pubs.acs.org/jmc Article Rationally Modified Antimicrobial Peptides from the N‑Terminal Domain of Human RNase 3 Show Exceptional Serum Stability Daniel Sandín, Javier Valle, Belén Chaves-Arquero, Guillem Prats-Ejarque, María Nieves Larrosa, Juan José González-López, María Á ngeles Jiménez, Ester Boix,* David Andreu,* and Marc Torrent* Cite This: J. Med. Chem. 2021, 64, 11472−11482 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Multidrug resistance against conventional anti- biotics poses an important threat to human health. In this context, antimicrobial peptides (AMPs) have been extensively studied for their antibacterial activity and promising results have been shown so far. However, AMPs tend to be rather vulnerable to protease degradation, which offsets their therapeutic appeal. Here, we demonstrate how replacing functional residues in the antimicrobial region of human RNase 3also named eosinophil cationic proteinby non-natural amino acids increases stability in human serum. These changes were also shown to reduce the hemolytic effect of the peptides in general terms, whereas the antimicrobial activity was reasonably preserved. Digestion profiles enabled us to design new peptides with superior stability and lower toxicity that could become relevant candidates to reach clinical stages. ■ INTRODUCTION The increasing spread of antibiotic-resistant bacteria is putting In recent years, several limitations of AMPs, including industrial scale up or high production costs, have been our health system at risk.1−3 Misuse and abuse of antibiotic satisfactorily addressed.25 Yet, two main drawbacks still prescriptions in humans and the extended use in animal preclude their transference to clinics: (i) low stability to feeding are leading causes of antibiotic resistance.4 In this serum proteases and (ii) cytotoxicity to host cells.26 scenario, in addition to awareness campaigns to curb antibiotic Here, we designed and tested an optimized fragment from overconsumption, new antimicrobial drugs are urgently the ECP antimicrobial region (hECP30 peptide) using a needed. rational, structure-guided design with enhanced serum stability Among the newest emergent agents against resistant and low cytotoxicity effects.27,28 First, we analyzed the bacterial infections, antimicrobial peptides (AMPs) are very promising candidates.5,6 AMPs are produced by many digestion pattern of hECP30 and identified its most vulnerable organisms to kill pathogens and modulate the host response points for protease degradation. Following Arg replacement against infections.7−9 Their mode of action is based on with non-proteinogenic surrogates (Figure 1), we created a bacterial membrane destabilization in a fast and unspecific highly stable fragment with a half-life above 6 h in the presence manner,10 although intracellular interactions (i.e., targeting of human serum. These peptide analogs have similar protein synthesis or DNA replicating pathways) have also been antimicrobial activity against Gram-negative bacterial strains, reported.11 The human RNase A protein family includes including clinical isolates, but low toxicity compared to the proteins with a wide functional repertoire, besides its main original peptide. We envision that such modified versions of ribonuclease activity.12−14 Antimicrobial, antihelmintic, anti- tumor, and cytotoxic actions have been extensively stud- hECP30 may be suitable candidates for the design of new ied.15−19 Within this family, human RNase 3, also called antimicrobial drugs. eosinophil cationic protein (ECP), has a potent antimicrobial activity against both Gram-negative and Gram-positive Received: May 1, 2021 bacteria, stronger against the former group.20,21 Previous Published: August 3, 2021 studies located the antimicrobial domain of ECP on the N- terminal region,22,23 which could be trimmed down by structure-based design without significant loss of antimicrobial activity.24 © 2021 American Chemical Society https://doi.org/10.1021/acs.jmedchem.1c00795 11472 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 2. Peptide Half-Life Time in Incubations with 50% (v/v) Human Serum peptide half-life time (min) 1 12.3 ± 0.4 2 29.1 ± 0.5 3 20.6 ± 3.6 4 16.6 ± 0.5 5 >480 6 >480 7 >480 as responsible for this fragment. On this basis, we argued that shielding of the Pro2-Phe3 peptide bond should prevent or minimize protease cleavage. To test this hypothesis, we Figure 1. Structure of the amino acids chosen to replace Arg in the synthesized two peptides where Pro2 was mutated to D-Pro ECP-derived peptide. and Orn was either in the L- or D-configuration (Op (6) and ■ Op (7) analogs, respectively, Table 1). Both peptides displayed a strong improvement in stability, compared to 1 and 2. The RESULTS t1/2 > 480 min observed for peptides 6 and 7 represents a 30- Rational Design of ECP Analogs and Stability in fold stability increase over 1 (Table 2). Serum. hECP30 (1, Table 1) is a 30-amino acid peptide with Antimicrobial Activity. The antimicrobial activity of all strong antimicrobial activity against Gram-negative bacteria, peptides was measured and expressed as the minimal inhibitory both in planktonic culture and biofilms.29 It includes six Arg concentration (MIC) against several bacterial strains. As ECP residues, 20% of the total in its sequence. Arg and Lys are and 1 are most active against Gram-negative bacteria, MIC was common targets for trypsin-like serine proteases.30 To avoid determined against such bacterial species, including Escherichia inactivation by protease cleavage, non-natural amino acid coli, Acinetobacter baumannii, Pseudomonas sp., Salmonella substitution is often used.31 In this case, replacement of all Arg enterica, Klebsiella pneumoniae, and Shigella f lexneri. The results residues in 1 by non-proteinogenic, cationic Orn, Dab, and confirmed the high antimicrobial activity of 1 against all Gram- Har improved stability in a moderate (∼2-fold) but significant negative strains (Table 4). Arg replacement with non- way (Table 2). proteinogenic amino acids slightly reduced the activity, The incubation profile of 1 with human serum shows especially for 3 (Table 4). Analog 5, which lacks the first extensive degradation, even at short times (Figure 2A). two N-terminal residues, showed a marked reduction of Although the three analogs are also rapidly degraded, they activity against all strains, suggesting that these two residues generate a second peak that is very stable over time, only are essential. Analog 7 had similar activity than the original further degraded at much longer incubation times (Figure 2 peptide while 6 performed worse against some strains. and Figures S1 and S2). These results suggest that Arg Altogether, these results suggested that structural differences replacement protects all susceptible peptide bonds except one. between 6 and 7 affect antimicrobial activity. All peptides show Most of the digestion subproducts could be identified by MS a minimal bactericidal concentration (MBC) within the same (Table 3), including the fragment with high stability (over 2 range as the MIC (Table S1). Results obtained by killing curve h), which was assigned to the original 1 peptide minus the first analysis for analogs 1, 3, 6, and 7 also support these two N-terminal amino acids, Arg1-Pro2. To confirm this observations (Figure S3). In any event, most peptides display assignment, Des-OP (5, Table 1), a peptide lacking Arg(Orn)- similar or slightly lower antimicrobial activity than LL-37, a Pro at the N-terminus and with Arg to Orn replacements at the well-known reference AMP (Table 4). When peptides were other five positions, was prepared and tested, showing indeed a preincubated with human serum and tested against Pseudomo- rather long half-life (>480 min). Prediction of the protease nas sp., we observed only a slight increase in the MIC values, degradation profile using a bioinformatic tool (https://web. suggesting that the peptide activity is barely affected by the expasy.org/peptide_cutter/) identified proline endopeptidase presence of serum proteins (Table S2). Table 1. Primary Structures, HPLC Retention Times, and Molecular Mass of hECP30 Synthetic Analogs molecular mass (Da) entry description sequencea HPLC retention time (min)b theory found 1 hECP30 RPFTRAQWFAIQHISPRTIAMRAINNYRWR 8.9 3757.4 3757.6 2 Orn analog OPFTOAQWFAIQHISPOTIAMOAINNYOWO 8.4 3505.2 3505.2 3 Dab analog XPFTXAQWFAIQHISPXTIAMXAINNYXWX 8.3 3420.9 3421.3 4 Har analog ZPFTZAQWFAIQHISPZTIAMZAINNYZWZ 9.1 3841.5 3842.2 5 Des[Orn1-Pro2] analog --FTOAQWFAIQHISPOTIAMOAINNYOWO 10.8 3293.9 3293.8 6 [Orn,1D-Pro2] analog OpFTOAQWFAIQHISPOTIAMOAINNYOWO 10.9 3505.2 3505.2 7 [D-Orn,1D-Pro2] analog opFTOAQWFAIQHISPOTIAMOAINNYOWO 10.8 3505.2 3505.2 a O, ornithine; X, 2,4-diaminobutyric acid; Z, homoarginine; D-amino acid residues in lower case. bAll peptides were analyzed by HPLC in a linear gradient of solvent B (acetonitrile) into solvent A (H2O) from 0 to 60% over 15 min. 11473 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 2. Peptide degradation profiles. 1 mM peptide stocks in water were incubated with human serum (1:1 v/v). Profiles shown are (A) hECP30 reference peptide 1, (B) peptide 2, and (C) peptide 7. The amount of peptide remaining after incubation at different times was quantified by HPLC analysis. Peak numbers in each panel correspond to fragments listed on Table 3. IP: intact peptide. Table 3. Sequence of the Original Orn Analog Peptide 2 and aeruginosa and S. typhimurium, the concentrations required to Digestion Fragments Generated in Serum Incubationsa inhibit bacterial growth were, on average, higher compared to the original peptide. Such behavior may be related to a higher fragment sequence net charge requirement to disrupt the cell membrane, as Orn is 1 OPFTOAQWFAIQHISPO------------- less basic than Arg.32 For E. coli and A. baumannii, MIC values 2 -----------------TIAMOAINNYOWO for analogs were similar, or even better, than the reference 3 OPFTOAQWFAIQHIS--------------- peptide and, in general terms, 7 was substantially more active 4 ------QWFAIQHISPOTIAMOAINNY--- than 6 (Table 5). It is also worth noting that both hECP30 and 5 --FTOAQWFAIQHISPOTIAMOAINNYOWO its D-amino acid analogs show high activity against A. intact peptide OPFTOAQWFAIQHISPOTIAMOAINNYOWO a baumannii clinical isolates (Table 5), even at submicromolar The fragments are classified in order of elution in HPLC analysis. concentrations. We also tested whether recurrent incubation of bacteria with peptides could induce resistance. To test for In addition to the above reference strains, we also performed antibiotic resistance, we incubated E. coli cells for 7 rounds of MIC assays on several clinical isolates. For some strains, i.e., P. evolution with concentrations below the MIC for the reference 11474 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 4. MIC (μM) and Percentage of Hemolysis of hECP30 (1) and Analogsa peptide E. coli A. baumannii Pseudomonas sp. S. enterica S. f lexneri hemolysis % (250 μM peptide) MRC-5 cytotoxicity (IC50, μM) 1 1.56 1.56 1.56 12.5 3.13 44 ± 4 23 ± 4 2 6.25 6.25 6.25 25 12.5 10 ± 2 113 ± 8 3 6.25 50 3.13 50 6.25 14 ± 6 182 ± 9 4 6.25 12.5 3.13 25 3.13 43 ± 1 7.6 ± 0.7 5 25 >100 25 >100 50 11 ± 4 > 300 6 3.13 12.5 1.56 50 50 1.1 ± 0.9 230 ± 40 7 3.13 6.25 1.56 25 6.25 3.6 ± 0.7 170 ± 20 LL-37 1.56 6.25 1.56 12.5 12.5 58 ± 7 64 ± 2 a LL-37 included for comparison. Table 5. MIC Values (μM) of hECP30 (1) and Analogs in Clinical Bacterial Strains strain peptide 1 peptide 6 peptide 7 E. coli 116878 6.25 25 6.25 E. coli cf073 3.13 6.25 3.13 P. aeruginosa 827651 25 25 100 P. aeruginosa 827632 12.5 >100 25 S. typhimurium 324 6.25 25 25 S. typhimurium 365 6.25 25 25 A. baumannii 3862 0.78 1.56 0.78 A. baumannii 3878 1.56 0.78 0.78 A. baumannii 3879 1.56 3.13 1.56 A. baumannii 3880 1.56 12.5 6.25 peptide 1 as well as analogs 6, 7, and LL-37 (1 μM) and ciprofloxacin (1 μM) as a positive control. We did not detect a significant increase in MIC for any of the compounds tested (Figure S4), whereas ciprofloxacin-treated cells increased their MIC from 1.5 to 5.0 μM after four cycles of incubation. Mechanism of Action. The mechanism of action was Figure 3. SEM images of E. coli in the mid-exponential phase (A) elucidated by membrane depolarization and scanning electron before treatment or after treatment with 10 μM of analogs (B) 1, (C) microscopy (SEM). To study membrane depolarization, we 2, (D) 6, (E) 7, or (F) LL-37. used bacterial cells stained with the lipophilic dye DiSC3(5). In this assay, when membranes are compromised after peptide incubation, an increase in fluorescence is detected. Peptides 1, the Har analog 4 showed lysis levels similar to 1 (42.86 ± 2, 6, and 7 displayed similar depolarization capacity although 1.29%), suggesting that cytotoxicity is strongly related to the kinetics was slightly slower for analog 6 (Figure S5). LL-37 cationic charge. Most interestingly, analogs 6 and 7 showed a was used as a positive control and showed a similar dye release, massive reduction in cytotoxicity (1.12 ± 0.93 and 3.59 ± confirming the ability of all analogs to disrupt the bacterial cell 0.69%, respectively), thus broadening the therapeutic window membrane (Figure S5). Membrane damage was also studied by relative to the reference peptide. SEM in live bacteria. Peptide incubation showed in all cases a The above results were compared to cytotoxicity assays in marked membrane damage, with blebs confirming that the MRC-5 cells using the MTT assay. Results confirmed the high structural integrity was compromised (Figure 3). The presence toxicity of peptides 1 and 4, with IC50 values of 23 and 7.6 μM, of blebs distributed all along the bacterial surface, the respectively. In line with the hemolytic results, the toxicity of disruption of the cell morphology, and the presence of debris analogs 2 and 3 was markedly reduced, with IC50 values outside the cell suggest a carpet-like mechanism, as observed increased up to 180 μM (Table 4). No toxicity was detected before for the ECP N-terminal domain,22 reinforcing the idea for analog 5, even at the highest concentration tested, that the mechanisms of action of these analogs are similar. concurring with its low antimicrobial activity. Finally, analogs Toxicity Analysis. The antimicrobial activity must not be 6 and 7 exhibited even lower toxicity than 5, with IC50 values analyzed on its own but assessed alongside peptide toxicity. It up to 230 μM. Such low toxicity, combined with a longer half- is well known that unspecific and potent antimicrobial activity life, motivated us to further study these peptides from a is often accompanied by high cytotoxicity against host structural point of view. cells.33,34 In our case, we tested cytotoxicity caused by the Peptide Structure. A preliminary evaluation of the peptide by measuring lysis of horse red blood cells as structure of peptides 1, 2, 6, and 7 was provided by circular monitored by hemoglobin release at 540 nm.35 The assay dichroism. Far UV spectra of peptides in aqueous solution or showed a high activity for reference peptide 1, with ∼50% lysis in the presence of SDS micelles (1 mM) were recorded. at 250 μM, similar to LL-37 (Table 4). In contrast, 2 and 3 Spectra for all four peptides indicated a random conformation showed an important reduction in toxicity, with only 10% in aqueous solutions (Figure S6 and Table S3). Upon addition hemolysis at the same concentration (Table 4). For its part, of SDS, the peptides adopted a more defined structure, mainly 11475 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 4. Bar plots of the ΔδHα values (ΔδHα = δHαobserved − δHαRC, ppm) as a function of sequence for peptides 1, 2, 6, and 7 in (A) aqueous solution and (B) DPC micelles at pH 4.4 and 25 °C. Notice that the sequence of peptide 1 is shown and that Arg residues are Orn in peptides 2, 6, and 7. α-helical, in percentages ranging from 45 to 57%, consistent solution but better defined (Figure S7 and Table S5). In the with previous results reported by our group for the ECP N- four peptides, the helix exhibits a hydrophobic patch formed by terminal domain (Table S3).22 residues F3, W8, I11, and I14 (Figure 6). The arrangement of Peptides 1, 2, 6, and 7 were studied in aqueous solution and these side chains seems to be slightly different in 1 compared dodecylphosphocholine (DPC) micelles by NMR. All four to the other analogs. The hydrophobic patch extends to D-Pro2 peptides exhibited a helical segment spanning residues 5−13 in the case of 6 and 7. ■ and a slight helical tendency at residues 16−27 in aqueous solution, based on the analysis of the conformational shifts DISCUSSION AND CONCLUSIONS (ΔδHα and ΔδCα; Figure 4, Table S4, and Figure S7). The replacement of original residues in peptides by non- According to the magnitudes of the conformational shifts, both proteinogenic or D-amino acids has been extensively studied helices become more populated in the presence of DPC, in over recent years to enhance AMP viability for clinical use.38 In tune with the notion that micelles tend to stabilize amphipathic AMPs, Arg and Lys are key cationic residues often involved in helices.36,37 On the whole, the peptides do not show any interactions with negatively charged bacterial membranes. significant differences in secondary structure, i.e., helices span However, these two residues are also a frequent target of serum the same residues and there are no great differences in their proteases, with ensuing peptide inactivation. In hECP30, Arg populations (Table S4). More specifically, in aqueous solution, represents 20% of the amino acid content; hence, we decided all four peptides show a well-defined N-terminal helix spanning to investigate whether its replacement by non-coded surrogate residues 5−13, the only ones with non-sequential NOEs, and a residues could improve peptide stability and therapeutic mainly disordered C-terminal tail (Figure 5A, Figures S8 and potential. S9, and Table S5). In contrast, in DPC micelles, the helix We first observed that substituting Arg by Orn, Dab, or Har populations are higher and are better defined for the entire could moderately stabilize hECP30 against proteolytic cleavage peptide length (Figures S8 and S9 and Table S5), with two in human serum (Table 2). This increase in half-life, up to 3- helical regions spanning residues 5−13 and 16−27 (Figure fold for analog 2, may be explained by impaired recognition of 5B). A closer inspection reveals interesting differences among Orn at the active site of proteases. Analyzing the degradation the four peptides, however, in the relative orientation of the profile by HPLC−MS, we found an extremely stable cleavage helices (Figure 5C,D), and at the N-termini, where the D- byproduct in the modified analogs. This fragment had lost only residues in 6 and 7 also give rise to some variations. A detailed the two residues (Orn and Pro) at the N-terminus; thus, we examination of side-chain packing at the N-terminal region hypothesized that peptide 5 might be an interesting analog, (residues 1−15, including helix 5−13) was performed on the largely impervious to proteolytic cleavage. Unfortunately, the DPC structures, essentially identical to those in aqueous improved stability was accompanied by low activity against 11476 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 5. Peptide NMR calculated structures. (A) Ensemble of the 20 lowest target function structures of peptide 1 in aqueous solution overlaid onto residues 5−13. Residues 5−13 in red and 16−30 in green. (B) Ribbon representation of the lowest target function structure for peptide 1 in DPC micelles. (C, D) Ensembles of the 20 lowest target function NMR calculated structures for peptide 1 (black), 2 (blue), 6 (green), and 7 (magenta) analogs in DPC micelles overlaid onto residues 5−13 (panel C) and onto residue 16−27 (panel D). bacterial strains, strongly suggesting that these first two that the mechanism of action of the analogs is similar to the residues are essential for antimicrobial action and cannot be original ECP N-terminal domain, suggesting that the peptides omitted. Discarding therefore 5 as a candidate and analyzing would display a carpet-like mechanism.22 Also, no antimicro- the in silico peptide degradation profile by known serum bial resistance was observed after cyclic incubations of bacteria proteases, we found that the Pro2−Phe3 bond was cleavable by with the peptides, suggesting that bacteria are less prone to a proline endopeptidase and hence decided to test two new developing resistance against these peptides compared to analogs with either Pro2 or both Orn1 and Pro2 replaced by the conventional antibiotics. corresponding D-enantiomers. As hypothesized, the new To measure the potential toxicity of the peptides, we analogs exhibited remarkable serum stability, improving over performed assays to define in vitro therapeutic windows. 30-fold the half-life (>6 h vs 12 min for reference peptide 1) Reference peptide 1 caused ∼50% hemolysis at 250 μM and thus confirming that shielding the Pro2−Phe3 peptide concentration (Table 4). Although this value may seem high, bond from cleavage was crucial for stability. This is a one must be reminded that it is about 100-fold the MIC in E. remarkable improvement when compared to similar studies.39 coli. Indeed, at lower concentrations, e.g., 10-fold the MIC, Having improved stability, we next tested the impact of hemolytic activity is below 10%. On the other hand, while these changes on antimicrobial activity. We had detected a analogs 2 and 3 show a marked decrease in hemolytic activity, slight decrease in both analogs 2 and 3, whereas the for 4, the antimicrobial and hemolytic activities almost replacement of Arg by Har was neutral in terms of coincide, again suggesting that the chemical nature of the antimicrobial activity. These findings are readily explainable cationic residues is highly relevant for the mechanism of action. in structural terms: Har is very similar to Arg, both having a Thus, the strong cationic nature of Arg and Har increases long side chain ending in a strongly cationic guanidine group, antimicrobial activity of both peptides, by strongly binding the whereas Orn and Dab have shorter side chains ending in less negatively charged bacterial membrane, whereas the long side basic amino functions (Figure 1). Importantly, shielding the chains also enhance, after charge neutralization, the hydro- Pro2−Phe3 bond by D-amino acid replacement in Orn analogs phobic nature of the residues, probably increasing hemolytic 6 and 7 improved both the half-life and the antimicrobial activity. Consistently with this hypothesis, Dab and Orn cause activity. Thus, MIC values for 7 were similar to the original a marked reduction in hemolysis (<5% at 250 μM in both peptide in almost all bacteria, both reference and clinical cases) and a slight decrease in activity which, upon N-terminal strains (Tables 4 and 5). These results suggest that bacteria protease shielding (analogs 6 and 7), rescues the antimicrobial may also use their own proteases to cleave peptides as a potency of the original peptide. protective strategy, as observed before for other AMPs.40 To investigate whether the amino acid substitutions Membrane depolarization assays and SEM images confirmed translated into peptide structure changes, we performed 11477 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 6. N-terminal regions (residues 1−15) of the NMR calculated structures for peptides 1, 2, 6, and 7 in DPC micelles. Backbone atoms are displayed in black, Arg and Orn side chains in blue, polar side chains in cyan, and aliphatic and aromatic side chains in green. The Hα proton of Pro and D-Pro residues is shown. NMR analysis on the most promising candidates (1, 2, 6, and Spain). Clinical strains were supplied by the Vall d’Hebron Hospital 7) and found that the overall structure is conserved, with two (Barcelona, Spain). MRC5 fibroblasts were obtained from the main α-helices spanning residues 5−13 and 16−27 (Figures 3 Cytometry and Cell Culture Facility (SCAC, UAB) and originally and 4). The two helices are weakly formed in aqueous solution purchased from ATCC. Peptide Synthesis. Peptides were assembled in the C-terminal (30−40 and 10−15% for helices 5−13 and 16−27, carboxamide form at a 0.1 mmol scale on a H-Rink Amide- respectively) but clearly defined in the presence of DPC ChemMatrix resin of 0.50 mmol/g substitution (PCAS BioMatrix, micelles (70−80%). This is consistent with most AMPs and Quebec, Canada) in a Prelude instrument (Gyros Protein previous studies on the antimicrobial domain of ECP24,41 in Technologies, Tucson, AZ) using Fmoc solid-phase peptide synthesis contact with the negatively charged bacterial membrane. Even (SPPS) protocols. After chain assembly, peptides were fully so, a slight conformational change in the second helix is deprotected and cleaved from the resin with TFA/H2O/triisopro- observed in 2, 6, and 7 relative to 1: the angle between the two pylsilane (95:2.5:2.5 v/v) for 90 min at RT with gentle agitation. helices increases upon replacing Arg by Orn, with peptides 2, Peptides were precipitated from the TFA solution by addition of 6, and 7 adopting a more extended conformation that may chilled diethyl ether followed by three centrifugations at 4800 rpm, 5 help explain the slightly lower activity of Orn analogs min, 4 °C, taken up in water and lyophilized. Crude peptides were checked by analytical RP-HPLC and LC−MS and purified by compared to the original peptide. preparative RP-HPLC. Analytical RP-HPLC was performed on an In light of the present results, we conclude that peptide 7 is a LC-20 AD instrument (Shimadzu, Kyoto, Japan) fitted with a Luna promising AMP lead. Its high antimicrobial activity and low C18 column (4.6 mm × 50 mm, 3 μm; Phenomenex) using linear cytotoxicity portray it as an excellent candidate to fight gradients of solvent B (0.036% TFA in ACN) into A (0.045% TFA in infections caused by Gram-negative bacteria. Moreover, its H2O) over 15 min, at 1 mL/min flow rate and with UV detection at remarkable in vitro serum stability suggests that it would 220 nm. Preparative RP-HPLC was performed on an LC-8 instrument survive in the organism for a prolonged time. (Shimadzu) fitted with a Luna C18 column (21.2 mm × 250 mm, 10 ■ μm; Phenomenex), using linear gradients of solvent B (0.1% TFA in ACN) into A (0.1% TFA in H2O) over 30 min, with a flow rate of 25 EXPERIMENTAL SECTION mL/min. MS analysis was performed on an LC−MS 2010EV Materials. Purified horse red blood cells were acquired from instrument (Shimadzu) fitted with an Aeris Widepore XB-C18 Thermo Fisher Scientific (Hampshire, England). Human serum was column (150 × 4.6 mm, 3.6 μm, Phenomenex), eluting with linear purchased from Sigma (St Louis, USA). E. coli was obtained from the gradients of B (0.08% formic acid (FA) in ACN) into A (0.1% FA in Coli Genetic Stock Center (BW25113). S. f lexneri (ATCC 29903), A. H2O) over 15 min at a 1 mL/min flow rate. Fractions of >95% HPLC baumannii (ATCC 15308), Pseudomonas sp. (ATCC 15915), and S. purity and with the expected mass by LC−MS were pooled and enterica (ATCC 14028) were obtained from the CECT (Valencia, lyophilized. Peptide purity was assessed by the area of the purified 11478 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article peptide peak relative to the total peak areas in the chromatogram. the dye to a final concentration of 0.4 μM and incubated in darkness Peptide stock solutions were prepared in sterile deionized water and from 20 to 30 min to obtain a stable baseline. The peptides were stored at −20 °C. added to the mixture and DiSC3(5) release was measured in a Varian Peptide Stability. To determine stability, 1 mM peptide stocks Cary Eclipse fluorescence spectrometer (Agilent, Santa Clara, were incubated with human serum in a 1:1 ratio. At various California) with excitation and emission at 625 and 666nm and 5 incubation times, aliquots were removed, and digestion was stopped nm and 10 nm excitation and emission slit, respectively. by ACN precipitation. After discarding serum proteins by Scanning Electron Microscopy (SEM). E. coli cell cultures were centrifugation, original peptides and proteolysis fragments in the grown in LB at 37 °C to the mid-exponential phase (OD600 = 0.4). 1 supernatant were analyzed by LC−MS. Solvents and methods are as mL of cell culture was incubated for 2 h with 10 μM peptides at room above. Results are the average of three independent studies. temperature. Samples were then prepared for analysis, as described Minimum Inhibitory Concentration (MIC) and Minimum previously.44 Briefly, the cell suspensions were filtered through 0.1 μm Bactericidal Concentration (MBC). The antibiotic activity was Nucleopore filters to retain the bacteria and then fixed with 2.5% defined by the minimum peptide concentration where the micro- glutaraldehyde in 100 mm Na-cacodylate buffer (pH 7.4) for 2 h at 4 organism is unable to grow. The assay was done in polypropylene 96- °C. Attached cells were post-fixed by immersing the filters in 1% well plates (Greiner, Frickenhausen, Germany) to avoid peptide osmium tetroxide in Na-cacodylate buffer for 30 min, rinsed in the binding to plate wells. Peptides were dissolved in water containing same buffer, and dehydrated in ethanol in ascending percentage 0.4% w/v bovine serum albumin (BSA) and 0.02% v/v glacial acetic concentrations [31, 70, 90 (×2), and 100 (×2)] for 15 min each. The acid to prevent self-aggregation, following the reference protocol by filters were mounted on aluminum stubs and coated with gold− Wiegand et al.,42 based on the classical microtiter broth dilution palladium in a sputter coater (K550; Emitech, East Grinsted, UK). recommended by the National Committee of Laboratory Safety and The filters were viewed at 15 kV accelerating voltage in a EVO MA 10 Standards (NCLSS). Initial bacteria inoculum was adjusted to 5 × 105 scanning electron microscope (Zeiss, Oberkochen, Germany). CFU/mL in Mueller-Hinton (MH) medium. Incubations were kept Cytotoxicity in Mammalian Cells. MRC-5 cells were grown in at 37 °C for 24 h. Results are the average of three independent Eagle’s minimum essential medium (MEMα), supplemented with studies. MIC assays in the presence of serum were performed by 10% fetal bovine serum (FBS). 96-well plates were seeded with 3 × incubating the peptide with human serum at a 1:1 ratio. Then, 106 104 cells/well and cultured overnight in order to get cells adhered into cfu/mL of Pseudomonas sp. were incubated with serial dilutions of the the plate and then incubated with two-fold peptide serial dilutions. peptides for 4 h. To determine the MBC, 50 μL from all wells were After 4 h of incubation, the medium containing the peptides was seeded into Petri plates and incubated for 24 h. The MBC replaced by MEMα supplemented with FBS and 3-(4,5-dimethylth- concentration was attributed to the lowest concentration without iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 0.4 mg/mL detectable bacterial growth for each peptide. and incubated for 150 min. Detection of formazan crystals in the Antimicrobial Resistance. To test whether recurrent incubation living cells was done by cell disruption by addition of 200 μL of of bacteria with peptides could induce resistance, E. coli (107 cfu/mL) dimethyl sulfoxide (DMSO) followed by absorbance measuring at were incubated with peptides (1 μM) or ciprofloxacin (1 μg/mL) for 600 nm in a Tecan Infinite F Nano+ microplate reader (Tecan, 1 h. After incubation, cells were centrifuged, washed, and grown Germany). Results are the average of three independent studies. overnight in Mueller-Hinton medium. The cycle of incubation and Circular Dichroism. The CD spectrum of peptides 1, 2, 6, and 7 growth was repeated in a cyclic manner for 7 days, and each day, was measured in 5 mM sodium phosphate pH 7 or 5 mM sodium cultures were tested for MIC to detect resistance. phosphate buffer pH 7 with 1 mM SDS to simulate a membrane Killing Curve Assay. Killing curves were followed using the live/ environment. Peptide final concentration was 10 μM. Samples were dead bacterial viability kit (Invitrogen, Oregon, USA). Briefly, fresh analyzed in a Jasco J-815 CD spectropolarimeter (Jasco, Easton, bacteria cultures (E. coli, A. baumannii, and Pseudomonas sp.) were Maryland) in 0.2 mm quartz cuvettes (Hellma, Germany). Far UV brought to a concentration of approximately 4 × 108 cfu/mL for the spectra were measured from 260 to 190 nm and analyzed using the assay. In a polypropylene 96-well plate (Greiner, Frickenhausen, CDSSTR method to predict the secondary structure.45 Germany), 100 μL of a 3:2 mixture of bacteria and live/dead reagent NMR Spectroscopy. NMR samples were prepared by dissolving in phosphate-buffered saline (PBS), incubated previously for 15 min, the lyophilized peptides at about 1 mM concentration in aqueous was added to each well containing 50 μL of 1:2 serially diluted solution (H2O/D2O 9:1 v/v or pure D2O) or in DPC micelles (50 peptide in PBS (concentration range from 100 μM to 0.2 μM). The mM [D38]-DPC in H2O/D2O 9:1, v/v or in pure D2O). The pH was fluorescence emission was continuously recorded for 12 h with a measured using a glass microelectrode and adjusted to 4.4 by addition Tecan Infinite F Nano+ microplate reader (Tecan, Germany) using of NaOD or DCl. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate an excitation wavelength of 485 nm. Emission wavelength for SYTO9 (DSS) at a 0.1−0.2 mM concentration was added as internal reference was set to 530 nm (green, living cells) and 630 nm for propidium for the 1H chemical shifts. iodide (red, dead cells). Results are the average of three independent As previously reported,46 a Bruker Avance-600 spectrometer studies. equipped with a cryoprobe was used to record NMR spectra: 1D 1 Hemolytic Activity. The peptide toxicity was determined H, 2D 1H,1H-COSY (phase-sensitive two-dimensional correlated detecting the disruption of horse red blood cells, following a previous spectroscopy), 1H,1H-TOCSY (total correlated spectroscopy), 1H,1H- assay report.35 10-fold diluted cells in PBS were centrifuged three NOESY (nuclear Overhauser enhancement spectroscopy), and 1 times at 800g to eliminate the hemoglobin from disrupted cells. After H-13C-HSQC (heteronuclear single quantum coherence) at 13C the last resuspension, erythrocytes were incubated with the peptides natural abundance. TOCSY and NOESY mixing times were 60 and for 4 h at 37 °C. The detection was done measuring the supernatant 150 ms, respectively. Data were processed using the TOPSPIN absorbance in a VICTOR3 multilabel plate reader (Perkin Elmer, software (Bruker Biospin, Karlsruhe, Germany). Waltham, Massachusetts) at 540 nm in a microplate reader. SPARKY software47 was used to analyze the NMR spectra. The 1H Percentage of hemolysis was calculated through a pattern done with chemical shifts were assigned by analysis of the 2D homonuclear dilutions of total cell lysis done with a 1% Triton X-100 buffer. Results spectra using the well-established sequential assignment method- are the average of three independent studies. ology,48 and the 1H-13C-HSQC spectra were analyzed to assign the 13 Bacterial Membrane Depolarization. Membrane depolariza- C chemical shifts. The assigned chemical shifts have been deposited tion on E. coli was performed by the quenching and later release of at the BioMagResBank (http://www.bmrb.wisc.edu) with accession DiSC3(5) (Fisher, Hampshire, England) as described in Zhang et al., codes BMRB ID: 50486-50493. with some modifications.43 E. coli fresh cultures were brought to The conformational shifts for Hα protons (ΔδHα, ppm) and Cα exponential growth (0.2−0.4 OD), washed twice with 5 mM HEPES, carbons (ΔδCα, ppm) were obtained using the following equations: 20 mM glucose, and 100 mM KCl, pH 7.2, and finally resuspended ΔδHα = δHαobserved − δHαRC, ppm and ΔδCα = δCαobserved − δCαRC, with the same buffer up to an OD of 0.05. Samples were mixed with ppm; where δHαobserved and δCαobserved are, respectively, the Hα and 11479 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Cα chemical shifts observed for the peptides, and the random coil Javier Valle − Department of Experimental and Health values δHαRC and δCαRC were taken from ref 49. Sciences, Universitat Pompeu Fabra, Barcelona Biomedical Helix populations were estimated from the 1Hα and 13Cα chemical Research Park, Barcelona 08003, Spain shifts as previously described.46 Belén Chaves-Arquero − Departamento de Química-Física Peptide structures were calculated using the iterative procedure for automatic NOE assignment integrated in the CYANA 3.98 Biológica, Instituto de Química Física Rocasolano (IQFR- program.50 This algorithm consists of seven cycles of combined CSIC), Madrid 28006, Spain automated NOE assignment and structure calculation, in which 100 Guillem Prats-Ejarque − Department of Biochemistry and conformers were computed per cycle. The experimental input data Molecular Biology, Universitat Autònoma de Barcelona, comprises the lists of assigned chemical shifts, and NOE integrated Cerdanyola del Vallès 08193, Spain cross-peaks present in 150 ms NOESY spectra, plus the ϕ and ψ María Nieves Larrosa − Servei de Microbiologia, Hospital dihedral angle restraints. The NOE cross-peaks were integrated using Universitari Vall d’Hebron, Barcelona 08035, Spain; the automatic integration subroutine of the SPARKY software.47 The Departament de Genètica i Microbiologia, Universitat TALOSn webserver51 was used to obtain the dihedral angle restraints Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain from the1H and13C chemical shifts. The final structure of each peptide is the ensemble of the 20 lowest target function conformers calculated Juan José González-López − Servei de Microbiologia, in the last cycle. These ensembles were visualized and examined using Hospital Universitari Vall d’Hebron, Barcelona 08035, the MOLMOL program.52 The coordinates for the calculated Spain; Departament de Genètica i Microbiologia, Universitat structures are available upon request from the authors. Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain María Á ngeles Jiménez − Departamento de Química-Física ■ ASSOCIATED CONTENT * Supporting Information sı Biológica, Instituto de Química Física Rocasolano (IQFR- CSIC), Madrid 28006, Spain; orcid.org/0000-0001- 6835-5850 The Supporting Information is available free of charge at Complete contact information is available at: https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00795. https://pubs.acs.org/10.1021/acs.jmedchem.1c00795 Peak integration data for all peptides after incubation with human serum; degradation profiles for peptides 3 Author Contributions and 4; A. baumannii survival ratio after incubation with E.B., D.A., and M.T. designed, directed, obtained funding, and peptides; antimicrobial resistance; membrane depolari- coordinated the study. D.S. performed the synthesis of the zation after incubation with peptides; circular dichroism peptides, supervised by J.V.; the antimicrobial assays, assisted spectra for peptides in the presence and absence of SDS by G.P.E., M.N.L., and J.J.G., and the remaining experimental micelles; ΔδCα values for peptides 1, 2, 6, and 7 in work other than the NMR experiments, carried out by B.C.-A., aqueous solution and DPC micelles; NMR solution supervised by M.A.J., who analyzed the results and contributed structures for peptides 1, 2, 6, and 7; NMR solution the corresponding text, tables, and figures. D.S. wrote an initial structures for C-Terminal regions of peptides 1, 2, 6, and version of the paper, subsequently edited by D.A. and M.T. All 7; MBC values; antimicrobial activity in the presence of authors contributed to editing and revising the final version of human serum for peptides 1, 2, 3, and 4; predicted the manuscript. secondary structure of peptides; averaged ΔδHα and Funding ΔδCα values for peptides 1, 2, 6, and 7 in aqueous This work was supported by the Spanish Ministerio de Ciencia, solution and DPC micelles; structural statistics param- Innovación y Universidades: SAF2015-72518-EXP, SAF2017- eters for peptide structures 1, 2, 6, and 7; HPLC traces 82158-R and RYC-2012-09999 to M.T.; CTQ2017-84371-P to and MS spectra for all peptides (PDF) M.A.J.; AGL2014-52395-C2; AGL2017-84097-C2-2-R to D.A. Molecular formula strings (CSV) and PID2019-106123GB-I00 to E.B. Additional financial support from the European Society for Clinical Microbiology ■ and Infectious Disease, ESCMID 2016, to M.T., the Secretaria AUTHOR INFORMATION d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya, 2019 LLAV Corresponding Authors 00029 to M.T. and 2016 PROD 00060 to E.B. The “Mariá de Ester Boix − Department of Biochemistry and Molecular Maeztu” Program for Units of Excellence in R&D from the Biology, Universitat Autònoma de Barcelona, Cerdanyola del Spanish Ministry of Innovation and Competitiveness (MINE- Vallès 08193, Spain; orcid.org/0000-0003-1790-2142; CO) for work at Pompeu Fabra University (D.A.) is Email: ester.boix@uab.cat acknowledged. This work was supported, in part, by the David Andreu − Department of Experimental and Health European Regional Development Fund ‘A Way to Achieve Sciences, Universitat Pompeu Fabra, Barcelona Biomedical Europe’ [Spanish Network for Research in Infectious Diseases Research Park, Barcelona 08003, Spain; orcid.org/0000- (Grant No. RD16/0016/0003)]. D.S. and B.C.-A. are 0002-6317-6666; Email: david.andreu@upf.edu recipients of pre-doctoral FPI scholarships (PRE2018-083243 Marc Torrent − Department of Biochemistry and Molecular and BES-2015-073383, respectively) from the Spanish Biology, Universitat Autònoma de Barcelona, Cerdanyola del Ministerio de Ciencia, Innovación y Universidades. Vallès 08193, Spain; orcid.org/0000-0001-6567-3474; Notes Email: marc.torrent@uab.cat The authors declare no competing financial interest. Authors Daniel Sandín − Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Cerdanyola del ■ ACKNOWLEDGMENTS The NMR experiments were performed in the “Manuel Rico” Vallès 08193, Spain NMR laboratory, LMR, CSIC, a node of the Spanish Large- 11480 https://doi.org/10.1021/acs.jmedchem.1c00795 J. Med. Chem. 2021, 64, 11472−11482 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scale National Facility ICTS R-LRB. CD and Fluorescence (17) Maeda, T.; Kitazoe, M.; Tada, H.; de Llorens, R.; Salomon, D. measurements were performed at the Laboratory of S.; Ueda, M.; Yamada, H.; Seno, M. Growth Inhibition of Mammalian Luminescence and Biomolecule Spectroscopy (LLEB) and Cells by Eosinophil Cationic Protein. Eur. J. Biochem. 2002, 269, SEM was performed at the Servei de Microscopia (SM) under 307−316. (18) Malik, A.; Batra, J. K. Antimicrobial Activity of Human the supervision of Dr. Alex Sánchez, at the Universidad Eosinophil Granule Proteins: Involvement in Host Defence against Autònoma de Barcelona. ■ Pathogens. Crit. Rev. Microbiol. 2012, 38, 168−181. (19) Torrent, M.; Pulido, D.; Valle, J.; Nogués, M. V.; Andreu, D.; ABBREVIATIONS Boix, E. Ribonucleases as a Host-Defence Family: Evidence of Evolutionarily Conserved Antimicrobial Activity at the N-Terminus. AMP, antimicrobial peptide; ECP, eosinophil cationic protein; Biochem. J. 2013, 456, 99−108. ACN, acetonitrile; MIC, minimum inhibitory concentration; (20) Bystrom, J.; Amin, K.; Bishop-Bailey, D. Analysing the Orn, ornithine; Dab, 2,4-diaminobutyric acid; Har, homo- Eosinophil Cationic Protein - a Clue to the Function of the arginine; PBS, phosphate buffered saline; MTT, 3-(4,5- Eosinophil Granulocyte. Respir. Res. 2011, 12, 10. dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (21) Boix, E.; Salazar, V. A.; Torrent, M.; Pulido, D.; Nogués, M. 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