The wasp venom antimicrobial peptide polybia‐CP and its synthetic derivatives display antiplasmodial and anticancer properties

Abstract The wasp venom‐derived antimicrobial peptide polybia‐CP has been previously shown to exhibit potent antimicrobial activity, but it is also highly toxic. Previously, using a physicochemical‐guided peptide design strategy, we reversed its toxicity while preserving and even enhancing its antibacterial properties. Here, we report on several additional unanticipated biological properties of polybia‐CP and derivatives, namely their ability to target Plasmodium sporozoites and cancer cells. We leverage a physicochemical‐guided approach to identify features that operate as functional hotspots making these peptides viable antiplasmodial and anticancer agents. Helical content and net positive charge are identified as key structural and physicochemical determinants for antiplasmodial activity. In addition to helicity and net charge, hydrophobicity‐related properties of polybia‐CP and derivatives were found to be equally critical to target cancer cells. We demonstrate that by tuning these physicochemical parameters, it is possible to design synthetic peptides with enhanced submicromolar antiplasmodial potency and micromolar anticancer activity. This study reveals novel and previously undescribed functions for Polybia‐CP and analogs. Additionally, we demonstrate that a physicochemical‐guided rational design strategy can be used for identifying functional hotspots in peptide molecules and for tuning structure–function to generate novel and potent new‐to‐nature therapies.


| INTRODUCTION
The role of peptides as antimicrobial agents has been extensively described in the literature. [1][2][3][4][5] New strategies for the design and development of these molecules, [6][7][8] combined with the rising resistance of microorganisms to standard antibiotics, 9,10 are boosting worldwide interest and studies on antimicrobial peptides (AMPs). For example, recent reports have described the design of AMPs with broadspectrum activity, particularly amphipathic cationic peptides. 11 Exploring the multifunctional properties of these molecules may lead to candidate molecules that simultaneously kill resistant microorganisms, viruses, 12 parasite infections, [13][14][15][16] and cancer cells. 17,18 Torres et al. 6 using a physicochemical feature-guided design of polybia-CP (Pol-CP-NH 2 : Ile-Leu-Gly-Thr-Ile-Leu-Gly-Leu-Leu-Lys-Ser-Leu-NH 2 ), identified functional determinants that were key for converting a toxic wasp venom peptide into nontoxic variants with enhanced antimicrobial activity against fungi, Gram-positive and Gram-negative bacteria by destabilizing the membrane of those microorganisms. The structure-guided design of active derivatives of Pol-CP-NH 2 involved reprogramming peptide features to favor the interaction between AMPs and negatively charged biomembranes.
Here, we describe the previously unrecognized ability of Pol-CP-NH 2 and analogs to also target the malaria parasite and cancer cells.
These results are significant, as malaria is among the deadliest parasitic infectious diseases according to the World Health Organization, threatening the lives of approximately half of the world's population.
Pregnant women and children under 5 years of age are the most common victims of this disease. 19 Currently, there are limited treatment options available for earlier stages of the disease 20 and most of the strains are resistant to standard antibiotics. Thus, the best alternative for treating malaria involves preventing infection and monitoring relevant vectors. The anticancer activity of the peptide is also highly F I G U R E 1 Schematic of the naturally occurring wasp venom peptide polybia-CP with (a) antimicrobial and prospective antiplasmodial and anticancer activities, by (b) tuning physicochemical features responsible for peptide-membrane interactions relevant, as cancer is a major public health problem worldwide and the second leading cause of death in the United States. 21 Alternatives for cancer treatment have been sought over the last decades, but effective broad-spectrum methods have not been reported.
To identify the physicochemical determinants driving these biological properties, we synthesized (Supplementary Table 1) and evaluated the effectiveness of the designed synthetic analogs against Plasmodium sporozoites and cancer cell lines (Figure 1a). Our results indicate that physicochemical feature optimization aimed at enhancing the targeting of negatively charged membranes such as those of parasites and cancer cells may provide a viable strategy for treating such diseases (Figure 1b).

| RESULTS AND DISCUSSION
Pol-CP-NH 2 is a potent AMP with in vitro and in vivo activity against bacteria and fungi. 6 Here, we identified additional biological properties of this peptide and its synthetic analogs through their ability to target malarial sporozoites and cancer cells. In order to analyze the potential of Pol-CP-NH 2 as a triple antimicrobial, antiplasmodial and anticancer agent, we leveraged the systematic design approach proposed by Torres et al., where the main physicochemical properties of the peptides were optimized to achieve increased interactions with negatively charged membranes 6 and minimize potential enzymatic degradation by avoiding certain motifs and amino acid residues that are targeted by proteases present in blood serum. Briefly, here we engineered specific substitutions into the template sequence of Pol-CP-NH 2 in order to elucidate the structure-function relationships underlying biological function. The modifications were rationally proposed by fine-tuning physicochemical functional determinants commonly responsible for activity against negatively charged membranes, such as hydrophobicity, hydrophobic moment, net positive charge, amphipathicity, and helical propensity. 6 The substitutions generated identified physicochemical activity determinants that were important for peptide-membrane interactions.
Hydrophobicity and hydrophobic moment effects on the biological activities of the peptides were evaluated through substitutions by Leu and Phe residues. The aliphatic residue Leu was chosen because of its higher propensity for adopting helical structures compared to other aliphatic or aromatic hydrophobic residues. 22 Leu residues are also common in wasp venom peptide sequences. 23 Although Phe presents higher hydrophobicity and, in some cases, potentially toxicity toward eukaryotic cells, 24 its hydrophobicity is not as high as tryptophan. Thus, by introducing Phe into the original aliphatic residues from the hydrophobic face, it is possible to evaluate the effect of the aromatic residue on structure and biological function. Additionally, unlike Trp, Phe residues are not major components of AMPs, 25 which are typically cytotoxic, and are therefore better candidates for the design of potential therapeutic agents.
The net charge was analyzed by substituting residues on both faces of the amphipathic helical structure by Lys residues that are frequently found in wasp venom peptides. 23 Lys was chosen instead of Arg due to its superior flexibility and lower propensity in potentially toxic cell penetrating peptides. 26 Effects exerted by hydrophobicityrelated and charge-related substitutions to the helical propensity of the peptides were evaluated in parallel, since structure is crucial to the biological activities of peptides.
To assess the antiplasmodial activity of Pol-CP-NH 2 and its derivatives, the molecules were incubated with Plasmodium gallinaceum sporozoites. The avian malaria parasite, P. gallinaceum, was chosen as the plasmodium model for this study because it presents lower risk and it is highly similar 27,28 to existing Plasmodium falciparum models responsible for human malaria. 29 The template and designed peptides were screened against P. sporozoites in the range of concentrations at which they presented antimicrobial activity against bacteria and fungi (0.39-6.25 μmol L −1 ). 6 Generally, naturally occurring small cationic peptides that are active against bacteria are not as active against Plasmodium. 13 In fact, Pol-CP-NH 2 did not exhibit antiplasmodial activity at the range of concentrations tested. However, the synthetic peptides designed displaying increased net positive charge showed higher antiplasmodial activity compared to other synthetic peptides described in the litera- Plasmodium protozoa, followed by destabilization of the lipid bilayer. 6 The cationic and helical analogs were even more active against the Plasmodium parasite than against bacteria and fungi. At the range of concentrations at which the peptides were active (nanomolar range), they did not exert cytotoxic or hemolytic activities. 6 Analogs with increased hydrophobicity, which were also the ones

| CONCLUSION
AMPs represent promising alternatives to conventional therapies to combat a number of global health problems, including antibiotic resistance, 5,11 neglected infectious diseases, and cancer. However, the development of AMPs has been limited by the lack of methods for cost-effective 38 and rational 39 design. Although some alternative methods to overcome these limitations have been proposed, 2,3 we are far from understanding the structure-activity relationship (SAR) of these agents, which would provide a more substantial basis for their rational design and accelerate their translation into the clinic.
In this study, we leveraged a technique involving the structure-

DISCLOSURE OF INTERESTS
The authors declare no competing financial interests.