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IsCT1‐NH2 is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α‐helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)‐based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out.Peptides with a substitution at position 3 or 5 ([L]3‐IsCT1‐NH2, [K]3‐IsCT1‐NH2, or [F]5‐IsCT1‐NH2) showed no significant change in an activity relative to IsCT1‐NH2. The addition of a proline residue at position 8 ([P]8‐IsCT1‐NH2) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13‐50 μmol L−1), and the addition of a tryptophan residue at position 1 ([W]1‐IsCT1‐NH2) increased the hemolytic activity (MHC = 1.56 μmol L−1) without an improvement in antimicrobial activity. The analog [A]1[F]5[K]8‐IsCT1‐NH2, which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 μmol L−1) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.

Multidrug-resistant bacteria represent a global health problem increasingly leading to infections that are untreatable with our existing antibiotic arsenal. Therefore, it is critical to identify novel effective antimicrobials. Venoms represent an underexplored source of potential antibiotic molecules. Here, we engineered a peptide (IsCT1-NH2) derived from the venom of the scorpion Opisthacanthus madagascariensis, whose application as an antimicrobial had been traditionally hindered by its high toxicity. Through peptide design and the knowledge obtained in preliminary studies with single and double-substituted analogs, we engineered IsCT1 derivatives with multiple amino acid substitutions to assess the impact of net charge on antimicrobial activity and toxicity. We demonstrate that increased net charge (from +3 to +6) significantly reduced toxicity toward human erythrocytes. Our lead synthetic peptide, [A]1[K]3[F]5[K]8-IsCT1-NH2 (net charge of +4), exhibited increased antimicrobial activity against Gram-negative and Gram-positive bacteria in vitro and enhanced anti-infective activity in a mouse model. Mechanism of action studies revealed that the increased antimicrobial activity of our lead molecule was due, at least in part, to its enhanced ability to permeabilize the outer membrane and depolarize the cytoplasmic membrane. In summary, we describe a simple method based on net charge tuning to turn highly toxic venom-derived peptides into viable therapeutics.

VmCT1, a linear helical antimicrobial peptide isolated from the venom of the scorpion Vaejovis mexicanus, displays broad spectrum antimicrobial activity against bacteria, fungi, and protozoa. Analogs derived from this peptide containing single Arg‐substitutions have been shown to increase antimicrobial and antiparasitic activities against Trypanossoma cruzi. Here, we tested these analogs against malaria, an infectious disease caused by Plasmodium protozoa, and assessed their antitumoral properties. Specifically, we tested VmCT1 synthetic variants [Arg]3‐VmCT1‐NH2, [Arg]7‐VmCT1‐NH2, and [Arg]11‐VmCT1‐NH2, against Plasmodium gallinaceum sporozoites and MCF‐7 mammary cancer cells. Our screen identified peptides [Arg]3‐VmCT1‐NH2 and [Arg]7‐VmCT1‐NH2 as potent antiplasmodial agents (IC50 of 0.57 and 0.51 μmol L−1, respectively), whereas [Arg]11‐VmCT1‐NH2 did not show activity against P. gallinaceum sporozoites. Interestingly, all peptides presented activity against MCF‐7 and displayed lower cytotoxicity toward healthy cells. We demonstrate that increasing the net positive charge of VmCT1, through arginine substitutions, modulates the biological properties of this peptide family yielding novel antiplasmodial and antitumoral molecules.

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