufacturing of non-toxic, antibacterial, and antiviral peptides.