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Tryptic Stability of Synthetic Bactenecin Derivatives Is Determined by the Side Chain Length of Cationic Residues and the Peptide Conformation Mojtaba Bagheri, Shima Arasteh, Evan F. Haney, and Robert E.W. Hancock J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01740 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 9, 2016

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Journal of Medicinal Chemistry

Tryptic Stability of Synthetic Bactenecin Derivatives Is Determined by the Side Chain Length of Cationic Residues and the Peptide Conformation Mojtaba Bagheri,*† Shima Arasteh,† Evan F. Haney,‡ and Robert E. W. Hancock*‡ †

Peptide Chemistry Laboratory, Institute of Biochemistry and Biophysics, University of Tehran,

16 Azar Street, 1417614335 Tehran, Iran;

‡

Centre for Microbial Diseases and Immunity

Research, University of British Columbia, 2259 Lower Mall Research Station, Vancouver, BC V6T 1Z4, Canada

KEYWORDS. antimicrobial peptides, bioavailability, proteolytic stability, molecular modeling, isothermal titration calorimetry

ACS Paragon Plus Environment

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ABSTRACT

Synthetic bactenecins 1 (HHC-10) and 10 (HHC-36), with excellent activities against bacterial superbugs, display low tryptic stability. To investigate factors influencing this stability, a series of 1/10 derived peptides bearing arginine and lysine analogs with varied methylene chains as well as all-D-isomers were synthesized. Whereas incorporation of D-/L-nonproteinogenic amino acids into the turn-forming peptides did not dramatically affect the antimicrobial activities, the degree of peptide cleavage decreased significantly in peptides with the shortest length of cationic side chain, and was influenced by the relative conformational stabilities of the turn structure and the stereoselectivity of tryptic digestion. The site of enzymatic cleavage was located at the less conformationally hindered position distant from the turn motif. Isothermal titration calorimetry showed strong and weak constant increments in the generated heat of enzymatic reaction of unstable and slowly degradable peptides with trypsin, respectively, and suggested a one-site binding model for the enthalpy-driven all-D-peptide-trypsin interactions.


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INTRODUCTION Antimicrobial peptides (AMPs) play important roles in the innate and acquired immune system of many organisms and have a broad spectrum of activity against bacteria with often weakly cytotoxic effects. These positively charged, amphipathic peptides act either directly by disrupting the barrier function of the cell membrane ultimately leading to bacterial cell death, or indirectly through modulation of the immune response (e.g., induction of chemokines, suppression of pro-inflammatory cytokines, promotion of chemotaxis).1 Unfortunately, low bioavailability and poor metabolic stability of AMPs have limited their effectiveness as orally administered antibiotics.2 Despite the most widely employed engineering approaches for minimizing peptide proteolytic digestion, including modifying peptide structural conformation (e.g., C- and N-terminal modification, side chain modifications, Nα-alkylation, synthesis of all-Disomers), and/or changing peptide conformation (e.g., peptide cyclization, backbone modifications, β-/α,β-peptides),2 few systematic investigations on the proteolytic stability of AMPs have been performed that focus on the relationship between the peptide conformation and the enzymes’ substrate specificity to enable understanding of the modes of binding of AMPs to proteases.3-5 Short AMPs offer several advantages over longer peptides, most notably easy synthesis and simple chemical modification by the introduction of non-proteinogenic amino acids.6-8 1 (HHC10) and 10 (HHC-36) are short AMPs containing primarily lysine (Lys), arginine (Arg) and tryptophan (Trp) residues (Table 1), and have demonstrated potent and broad-spectrum antimicrobial activities against multidrug-resistant superbugs in the low micromolar range in vitro.9 Additionally, they protected mice against invasive Staphylococcus aureus infections


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in vivo.9 These characteristics make 1 and 10 promising lead structures for further development as antibacterial therapeutics. However, due to the presence and abundance of Lys and Arg residues in their sequences, these peptides are highly susceptible to proteolytic digestion by trypsin-like serine proteases which are the most abundant family of peptidases in humans and are involved in various biological processes including digestion, coagulation and immunity.10 To address this issue, the substrate specificity of trypsin with respect to the structural determinants of the enzyme binding pockets (S and S’) and their preferences for the peptide substrate side chains (P and P’ sites) should be taken into consideration to systematically design active peptide variants with improved protease stability. Trypsin is characterized by high substrate specificity, mainly determined by an electrostatic interaction between aspartate (Asp)-189 in the S1 binding pocket with P1-Arg/Lys residues, and very broad S’ specificity; i.e., S1’/S3’ and S2’ with slight preferences for hydrophobic and positively charged residues, respectively.11,12 In this study, a design principle based on the S1 site preference was used to synthesize a set of 1 and 10 peptide analogs carrying substitutions of Arg and Lys with non-proteinogenic amino acid variants (Table 1). The substituents preserved the cationicity of the Arg and Lys residues but differ in terms of side chain length by varying the number of methylene groups. They include L-ornithine (Orn) and (2S)-2,4-diaminobutyric acid (Dab) as Lys mimics, and L-homoarginine (Har) as an Arg mimic with shortened and elongated side chains, respectively. Lys or Arg in the parent sequences were either substituted by their corresponding mimics or entirely replaced with Lys or Arg mimics to test whether variation in the cationic amino acids side chain length had any influence on the peptide structure, bioactivity and stability towards trypsin degradation. Also, peptides 4 and 13, where Arg and Lys residues were interchanged within the sequences, were synthesized to study amino acid preferences for


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the S/S’ subsites in trypsin. Moreover, the stereoselective action of trypsin was evaluated using all-D-peptides (2 and 11) and the retro-inverso sequences (3 and 12). The results of this study revealed important insights that can be applied to the design and development of AMPs with improved bioavailability and increased metabolic stability.


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RESULTS AND DISCUSSION Synthesis and Chemical Characterization of the Peptide Derivatives. The peptide library was synthesized manually by solid-phase peptide synthesis (SPPS),8 purified by reversed-phase high performance liquid chromatography (RP-HPLC) and further characterized by mass spectrometry (MS) (Table 1, see Figures S1 and S2 in the Supporting Information). Whereas the peptide variants of 1 were more amphipathic/hydrophobic than the analogs of 10 (compare tR = ~ 27 min with tR = ~ 25 min for the former and the latter, respectively), all the peptides derived from the same parent sequence had almost identical tR values independent of the substituents chemical properties and the peptide design principles. Antimicrobial Assay. The antimicrobial activities of the peptides against Gram-negative and Gram-positive bacterial species were evaluated in vitro (Table 1). While the peptides derived from 1 were largely more active than the derived sequences from 10, in general all the peptide analogs demonstrated comparable activities to their parent sequences. This was well correlated with the more pronounced hydrophobicity of 1 compared with 10 as well as identical tR values among the peptides in the same group. Exceptions to this included 13 and 15 with weaker activity against A. baumannii, B. subtilis, and S. aureus as well as all-Har-incorporated peptides (i.e., 7 and 16), which exhibited a 4-fold improvement in anti-B. subtilis activity. Overall, these results indicate that substitution of cationic residues in both peptides had a very minor influence on their antimicrobial potency. Structural Characterization of the Peptides. All of the peptides were structurally characterized by circular dichroism (CD) spectroscopy and their conformations were modeled using molecular dynamics (MD) simulations.


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CD showed a strong negative band at ~ 200 nm and a weak positive band at 225-230 nm for the peptides derived from 1 as well as an extra well-resolved shoulder at ~ 217 nm for 7 at 200 µM (Figure 1). At this concentration, all the L-peptides derived from 10 revealed two negative bands at ~ 200 nm and ~ 225 nm, with the former band being stronger (and vice versa for 16). In addition, the spectra of all the peptides in 50% 2,2,2-trifluoroethanol (TFE) were recorded resulting in major spectral changes for both the 1 and 10 derived sequences (Figure 1). Interestingly, the spectral characteristics of the derivatives of 1 become similar to those of peptides derived from 10 in the buffer, and all the spectra demonstrated an increase in the intensity of the band at ~ 223 nm at the expense of the ~ 200 nm band. This effect was particularly pronounced for the 10 derived sequences. Indeed, the ellipticity contributions in the CD spectra of these Trp-rich peptides could be attributed to the superimposed effects of the amide backbone and the probable Trp-Trp exciton coupling as a result of specific stacking interaction between Trp residues (seen as two positive or negative absorption bands at 199202 nm and 225-230 nm)13,14 making the interpretation of spectra complex. However, the data presented in Figure 1 are in good agreement with the existence of turn structures found for the parent 1 and 10 sequences in a recent study15 as well as in Trp-rich indolicidin analogs.13 Two strong positive bands at ~ 200 nm and ~ 223 nm appear for 11 and 12, which look like a mirror image of the L-peptide CD spectra. MD simulations of the peptides in water supported these findings (Figure 2). Analysis of the simulations at 310 K and the distribution of Φ and Ψ dihedral angles in Ramachandran plots suggested that almost all the L-peptides adopt turn structures, but the turn-forming residues differed among the sequences (Figure 3, see Figures S3 and S4 in the Supporting Information).


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Exceptions were 16 and the D-peptides that formed αR and αL helices, respectively (Figure 2, Figures S3 and S4). Furthermore, the concentration dependence profile of the CD spectra for 7, 11, 12, and 16 (from 400-25 µM in two-fold dilutions) were recorded to evaluate the self-association properties of the peptides (Figure 4). The excitonic coupling effects on the CD spectra for the peptides in buffer were stronger at lower concentrations leading to the reorientation of the peptide side chains and stronger Trp-Trp stacking. This interpretation of the CD spectra is supported by the results of the MD studies for the peptides (Figure 2B). However, the positive molar ellipticity of the peptides decreased at higher concentrations and a high red shift was observed in the adsorption bands at ~ 200 nm and ~ 223 nm, which are indicative of peptide oligomerization.16,17 These changes were more pronounced for 7 and in fact, the adsorption bands largely disappeared at the highest peptide concentration of 400 µM in the buffer, suggesting that this peptide is the most aggregation prone sequence. These observations pointed to different peptide structure for 7 compared with 11, 12, and 16: the type II’ β-turn-forming for the former as supported by the MD simulations (Figure 2). Similar changes in the CD spectra was seen for the peptides in TFE, but the reduction in the adsorption bands was less pronounced than the spectral changes seen in the buffer, suggesting that these peptides are less susceptible to aggregation under these conditions. Taken together, these observations are consistent with the recent results on the self-association of 1 and 10.15 Enzymatic Assay. The in vitro stability of the peptides was evaluated by analytical-HPLC and MS analysis of peptide fragments produced during the course of enzymatic digestion with trypsin. Although substitution of Arg with Orn and Har in antimicrobial apidaecin 1b extended


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serum half-life of the peptide,18 the tryptic stability of the 1 and 10 derivatives was not much improved when the cationic residues were exchanged with the same Arg/Lys-mimics (Table 2, Figures S5 and S6 in the Supporting Information). In contrast, replacing the Arg/Lys in 10 (that has one more positive charge than 1) with Dab resulted in a stable analog; i.e., type II β-turnforming 14, compared with type VIII β-turn forming 5. Moreover, the cleavage of the peptide bond in 14 occurred slowly overnight between Dab-1 and Dab-2 (Table 2). Most likely, the formation of the energetically favored type II β-turn motif, and its prominent impact on the peptide structural stability,19 together with the shorter side chain of the Dab residues, played an important role in improving the resistance of 14 to the proteolytic action of trypsin. The early degradation products by trypsin of most of the other peptide derivatives showed that the predominant cleavage sites were located at the N terminus, with the notable exception being 8 (Table 2). No exact information on the cleavage sites for 9 and the fast degradable 1, 4, and 10 could be obtained. As expected, all of the D-peptides (2, 3, 11 and 12) were impervious to the enzymatic activity of trypsin, validating the stereoselectivity of trypsin hydrolysis. Thermodynamics of the Peptide-Trypsin Interactions. The enzymatic digestion profiles were correlated with the thermodynamics of peptide-trypsin interactions using isothermal titration calorimetry (ITC). Fast degradation of unstable peptides made of natural L-amino acids as well as all-Har-incorporated peptides was accompanied by strong exothermic heat of enzymatic reaction with slight changes in the calorimeter traces (see Figure S7 in the Supporting Information). This indicated that the heat of reaction was dominated by amide bond cleavage rather than any physical interaction between the peptide and the enzyme. In contrast, the constant stepwise enthalpy assessed upon binding to trypsin of all-Orn and all-Dab peptides, particularly the latter, was negligible and pointed to weak interactions. Interestingly, the stable peptides


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composed of D-amino acid subunits exhibited distinctly different ITC traces compared with the L-peptides (Figure S7). The enthalpy-driven stoichiometric peptide-trypsin interactions were consistent with a one-site binding model. Molecular Docking. Support for the models of the peptide-trypsin interactions was provided by docking experiments. In agreement with the MS analysis, the docking studies showed that the peptide bonds located at the N terminus of the L-peptides were most affected by trypsin (see Figures S9, and S10 in the Supporting Information). The exceptions were unstable 1 and 4, which were specifically cleaved from the carboxyl end and in the middle of the peptide, respectively. P1-Orn5 and P1’-Trp6 in 6 were detected as substrates for the S1/S1’ enzyme binding pocket. Taken together with the peptide structural variations (Figure 1-3), it appears that trypsin cleaved the less sterically hindered amide bonds. The cleavage site was especially far away from the existing turn motif in peptides bearing a turn structure (Figure 3).20 Also, no cationic residues in the D-peptides fit into the S1 substrate binding site, emphasizing their tryptic stability. The low relative stability of the poorly-folded turn-structure might have been the reason for the lack of correlation between the structure and mode of enzymatic cleavage for 8, 9, and 15. With respect to the S/S’ preferences profile, the docking models indicated slight substrate specificity of S1’ for Trp and only a minor role for other binding pockets (Sn/Sn’ where n > 1) in trypsin (Figures S9, and S10). CONCLUSION In summary, we have shown that independent of the number of Arg/Lys residues in the sequences, the tryptic stability of the synthetic bactenecin derivatives increased as the length of the cationic amino acid side chain decreased, as supported by the lowest degree of HHC-peptide


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degradation and the minimal number of the peptide fragments generated from tryptic digestion. Additionally, the enzymatic hydrolysis of the peptides was affected by the relative stability of the peptide structure. This became more evident when we compared the early tryptic digestion of 14; forming a stable type II β-turn motif, with the digestion products of type VIII β-turn 5. Also, the stability of the all-D-peptides demonstrated the stereoselectivity of the enzymatic action of trypsin. Importantly, the antimicrobial activities of the peptide analogs against various bacterial strains did not dramatically change compared with the parent peptides. Altogether, the outcomes of this study may serve in designing effective AMPs with increased range of physicochemical properties for use against multi-resistant bacterial infections.


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EXPERIMENTAL SECTION Compounds. The Rink Amide-MBHA, the activating reagents 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), and the 9-fluorenylmethoxycarbonyl (Fmoc)-N-protected amino acids, such as Fmoc-Arg (2,2,4,6,7-pentamethyldihydrobenzofuran5-sulfonyl [Pbf])-OH, Lys (tert-butyloxycarbonyl [Boc])-OH, Fmoc-Trp (Boc)-OH (in D- or Lisomers) as well as Fmoc-Dab (Boc)-OH, Fmoc-Orn (Boc)-OH, Fmoc-Har (Pbf)-OH were purchased from GL Biochem, Ltd., China. Trypsin was obtained from Sigma-Aldrich, Germany. Acetic acid (Fisher, U. K.) was obtained from the noted provider. N,N'-diisopropylethylamine (DIEA), disodium monohydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4),

sodium

fluoride

(NaF),

piperidine,

trifluoroacetic

acid

(TFA),

TFE,

tris(hydroxymethyl) aminomethane (Tris) hydrochloride, ammonium bicarbonate [(NH4)HCO3], and solvents were used as obtained from Merck, Germany. Sterile 96-well microtiter plates was from Costar, U. S. Peptide Synthesis, Purification and RP-HPLC/Mass characterization. Peptides were synthesized manually by SPPS using the standard Fmoc / tert-butyl protocol.8 Double coupling (each for 40 min) syntheses were carried out on Rink Amide-MBHA (cross linker: 1% divinyl benzene, parity grade: 0.3-0.8 mmol.g-1, particle size 100-200 mesh) using Fmoc-protected amino acids (3 equiv, 0.3 M) and HBTU (2.9 equiv, 0.3 M) as coupling reagent in the presence of DIEA (6 equiv, 1.2 M) in dimethylformamide (DMF). Fmoc-removal was performed twice with 20% piperidine in DMF for 5 min. Washing steps were carried out with DMF. The final cleavage of the peptide from the resin and deprotection of amino acids side chain were achieved by incubation in a mixture of 5% phenol in TFA for 3 hours. Then, peptides were precipitated from cold diethyl ether. The crude peptides were dissolved in water / acetonitrile (ACN)


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1/1 (v/v), and were purified by RP-HPLC (Agilent 1260 infinity, U. S.) operating at 220 nm to give final products ≥ 95% pure. Chromatographic characterization was performed on analytical RP-HPLC system (Waters (U. S.)) operating at 220 nm using a Nova-Pak C18 Cartridge, 60 Å, 4 µm, 4.6 mm × 250 mm column (Waters, U. S.). Sample concentration was 1 mg of peptide/mL in water / ACN 1/1 (v/v). The mobile phase A was 0.1% TFA in water, and phase B was 0.1% TFA in water / ACN 1/4 (v/v). tR of the peptides was determined using a linear gradient of 5 to 95% phase B over 40 min at room temperature. The peptides were further characterized by MS with electrospray ionization operated in the positive ionization mode using an Agilent 6410 A Triple Quadrupole LC/MS (U. S.) to confirm the correct molar masses. Antimicrobial activity. Antimicrobial activity of the peptides was tested in sterile 96-well polypropylene microtiter plates against two Gram-negative strains; i.e., E. coli (O157:H7), A. baumannii (ATCC 17978), and two Gram-positive strains; i.e., B. subtilis (C626), and S. aureus (C622) using a modified broth microdilution method in Mueller-Hinton broth (MHB) similar to that described previously.9 Briefly, the peptides were dissolved and stored in glass vials. Serial dilutions of the peptides to be assayed were performed in water at 2-fold the desired final concentration. 50 µL of the 2-fold concentrated peptides were added to each well of the microtiter plate and 50 µL of a bacterial suspension in 2 × concentrated MHB was added to each well. Bacteria were added to the plate to a final concentration of 2˗7 × 105 CFU.mL-1. The plates were incubated overnight at 37 °C and the minimal inhibitory concentration (MIC) was recorded as the concentration at which no growth was observed.


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CD spectroscopy. Stock solutions of peptides were prepared in phosphate buffer (400 µM, 10 mM NaH2PO4/Na2HPO4, 154 mM NaF, pH 7.4). This solution was further diluted with either phosphate buffer or mixed 1:1 with TFE (a chemical agent inducing secondary structures in peptides/proteins)21 to reach the desired peptide concentration (200 µM) for the CD analysis. For the concentration dependence of the CD spectra studied for 7, 11, 12, and 16 as the representatives of the synthetic bactenecin analogs, the peptide concentrations ranged from 400 µM to 25 µM in two-fold dilutions. The measurements were carried out on a CD spectrometer (Aviv Biomedical, Inc. U. S.) between 190 and 260 nm at room temperature using a cuvette with pathlength of 1.0 mm. Peptide spectra were corrected by subtracting the spectra of the phosphate buffer alone or mixed with TFE. The results are presented as the mean residue ellipticity [Θ] with units of deg.cm2.dmol-1. Enzymatic Assay. A fresh stock solution of trypsin from bovine pancreas (salt-free, lyophilized powder, ≥ 10,000 BAEE units/mg protein) was prepared by dissolving 1 mg of the enzyme in 50 mL of 0.1 M (NH4)HCO3 buffer, pH = 8.2. Aliquots of peptide solution at concentration ranging from 2˗5 mM in buffer, 150 µL of the trypsin solution and the buffer were pipetted into Eppendorf tubes to give a final volume of 1.5 mL. The final peptides concentration was 60 µM. At this concentration the risk of peptides oligomerization, which happened at high concentrations, is relatively low and the resulting peaks in HPLC and MS corresponding to the products of enzymatic cleavage are detectable. The mixture was incubated with gentle shaking in an Eppendorf thermomixer at 37 °C. The enzymatic reaction was followed 2 h and 24 h after the incubation. At each time interval, 50 µL of a quenching cocktail containing 99.99 % acetic acid and 0.1 % TFA was added to an Eppendorf tubes pre-filled with 200 µL-aliquot taken from the reaction mixture. The resulting sample was analyzed directly by a Waters RP-HPLC system and


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the Nova-Pak C18 Cartridge column as described above. Samples without the enzyme was used as control and analyzed further by the chromatography system. The mobile phase A, phase B, and the linear gradient were as mentioned earlier. The quenched samples were further subjected to MS (Agilent 6410 A Triple Quadrupole LC/MS) after 100-fold dilution. Direct-injection mass spectrometric method was used to prevent probable damage of the column performance. ITC. High-sensitivity ITC was performed on a VP-ITC (GE Healthcare, Sweden). All experiments were run at 37 °C to study peptide binding to trypsin. To determine peptide-enzyme interactions, a 0.1 mM solution of trypsin from bovine pancreas (salt-free, lyophilized powder, ≥ 10,000 BAEE units/mg protein) in the calorimeter cell was titrated with a 2 mM peptide solution (buffer: 50 mM Tris hydrochloride, 10 mM CaCl2, pH 8.2). The enzyme solution was degassed before ITC experiments. In a typical experiment, each titration step corresponds to the injection of 6 µL of peptide solution (except the 10 µL-injections for 5/6, and 15 µL-injection for 13) into the enzyme solution. The content of the sample cell was stirred continuously at 310 rpm. Because of a small loss of titrant during the mounting of the syringe and the equilibration stage preceding the actual titration, we set the first injection volume to 3 µL and excluded the first peak from data analysis. Time spacings were long enough to allow the ITC signal to return to the baseline value. The measured heat of binding either decreased or remained unchanged with consecutive peptides injections. Baseline correction, peak integration and adjustment were done using VP-ITC ORIGIN 5.0 (MicroCal Software, Northampton, U. S.) as described by the manufacturer. Control experiments injecting peptides into buffer without trypsin were recorded and subtracted from ITC signals for the corresponding samples. If applicable, the non-linear least square analysis with the “one set of sites” binding model was used to fit the peptides binding to trypsin and derive the binding isotherms.


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Molecular Modeling. The GROningen MAchine for Chemical Simulations (GROMACS) simulation package22 and the Chemistry at HARvard Macromolecular Mechanics (CHARMM) force field23 were used for the peptides and trypsin MD simulation in water. The crystal structure of trypsin (PDB ID: 4I8G) was retrieved from the protein data bank (http://www.rcsb.org./pdb). Peptide acid models (“.pdb” file) were constructed using Hyperchem 8 software (Hypercube Inc., U. S.). The random-coil structure was selected as the initial structure while generating the “.pdb” files. The parameters for the non-proteinogenic and D-amino acids were added to the CHARMM force field for the peptide analogs using the SwissSidechain database (http://www.swisssidechain.ch/index.php).23 While generating the “.top” file, the peptide C terminal carboxyl was further capped with an NH2 group in GROMACS. Missed bonds, dihedrals, pairs and angles were manually added to the “.top” file. Then, the peptides and trypsin were independently solvated in a cubic box filled with TIP3P water molecules. The peptides and trypsin structures were minimized in separate experiments, after which the system was rendered neutral by the addition of a sufficient number of sodium or chloride ions with respect to the total charge of the system. The MD simulations were performed using standard parameters under the NPT ensemble (T = 310 K, p = 1 atm) until a root-mean-square deviation of atomic positions (RMSD) values less than 0.4 nm was reached, which represents the existence of stable peptide/protein structures in water. The temperature of the system was coupled to a V-rescale thermostat to generate the Maxwell-Boltzmann distribution at 310 K with a coupling time of 0.1 ps. The pressure was also coupled to Parrinello-Rahman at 1 atm. Simulations were done with the time step of 2 fs. MD simulations were performed for a maximum of 20 ns in order to approach the required RMSD < 0.4 nm. One hundred configurations were selected from the simulation at each 200 ps time intervals and subjected to cluster analysis. Cluster analysis was


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done over the whole MD trajectory with a 0.1 nm cutoff on Cα atoms, which is a good value especially for peptides with flexible structure. Clusters with the highest percentage of occurrence for each peptide/protein were selected and used to build the models for peptide/trypsin structural analysis and docking experiments. Docking experiments were done using Hex Protein Docking program HEX 8.0.0 software between

the

each

independent

peptide

and

trypsin

models

as

described

in

http://hex.loria.fr/hex.php.24 Information on trypsin binding pockets (shown in Figure S8 in the Supporting Information) was taken from ref. 10. Afterwards, the energetically favored docked models were used further for an extra 1 ns MD simulation in water under the same setup conditions as above. Visualization and analysis of the peptides and trypsin structure as well as docked systems was performed using visual molecular dynamics (VMD) 1.9.2.25


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ASSOCIATED CONTENT Supporting Information. Details of HPLC and MS characterization of the peptides and the products of enzymatic cleavages, Ramachandran plots of the amino acids residues in the sequences, structure of trypsin and molecular docked models of the peptides with the enzyme. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors Dr. Mojtaba Bagheri, Peptide Chemistry Laboratory, Institute of Biochemistry and Biophysics, University of Tehran, 16 Azar Street, 1417614335 Tehran, Iran, E-mail: [email protected], Tel: +98 (21) 6696 9255; Fax: +98 (21) 6640 4680 Dr. Robert E. W. Hancock Centre for Microbial Diseases and Immunity Research, University of British Columbia, 2259 Lower Mall Research Station, Vancouver, BC V6T 1Z4, Canada, Email: [email protected], Tel: +1 (604) 822 2682; Fax: +1 (604) 827 5566 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Dr. Farhad Bani from University of Tehran for his help with the CD experiments. Support from Iran National Science Foundation (funding reference number 91058943) and University of Tehran to M.B. are gratefully acknowledged. This research was also supported by a grant from the Canadian Institutes of Health Research (CIHR) to R.E.W.H (funding reference


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number MOP-74493). E.F.H was supported by a postdoctoral fellowship from the CIHR. R.E.W.H holds a Canada Research Chair.

REFERENCES 1. Fjell, C. D.; Hiss, J. A.; Hancock, R. E. W.; Schneider, G. Designing Antimicrobial Peptides: Form Follows Function. Nature Rev. Drug Discov. 2012, 11, 37–51. 2. Pollaroa, L.; Heinis, C. Strategies to Prolong the Plasma Residence Time of Peptide Drugs. Med. Chem. Commun. 2010, 1, 319–324. 3. Howell, S. M.; Fiacco, S. V.; Takahashi, T. T.; Jalali-Yazdi, F.; Millward, S. W.; Hu, B.; Wang, P.; Roberts, R. W. Serum Stable Natural Peptides Designed by mRNA Display. Sci. Rep. 2014, 4, 6008. 4. Svenson, J.; Vergote, V.; Karstad, R.; Burvenich, C.; Svendsen J. S.; de Spiegeleer, B. Metabolic Fate of Lactoferricin-Based Antimicrobial Peptides: Effect of Truncation and Incorporation of Amino Acid Analogs on the In Vitro Metabolic Stability. J. Pharmacol. Exp. Ther. 2010, 332, 1032–1039 5. Knappe, D.; Henklein, P.; Hoffmann, R.; Hilpert, K. Easy Strategy to Protect Antimicrobial Peptides from Fast Degradation in Serum. Antimicrob. Agents Chemother. 2010, 54, 4003–4005. 6. Bagheri, M. Cationic Antimicrobial Peptides (AMPs): Thermodynamic Characterization of Peptide-Lipid Interactions and Biological Efficacy of Surface-Tethered Peptides. ChemistryOpen. 2015, 4, 389–393


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7. Bagheri, M.; Keller, S.; Dathe, M. Interaction of W-substituted Analogs of CycloRRRWFW with Bacterial Lipopolysaccharides: the Role of the Aromatic Cluster in Antimicrobial Activity. Antimicrob. Agents Chemother. 2011, 55, 788–797 8. Bagheri M.

Synthesis

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Cyclic

Antimicrobial Arginine and Tryptophan-Rich Peptides with Selectivity for GramNegative Bacteria. In Methods in Molecular Biology, vol. 618; Giuliani, A., Rinaldi, A. C., Eds.; Humana Press: New York, 2010; pp. 87–109. 9. Cherkasov, A.; Hilpert, K.; Jenssen, H.; Fjell, C. D.; Waldbrook, M.; Mullaly, S. C.; Volkmer, R.; Hancock, R. E. W. Use of Artificial Intelligence in the Design of Small Peptide Antibiotics Effective Against a Broad Spectrum of Highly Antibiotic-Resistant Superbugs. ACS Chem. Biol. 2009, 4, 65–74. 10. Page, M. J.; Di Cera, E. Serine peptidases: classification, structure and function. Cell Mol. Life Sci. 2008, 65, 1220–1236. 11. Ma, W.; Tang, C.; Lai, L. Specificity of Trypsin and Chymotrypsin: Loop-MotionControlled Dynamic Correlation as a Determinant. Biophys. J. 2005, 89, 1183–1193 12. Kurth, T.; Grahn, S.; Thormann, M.; Ullmann, D.; Hofmann, H. J.; Jakubke, H. D.; Hedstrom, L. Engineering the S1' Subsite of Trypsin: Design of a Protease Which Cleaves between Dibasic Residues. Biochemistry. 1998, 37, 11434–11440. 13. Ladokhin, A. S.; Selsted, M. E.; White, S. H. CD Spectra of Indolicidin Antimicrobial Peptides Suggest Turns, not Polyproline Helix. Biochemistry. 1999, 38, 12313–12319.


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14. Grishina, I. B.; Woody, R. W. Contributions of Tryptophan Side Chains to the Circular Dichroism of Globular Proteins: Exciton Couplets and Coupled Oscillators. Faraday Discuss. 1994, 99, 245–262. 15. Nichols, M.; Kuljanin, M.; Nategholeslam, M.; Hoang, T.; Vafaei, S.; Tomberli, B.; Gray, C. G.; DeBruin, L.; Jelokhani-Niaraki, M. Dynamic Turn Conformation of a Short Tryptophan-Rich Cationic Antimicrobial Peptide and Its Interaction with Phospholipid Membranes. J. Phys. Chem. B. 2013, 117, 14697–14708. 16. Dai, X.; Eccleston, M. E.; Yue, Z.; Slater, N. K. H.; Kaminski, C. F. A Spectroscopic Study of the Self-Association and Inter-Molecular Aggregation Behaviour of pHResponsive Poly(L-Lysine iso-Phthalamide). Polymer. 2006, 47, 2689–2698. 17. Frère, V.; Sourgen, F.; Monnot, M.; Troalen, F.; Fermandjian, S. A Peptide Fragment of Human DNA Topoisomerase II Alpha Forms a Stable Coiled-Coil Structure in Solution. J. Biol. Chem. 1995, 270, 17502–17507. 18. Berthold, N.; Czihal, P.; Fritsche, S.; Sauer, U.; Schiffer, G.; Knappe, D.; Alber, G.; Hoffmann, R. Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens. Antimicrob. Agents Chemother. 2013, 57, 402–409. 19. Fu, H.; Grimsley, G. R.; Razvi, A.; Scholtz, J. M.; Pace, C. N. Increasing Protein Stability by Improving beta-turns. Proteins. 2009, 77, 491–498.


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20. Yang, S.; Proctor, A.; Cline, L. L.; Houston, K. M.; Waters, M. L.; Allbritton, N. L. βTurn Sequences Promote Stability of Peptide Substrates for Kinases within the Cytosolic Environment. Analyst. 2013, 138, 4305–4311. 21. Roccatano, D.; Colombo, G.; Fioroni, M.; Mark, A. E. Mechanism by Which 2,2,2Trifluoroethanol/Water Mixtures Stabilize Secondary-Structure Formation in Peptides: A Molecular Dynamics Study. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12179–12184. 22. Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; v. d. Spoel, D.; Hess, B.; Lindahl, E. GROMACS 4.5: a High-Throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics. 2013, 29, 845–854. 23. Gfeller, D.; Michielin, O.; Zoete, V. SwissSidechain: a Molecular and Structural Database of Non-Natural Sidechains. Nucleic Acids Res. 2013, 41, D327–D332. 24. Ghoorah, A. W.; Devignes, M. D.; Smaïl-Tabbone, M.; Ritchie, D. W. Protein Docking Using Case-Based Reasoning. Proteins. 2013, 81, 2150–2158. 25. Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. Graphics. 1996, 14, 33–38.


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Table 1. The sequences, secondary structure, in vitro antimicrobial activities, calculated and observed molecular masses, and RP-HPLC tRa values of the synthetic bactenecin peptides and their analogs Peptide code / Compound

X1

1d 2e 3e 4 5 6 7 8 9

Lys Lys NAg Arg Dab Orn Har Dab Orn

10d 11 12f 13 14 15 16 17 18 Polymyxin Bh Vancomycinh

Lys Lys NAg Arg Dab Orn Har Dab Orn -----

Mr [M+2H]+2 (Da)b MIC (µM) tR (min) Struct.c calc. obs. E. coli A. baumannii B. subtilis S. aureus Sequence = X1-X2-Trp-Trp-X1-Trp-Ile-X2-Trp-NH2 Arg 722.4 722.5 27.15 1.25 5 20 5 β-turnVIII Arg 722.4 722.5 NDf ND ND ND ND αL NA 722.4 722.5 ND ND ND ND ND αL Lys 722.4 722.5 24.93 1.25 5 20 2.5 β-turnIII 2.5 5 10 2.5 Dab 638.4 638.4 27.93 β-turnVIII Orn 666.4 666.5 27.10 β-turnI/VIII 1.25 10 10 5 2.5 5 5 2.5 Har 778.4 778.5 27.57 β-turnII’ Har 708.4 708.5 27.24 ND 1.25 5 20 5 Har 722.4 722.5 27.40 ND 1.25 5 20 5 Sequence = X1-X2-Trp-Trp-X1-Trp-Trp-X2-X2-NH2 Arg 743.9 744.0 24.99 ND 2.5 40 40 5 Arg 743.9 744.0 24.78 1.25 20 20 5 αL NA 743.9 744.0 24.79 1.25 ND 20 5 αL Lys 729.9 730.0 24.79 2.5 > 40 > 40 10 β-turnI Dab 631.9 631.9 24.58 2.5 80 20 5 β-turnII Orn 666.9 666.9 24.69 2.5 > 80 40 20 β-turnVIII Har 806.9 807.1 24.83 2.5 20 10 2.5 αR Har 736.9 737.0 25.30 ND 2.5 40 40 5 Har 750.9 751.0 25.21 ND 2.5 40 40 5 --------0.25 0.5 > 32 > 32 --------> 32 > 32 1 1 X2

a

Retention time. b Values are monoisotopic masses in positive mode. c The assigned structures with highest percentage of occurrence were derived from the cluster analysis of 20 ns MD simulation of the peptides in water and the allowed Φ and Ψ values of the peptide backbone in the Ramachandran plots (Figure 4 and Figure S3 and S4 in the Supporting Information). The type of turns was designated by the subscript number. 6 may exist in both type I or type VIII β-turn. αL and αR stand for left- and right-handed helix. d The peptide sequences for 1 and 10 were taken from reference 9. All other peptides (represented by code numbers 2-9 and 11-18) were generated according to the design principle explained in the text. e These peptides were used without any purification. f Not determined. g Not applicable. The sequences for 3 and 12 are Trp-Arg-Ile-Trp-Lys-Trp-TrpArg-Lys-NH2 and Arg-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Lys-NH2, respectively, which are composed of D-isomers of the amino acids. h The antibiotic control concentrations are in µg.mL-1. 1.25 µM of 1 and 2.5 µM of 10 are ~ 1.75 µg.mL-1 and ~ 3.5 µg.mL-1, respectively.


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Table 2. MS analysis of observed peptide fragments at the early (incubation time = 2 hours) and the late (incubation time = 24 hours) tryptic digestiona Peptide

Peptide fragments

incubation time = 2 hours

incubation time = 24 hours

1b

Trp6-Ile-Arg-OHc

2d 3d 4

no digestion no digestion UIe Dab2-Trp-Trp-Dab-Trp-Ile-Dab-Trp-NH2; Trp3-Trp-Dab-Trp-Ile-Dab-Trp-NH2 Orn2-Trp-Trp-Orn-Trp-Ile-Orn-Trp-NH2; Trp3-Trp-Orn-Trp-Ile-Orn-Trp-NH2; Trp6-Ile-Orn-Trp-NH2

Trp6-Ile-Arg-OH; Trp3-Trp-Lys-OH no digestion no digestion Lys2-Trp-Trp-Arg-OH Trp3-Trp-Dab-Trp-Ile-Dab-Trp-NH2; Trp3-Trp-Dab-OH

5 6 7

8

9b

10b 11 12 13 14 15

16 17 18

Trp6-Ile-Har-Trp-NH2 Dab1-Har-Trp-Trp-Dab-Trp-Ile-Har-OH; Har2-Trp-Trp-Dab-Trp-Ile-Har-OH; Trp3-Trp-Dab-Trp-Ile-Har-OH; Trp3-Trp-Dab-OH Orn1-Har-Trp-Trp-Orn-Trp-Ile-Har-OH; Har2-Trp-Trp-Orn-Trp-Ile-Har-OH; Trp3-Trp-Orn-Trp-Ile-Har-OH; Trp6-Ile-Har-Trp-NH2; Trp6-Ile-Har-OH; Trp3-Trp-Orn-OH UI no digestion no digestion Trp3-Trp-Arg-Trp-Trp-Lys-Lys-NH2; Trp3-Trp-Arg-Trp-Trp-Lys-OH; Trp6-Trp-Lys-OH no digestion Orn2-Trp-Trp-Orn-Trp-Trp-Orn-Orn-NH2; Trp3-Trp-Orn-Trp-Trp-Orn-Orn-NH2; Trp3-Trp-Orn-OH Har2-Trp-Trp-Har-Trp-Trp-Har-Har-NH2; Trp3-Trp-Har-Trp-Trp-Har-Har-NH2; Trp3-Trp-Har-Trp-Trp-Har-OH; Trp3-Trp-Har-OH (Trp6-Trp-Har-OH)g Har2-Trp-Trp-Dab-Trp-Trp-Har-Har-NH2 Trp3-Trp-Orn-Trp-Trp-Har-Har-NH2

Orn2-Trp-Trp-Orn-Trp-Ile-Orn-Trp-NH2; Trp6-Ile-Orn-Trp-NH2 Trp6-Ile-Har-Trp-NH2; Trp6-Ile-Har-OH UI

Trp3-Trp-Orn-Trp-Ile-Har-OH

UI no digestion no digestion NDf Dab2-Trp-Trp-Dab-Trp-Trp-Dab-Dab-NH2 ND Har2-Trp-Trp-Har-Trp-Trp-Har-Har-NH2 Trp3-Trp-Har-Trp-Trp-Har-OH Trp3-Trp-Har-OH (Trp6-Trp-Har-OH)g UI UI

a

The information for the peptide fragment sequences were obtained according to the peak lists of each mass spectra shown in Figure S6 in the Supporting Information. b No exact information on the cleavage sites for these peptides were obtained. c Numbers in subscript define the first residues at the intact peptides N-terminus at which the enzymatic cleavage occurred. The fragments with short sequences are an indication of the high degree of peptide degradation. d Impure peptides. e Unidentified. These peptides were digested by trypsin but as a result of their fast degradation no detectable sequences were identified. f Not determined. g As a result of the sequence symmetry in 16, two peptide fragment sequences are possible.


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Figures Legend

Figure 1. The CD spectra of the synthetic bactenecin derived peptides in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. Spectra were acquired on peptide samples prepared at the concentration of 200 µM.

Figure 2. Representative structures of (A) 1 and (B) 10 derived AMPs generated from cluster analysis of the peptides MD simulation in water. Values from left to right represent the total number of clusters, and the percentage of occurrence / average RMSD of the peptides structure, respectively. The colors in the peptide structures define as follow: Trp (orange), Ile (violet), Arg/Har (blue), Lys/Dab/Orn (cyan), and the peptide backbone (green).

Figure 3. Ramachandran plots of residues “i+1” and “i+2” and structures of different β-turn types in (A) 1 and (B) 10 peptides and their analogs. 1 (type VIII β-turn): i+1: Trp-3; i+2: Trp-4; 4 (type III β-turn): i+1: Arg-5; i+2: Trp-6; 5 (type VIII β-turn): i+1: Trp-4; i+2: Dab-5; 6 (type I or type VIII β-turn): i+1: Ile-7; i+2: Orn-8; 7 (type II’ β-turn): i+1: Har-5; i+2: Trp-6; 13 (type I β-turn): i+1: Trp-6; i+2: Trp-7; 14 (type II β-turn): i+1: Trp-4; i+2: Dab-5; 15 (type VIII β-turn): i+1: Orn-2; i+2: Trp-3. The values show the hydrogen bond distances in Angstrom. Carbon, hydrogen, nitrogen and oxygen were shown in black, yellow, blue and red colors.

Figure 4. The concentration dependence of the CD spectra for 7, 11, 12, and 16 in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. The colors representing the peptide concentration as follow: 25 µM (black), 50 µM (red), 100 µM (green), 200 µM (yellow), and 400 µM (blue).


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Figure 1. The CD spectra of the synthetic bactenecin derived peptides in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. Spectra were acquired on peptide samples prepared at the concentration of 200 µM.


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Figure 2. Representative structures of (A) 1 and (B) 10 derived AMPs generated from cluster analysis of the peptides MD simulation in water. Values from left to right represent the total number of clusters, and the percentage of occurrence / average RMSD of the peptides structure, respectively. The colors in the peptide structures define as follow: Trp (orange), Ile (violet), Arg/Har (blue), Lys/Dab/Orn (cyan), and the peptide backbone (green).


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Figure 3. Ramachandran plots of residues “i+1” and “i+2” and structures of different β-turn types in (A) 1 and (B) 10 peptides and their analogs. 1 (type VIII β-turn): i+1: Trp-3; i+2: Trp-4; 4 (type III β-turn): i+1: Arg-5; i+2: Trp-6; 5 (type VIII β-turn): i+1: Trp-4; i+2: Dab-5; 6 (type I or type VIII β-turn): i+1: Ile-7; i+2: Orn-8; 7 (type II’ β-turn): i+1: Har-5; i+2: Trp-6; 13 (type I β-turn): i+1: Trp-6; i+2: Trp-7; 14 (type II β-turn): i+1: Trp-4; i+2: Dab-5; 15 (type VIII β-turn): i+1: Orn-2; i+2: Trp-3. The values show the hydrogen bond distances in Angstrom. Carbon, hydrogen, nitrogen and oxygen were shown in black, yellow, blue and red colors.


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Figure 4. The concentration dependence of the CD spectra for 7, 11, 12, and 16 in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. The colors representing the peptide concentration as follow: 25 µM (black), 50 µM (red), 100 µM (green), 200 µM (yellow), and 400 µM (blue).


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Table of Contents Graphic


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Figure 1. The CD spectra of the synthetic bactenecin derived peptides in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. Spectra were acquired on peptide samples prepared at the concentration of 200 µM. 164x152mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Representative structures of (A) 1 and (B) 10 derived AMPs generated from cluster analysis of the peptides MD simulation in water. Values from left to right represent the total number of clusters, and the percentage of occurrence / average RMSD of the peptides structure, respectively. The colors in the peptide structures define as follow: Trp (orange), Ile (violet), Arg/Har (blue), Lys/Dab/Orn (cyan), and the peptide backbone (green). 218x281mm (300 x 300 DPI)


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Figure 3. Ramachandran plots of residues “i+1” and “i+2” and structures of different β-turn types in (A) 1 and (B) 10 peptides and their analogs. 1 (type VIII β-turn): i+1: Trp-3; i+2: Trp-4; 4 (type III β-turn): i+1: Arg-5; i+2: Trp-6; 5 (type VIII β-turn): i+1: Trp-4; i+2: Dab-5; 6 (type I or type VIII β-turn): i+1: Ile-7; i+2: Orn-8; 7 (type II’ β-turn): i+1: Har-5; i+2: Trp-6; 13 (type I β-turn): i+1: Trp-6; i+2: Trp-7; 14 (type II β-turn): i+1: Trp-4; i+2: Dab-5; 15 (type VIII β-turn): i+1: Orn-2; i+2: Trp-3. The values show the hydrogen bond distances in Angstrom. Carbon, hydrogen, nitrogen and oxygen were shown in black, yellow, blue and red colors. 1111x842mm (96 x 96 DPI)



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Figure 4. The concentration dependence of the CD spectra for 7, 11, 12, and 16 in the far-UV recorded in phosphate buffer (left panels) and in the presence of 50% (v/v) TFE (right panels) at 298 K. The colors representing the peptide concentration as follow: 25 µM (black), 50 µM (red), 100 µM (green), 200 µM (yellow), and 400 µM (blue). 353x702mm (300 x 300 DPI)


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Table of Contents Graphic 60x10mm (300 x 300 DPI)


Tryptic Stability of Synthetic Bactenecin Derivatives ... - ACS Publications

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