Examining Topoisomers of a Snake-Venom-Derived Peptide for Improved Antimicrobial and Antitumoral Properties

Ctn[15-34], the C-terminal section of crotalicidin (Ctn), a cathelicidin from a South American pit viper, is an antimicrobial and antitumoral peptide with remarkably longer stability in human serum than the parent Ctn. In this work, a set of topoisomers of both Ctn and Ctn[15-34], including the retro, enantio, and retroenantio versions, were synthesized and tested to investigate the structural requirements for activity. All topoisomers were as active as the cognate sequences against Gram-negative bacteria and tumor cells while slightly more toxic towards normal cells. More importantly, the enhanced serum stability of the D-amino-acid-containing versions suggests that such topoisomers must be preferentially considered as future antimicrobial and anticancer peptide leads.


Introduction
Since the discovery of penicillin by Fleming in 1928 [1] and the ensuing golden era of antibiotic development , the prospects for anti-infective therapies have become increasingly problematic due to the rise in antimicrobial resistance (AMR), first observed as the ability of E. coli to synthesize penicillinase that inactivated penicillin [2], later (1960) as the lack of response of 80% of S. aureus-infected patients to penicillin [3], and since then on to the current, widespread AMR crisis, an alarming predicament that is unanimously regarded as one of the most serious threats to global health [4][5][6][7].
In order to fight AMR, it is essential to have a good understanding of the mechanisms underlying infection, in particular those related to the interaction between anti-infective agents and pathogen targets. In this regard, antimicrobial peptides (AMPs) are emerging as promising therapeutic alternatives in the worrying context of global AMR for reasons that include, among others, their simple but effective mechanisms of action-different from traditional antibiotics and with a low likelihood of giving rise to resistance [8]broad spectrum (Gram-negative and -positive bacteria, fungi, protozoa, and viruses [9][10][11]), amenability to structural modification by versatile peptide engineering techniques, and affordable production costs. In addition, AMPs are also showing promise as potential antitumor agents [12]. Structurally, AMPs comprise a wide variety of motifs; although they share a generally cationic nature and a high content of hydrophobic residues, both features combine to promote amphipathic secondary structures that facilitate membrane interaction and ensuing biological action [13,14].
In peptide research, the topoisomer (the term topoisomer is used to denote compositionidentical (hence isomer) but three-dimensionally distinct (hence topo) variants of a given molecule, with structural changes translating into different bioactivity. Such a broad definition inevitably encompasses a wide variety of structural arrangements of identical molecular composition; in practice, the term is largely confined to the nucleic acid [21,22] and peptide/protein fields [23,24]) label is often used to describe the different spatial patterns multiple disulfide peptides can fold into as a result of different cysteine pairings. Less frequent but equally appropriate use of the term concerns peptide structures with altered sequence and/or stereochemical features within a fixed composition. This still rather broad category may be further narrowed down to peptides such as those in this work, i.e., with fully inverted amino acid sequence (retro isomers) or with the canonic sequence made up of residues of reversed chirality (enantio isomers), or with both features combined (retroenantio isomers). Research in this area over recent years has unveiled rather interesting features of these isomers, particularly the retroenantio ones [23][24][25][26][27].

Peptide Purification
Preparative HPLC runs were performed on a Luna C18 column (21.2 mm × 250 mm, 10 µm; Phenomenex), using linear gradients (15-50%) of solvent B (0.1% TFA in MeCN) into A (0.1% TFA in H 2 O), as required, at a flow rate of 25 mL/min. Fractions with adequate HPLC homogeneity (>95%) and the expected mass by LC-MS were pooled, lyophilized, and used in subsequent experiments. All the peptides were solved in water for resuspension.

NMR Spectroscopy
Samples were prepared by dissolving the lyophilized peptide (1-2 mg) in 0.5 mL of a fresh solution of 30 mM DPC-d38 (98% deuteration; Cambridge Isotope Laboratories, Inc Andover, MA) at pH 3.0 in either H 2 O/D 2 O (9:1 v/v) or pure D 2 O. Peptide concentrations were approximately 1 mM. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate was added as an internal reference. pH was measured with a glass microelectrode (not corrected for isotope effects) and adjusted, if necessary, by adding minimal amounts of NaOD or DCl.

NMR Structure Calculation
Structure calculations were performed with the CYANA 3.98 program [37,38]. Upper limit distance restraints were obtained from the NOE cross-peaks present in 2D NOESY spectra recorded at 25 • C and 35 • C, which were integrated using the automatic integration subroutine of the Sparky program (T. D. Goddard and D. G. Kneller, Sparky 3, NMR assignment program, University of California, San Francisco, CA, USA). In addition, restrictions for i, i + 4 hydrogen bonds were incorporated for the helical regions, as deduced from the δ Hα and δ Cα values (Figures S1 and S2). The standard iterative protocol for automatic NOE assignment implemented in CYANA 3.98 was used. It consists of seven cycles of combined NOE assignment and structure calculation of 100 conformers per cycle. For each peptide, the final NMR structure corresponds to the ensemble of the 20 conformers with the lowest target function value. MOLMOL [39] was used to visualize the structures of the peptides. Structures are available upon request from the authors.

Bacterial Strains and MIC
Assays were performed on three Gram-negative bacteria: E. coli (BW25113, Coli Genetic Stock Center), A. baumannii (ATCC 15308, CECT, Valencia, Spain), and Pseudomonas sp. (ATCC15915, CECT). The minimal inhibitory concentration (MIC) of each peptide was determined as described by Wiegand et al. [40]. Peptide stocks in water were added to polypropylene 96-well plates (Greiner, Frickenhausen, Germany) and serially diluted from 100 to 0.2 µM. Fresh bacteria were incubated at 37 • C in Mueller Hinton Broth (MHB, Condalab, Torrejón de Ardoz, Spain) up to exponential growth and diluted to a final inoculum of 5 × 10 5 CFU/mL. BSA and acetic acid at 0.04% (w/v) and 0.002% (v/v) final concentration, respectively, were added to avoid peptide self-aggregation. Plates were incubated for 20-22 h at 37 • C. Each peptide was tested in duplicate. MIC values were the lowest peptide concentration where bacterial growth was not detected.

Peptide Cytotoxicity
About 60,000 cells were added to different microfuge tubes containing 2-fold serial dilution of the peptide, with a final concentration in the range between 0.1-100 µM in RPMI containing 2% of FBS. After 30 min incubation at 37 • C and 5% of CO 2 , 100 mL of medium containing approximately 10,000 treated cells were transferred to a 96-well plate. Then, 15 µL of Cell Titer Blue dye (Promega, Madison, WI, USA) was added, and plates were reincubated for 24 h. Fluorescence was read at 4 and 24 h after dye addition in a Synergy HTX (BioTek, Winooski, VT, USA) reader, with λ exc = 560 nm and λ em = 620 nm. In the case of adherent cultures, 5000 cells/well were seeded in 96-well plates. After 24 h incubation at 37 • C and 5% of CO 2 , the medium was removed, and a new medium with 2% of FBS containing the various 2-fold dilution of the peptides was added. After a further 30 min, 15 µL of Cell Titer Blue dye was added to each well, and the measurement was done as above. Relative viability was calculated relative to a cell with only 2% FBS in the corresponding medium as a control (100% viability). All assays were conducted in triplicate.

Hemolytic Activity
Fresh human blood (10 mL) was collected in EDTA tubes and centrifuged at 1000× g for 10 min at 4 • C. After plasma removal, the pellet containing the erythrocytes was washed 3× with phosphate-buffered saline (PBS) and resuspended to an 8% (v/v) suspension in PBS. Then, 100 µL of the suspension was added to a final concentration of 4% (v/v) to microfuge tubs containing 2-fold serially diluted peptides (0.2-100 µM). The suspension was incubated for 30 min at 37 • C with agitation, then centrifuged for 2 min at 1000× g. The supernatant was transferred to 96-well plates, and hemoglobin was measured by OD at 540 nm in a Synergy HTX plate reader (BioTek). Positive and negative controls were 1% (v/v) Triton X-100 and untreated erythrocytes (4% v/v in PBS), respectively. Measurements were done in triplicate. Hemolysis was calculated as: (2)

Serum Stability
Briefly, 0.5 mL each of human serum (Sigma-Aldrich, St. Louis, MO, USA) and peptide (1 mM in water) were mixed and incubated for 24 h at 37 • C with stirring. Aliquots (0.1 mL) were taken at 0, 1, 5, 10, 30, 120, 360 and 1440 min and treated with 20 µL of trichloroacetic acid (15% (v/v) in water). The suspension was centrifuged for 30 min at 4 • C and 13,000 rpm, and the supernatant was analyzed by HPLC as described above.

Discussion
Bioactive peptides isolated from natural sources, including animal toxins, plant extracts, fungi, and bacteria, have long raised considerable interest for their manifold thera-
The enantiomers (D-amino acid versions) of natural bioactive peptides have been explored as a means to overcome the protease susceptibility of the native forms [20]. Unfortunately, as only a small fraction of biologically relevant interactions are chiralityindifferent, the application of strict enantiomers of bioactive peptides is confined to a few specific areas such as AMPs or cell-penetrating peptides (CPPs) [44,45]. In contrast, the retroenantio (re) approach [23], where the original bioactive sequence is reversed and made up of D-amino acids hence highly protease-resistant, has over recent years shown multiple examples of therapeutic promise. For instance, THRre, the retroenantio version of THR (THR is a peptide that interacts with the transferrin receptor but does not compete with transferrin [46,47]), has better blood-brain barrier (BBB)-crossing properties than its cognate, extensively degraded by proteases [24]. Our own group, applying the re approach, has recently developed an orally active BBB-crossing peptide candidate that minimizes the adverse cognitive effects of cannabinoid therapy while preserving analgesic properties [25].
While the enantio and retroenantio versions, in general, confirmed expectations of enhanced serum survival (Section 3.5, Figure 5), they were, unfortunately, slightly more toxic in eukaryotic cells than the original L-peptides. The most probable reason for this toxicity is that mammalian cells, unlike bacteria, do not have racemases directly converting D-into L-amino acid residues [49]; an alternative two-step conversion pathway has been postulated, i.e., (i) transformation to α-keto acids by D-amino acid oxidase and D-aspartate oxidase; (ii) transamination to L-amino acids [50]. Unfortunately, this sequence produces H 2 O 2 as a by-product, accumulation of which may result in lipid peroxidation and cell membrane damage [51].