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Discovery of a Membrane-Active, Ring-Modified Histidine Containing Ultrashort Amphiphilic Peptide That Exhibits Potent Inhibition of Cryptococcus neoformans Krishna K. Sharma,† Indresh Kumar Maurya,‡ Shabana I. Khan,§ Melissa R. Jacob,§ Vinod Kumar,∥ Kulbhushan Tikoo,∥ and Rahul Jain*,† †

Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, India ‡ Department of Microbial Biotechnology, Panjab University, Sector 25, Chandigarh, 160 014, India § National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ∥ Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, India S Supporting Information *

ABSTRACT: The new structural classes of ultrashort peptides that exhibit potent microbicidal action have potential as future drugs. Herein, we report that C-2 arylated histidines containing tripeptides His(2-Ar)Trp-His(2-Ar) exhibit potent antifungal activity against Cryptococcus neoformans with high selectivity. The most potent peptide 12f [His(2biphenyl)-Trp-His(2-biphenyl)] displayed high in vitro activity against C. neoformans (IC50 = 0.35 μg/mL, MIC = MFC = 0.63 μg/mL) with a selectivity index of >28 and 2 times higher potency compared to amphotericin B. Peptide 12f exhibited proteolytic stability, with no apparent hemolytic activity. The mechanism of action study of 12f by confocal laser scanning microscopy and electron microscopy indicates nuclear fragmentation and membrane disruption of C. neoformans cells. Combinations of 12f with fluconazole and amphotericin B at subinhibitory concentration were synergistic against C. neoformans. This study suggests that 12f is a new structural class of amphiphilic peptide with rapid fungicidal activity caused by C. neoformans.

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INTRODUCTION

the development of resistance to the pathogen, acute or chronic side effects, low clinical efficacy owing to poor pharmacokinetics, complex drug interactions with the host cells, poor bioavailability, and low therapeutic index created a need for the discovery of new structural classes of synthetic antifungal agents.6 The need is to discover new antifungal agents with potent fungicidal activity, target specificity, rapid time kill, unique mechanism of action, and absence of cross-resistance with the clinically available antifungal drugs.7 The antifungal drug discovery is considered more challenging compared to antibacterial drug discovery because fungi are metabolically similar to mammalian cells except cell wall, resulting in few pathogen specific targets.8 The quest for new antifungal development is important because extensively used antifungal drugs (amphotericin B and fluconazole) have developed resistance against the prevalent fungal pathogens such as Candida albicans and C. neoformans.9 In such a scenario,

In recent years, the incidences of invasive fungal infections such as cryptococcosis, candidiasis, and aspergillosis have increased rapidly and caused morbidity and mortality in immunocompromised situations such as burn, acquired immune deficiency syndrome (AIDS), cancer, and organ transplant.1,2 The infections caused by the yeast Cryptococcus neoformans is a human opportunistic fungal pathogen, responsible for approximately 600 000 deaths per year worldwide.3 In the past 2 decades, rapidly increasing antifungal drug resistance has emerged as a serious threat to public health and a major challenge in search of novel antifungals with distinct mode of actions. In disparity to the steep rise in invasive fungal infections, there is a dearth of novel and new classes of clinically approved antifungal activity specific drugs.4 The invasive antifungal treatments relied on five main classes of marketed drugs: azoles (fluconazole), polyenes (amphotericin B), fluoropyrimidines (5-fluorocytosine), allyamines (terbinafine), and echinocandins (caspofungin).5 Along with the limited repertoire of the marketed antifungals, the drawbacks such as © 2017 American Chemical Society

Received: March 29, 2017 Published: July 11, 2017 6607

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Figure 1. Generalized structures of the synthesized peptides.

Scheme 1. Synthesis of 2-Aryl-L-histidines (3a−e)a

a

Reagents and conditions: (i) 1 (1 equiv), ArB(OH)2 (2 equiv), AgNO3 (0.2 equiv), (NH4)2S2O8 (2 equiv), CF3CO2H (1.5 equiv), CH2CI2/H2O (1:1, v/v), 12−15 h, rt; (ii) 6 N HCl, 12 h, 100 °C.

exhibit potent antifungal activity.17,18 Because of the presence of imidazole side chain that displays buffering properties at physiological pH and amphiphilic character, heterocyclic amino acid histidine emerges as an obvious choice in the design of synthetic antifungal peptide motif.19 The tryptophan residue on the other hand provides lipophilicity and cell membrane interaction characteristics to the synthetic peptides.19 Upon the basis of above-mentioned observations, we envisioned using amphiphilic ring-modified L-histidine and lipophilic tryptophan as core amino acids to develop a new antifungal structural motif. Herein, we report the synthesis of ring-modified histidine and tryptophan (Trp) residues containing tripeptide motif as promising antifungal agents bearing +2/+3 charge, where additional lipophilicity was provided by the aromatic moiety placed at the C-2 position of histidine. We observed that incorporation of 2-aryl-Lhistidines in the tripeptide scaffold has greatly influenced the potency of synthetic peptides. The most potent antifungal peptide 12f was further examined for hemolysis, stability, time kill kinetics, mechanism of action, and its synergistic combination with marketed drugs against clinical isolates of human fungal pathogen C. neoformans. This investigation demonstrates that the presence of a lipophilic aromatic moiety at the C-2 position of the imidazole ring of histidine imparts desired amphiphilicity to His(2-Ar)-Trp-His(2-Ar) class of peptides, thereby increasing potency and cell membrane disruption properties.

cationic antimicrobial peptides (CAMPs) offer a promising alternative for the development of synthetic antifungals.10 In the past decade, several types of CAMPs have been reported to show efficacy against various fungal infections. The CAMPs are generally 6−50 amino acid residues in length and exist in structural conformations, including helix, sheet extended, and looped structures. They possess two common structural features: a net positive charge and amphiphilicity.11 The positive charge is known to facilitate interaction with the negatively charged microbial surface, while amphiphilicity allows an amphipathic secondary structure that permits the incorporation of CAMPs into the lipid bilayer of microbial membranes.12 It has been well established that CAMPs could alter the cell membrane functions through increased permeability, but it is not their sole mode of action. Peptides due to their large size have limited clinical potential because of high commercialization cost. This drawback has drawn attention toward the development of short synthetic peptides as clinically viable drugs.13 The regioselective functionalization of DNA-encoded amino acids leading to the synthesis of enantiomerically pure modified amino acids is receiving attention recently. These modified amino acids form ideal scaffolds in the synthesis of new structural classes of short synthetic peptides with high acceptability in drug discovery.14,15 The advent of ring-modified histidines as a core amino acid residue in short peptides has opened new avenues in the development of potential proteostable antimicrobial pharmacophores.15 In a nutshell, the short synthetic cationic peptides are ideally suited in search of novel synthetic antimicrobial agents because of their size, cost, and microbicidal mode of action. As a part of our ongoing interest in developing ultrashort CAMPs against infectious diseases, we investigated the minimum prerequisite features in reported antifungal peptides.16 It was observed that peptide motif bearing +2/+3 charge with sufficient lipophilicity results in promising antifungals. A number of histidine-rich natural CAMPs such as histatins (Hist-3 and Hist-5) and clavanins, and tryptophanrich CAMPs such as indolicidin and tritrpticin are known to

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RESULTS AND DISCUSSION Synthesis. Two structural types of peptides, one with a benzylamide group and another with a methyl ester group at the C-terminus, were synthesized (Figure 1). The synthesis required availability of building block amino acids in the suitably derivatized form. N-α-Trifluoroacetyl-2-aryl-L-histidine methyl esters (2a−e) were synthesized from N-α-trifluoroacetyl-L-histidine methyl ester (1) utilizing a regiospecific C−H arylation reaction in the presence of arylboronic acids under ambient conditions in moderate yields (Scheme 1).20 2-Aryl-Lhistidines (3a−e) possessing dual hydrophobic−hydrophilic 6608

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Scheme 2. Synthesis of His(2-Ar)-Trp-His(2-Ar)-NHBzl (8a−f)a

Reagents and conditions: (i) (Boc)2O, NaOH, 1,4-dioxane−water, 24 h; (ii) C6H5CH2NH2, DIC, HONB, 60 °C, 30 min, MW; (iii) 6 N HCl in MeOH, rt, 15 min; (iv) DIEA, Boc-Trp-OH, DIC, HONB, 60 °C, 30 min, MW; (v) DIEA, Boc-His(2-Ar)-OH (4a−f), DIC, HONB, 60 °C, 30 min, MW.

a

Scheme 3. Synthesis of His(2-Ar)-Trp-His(2-Ar)-OMe (12a−f)a

Reagents and conditions: (i) MeOH, HCl gas, 2 h; (ii) DIEA, Boc-Trp-OH, DIC, HONB, 60 °C, 30 min, MW; (iii) 6 N HCl in MeOH, rt, 15 min; (iv) DIEA, Boc-His(2-Ar)-OH (4a−f), DIC, HONB, 60 °C, 30 min, MW.

a

Boc-L-His(Ar)-NHBzl derivatives (5a−f) were deprotected in the presence of 6 N HCl in methanol (MeOH) for 15 min at ambient temperature. The resulting intermediates were neutralized using N,N-diisopropylethylamine (DIEA) and subsequently coupled with Boc-Trp-OH in the presence of coupling reagent combination of DIC and HONB at 60 °C for 30 min under MW irradiation to produce dipeptides (6a−f) in high yields. The dipetides (6a−f) were deprotected using a solution of 6 N HCl in methanol (MeOH) for 15 min at ambient temperature. The deprotected dipeptides were neutralized in situ using DIEA and allowed next coupling step immediately with Boc-L-His(Ar)-OH (4a−f) in the presence of DIC and HONB under MW irradiation at 60 °C for 30 min to afford first set of desired tripeptides (7a−f). The

character were obtained by deprotection of 2a−e under acidic conditions at 100 °C for 12 h (Scheme 1). The latter compounds 3a−e upon reaction with di-tert-butyl dicarbonate under basic conditions for 24 h afforded Boc-LHis(Ar)-OH (4a−f) in excellent yields (Scheme 2). The Bocprotected derivatives 4a−f upon reaction with benzylamine in the presence of coupling reagent 1,3-diispropylcarbodiimide (DIC) and auxiliary nucleophile N-hydroxy-5-norbornene-2,3dicarboximide (HONB) at 60 °C for 30 min under microwave (MW) irradiation produced key intermediates Boc-L-His(Ar)NHBzl (5a−f) in high yields (Scheme 2). The designed peptides (series 1 and 2) were synthesized by adapting a rapid and highly efficient microwave (MW)mediated solution phase peptide synthesis protocol.21 First, 6609

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Table 1. In Vitro Antifungal Activities of Peptidesa

C. neoformans (μg/mL) peptide

R

R1

R2

IC50

MIC

MFC

cytotoxicity (CTX) (μg/mL)

selectivity index (SI)

7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 11a 11b 11c 11d 11e 11f 12a 12b 12c 12d 12e 12f amphotericin B

H C6H5 4-OCH3-C6H4 4-CH3-C6H4 4-C(CH3)3-C6H4 4-C6H5-C6H4 H C6H5 4-OCH3-C6H4 4-CH3-C6H4 4-C(CH3)3-C6H4 4-C6H5-C6H4 H C6H5 4-OCH3-C6H4 4-CH3-C6H4 4-C(CH3)3-C6H4 4-C6H5-C6H4 H C6H5 4-OCH3-C6H4 4-CH3-C6H4 4-C(CH3)3-C6H4 4-C6H5-C6H4

NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl NHBzl OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe

Boc Boc Boc Boc Boc Boc H H H H H H Boc Boc Boc Boc Boc Boc H H H H H H

0.80 1.40 14.0 12.61 >20 >20 8.1 >20 7.2 5.56 2.60 1.40 >20 9.14 15.21 2.68 1.27 >20 9.81 3.71 2.47 1.67 0.83 0.35 0.69

1.25 2.50 >20 >20

1.25 2.50 >20 >20

>12.5 >7.1

10

10

>20 10 5 2.50

>20 10 5 2.50

>20 >20 5 5

>20 >20 5 5

20 10 5 2.50 1.25 0.63 1.25

20 10 5 5 1.25 0.63 1.25

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

>1.2 >1.4 >1.8 >3.8 >7.1 >1.1 >3.7 >7.9 >1.0 >2.7 >4.0 >6.0 >12.0 >28.6

a IC50, the concentration (μg/mL) that affords 50% inhibition of growth; MIC, minimum inhibitory concentration is the lowest test concentration (μg/mL) that allows no detectable growth; MFC, minimum fungicidal concentration is the lowest test concentration (μg/mL) that kills the organism; selectivity index (CTX/IC50)

Table 2. In Vitro Antibacterial Activity of Peptidesa S. aureus (μg/mL)

MRSA (μg/mL)

peptide

IC50

MIC

MBC

CTX (μg/mL)

SI

IC50

MIC

MBC

CTX (μg/mL)

SI

7b 8e 12e 12f ciprofloxacin

7.60 3.72 4.33 2.56 0.08

>20 10 10 >20 0.25

>20 20 >20 >20 0.50

>10 >10 >10 >10

>1.3 >2.68 >2.3 >3.9

10.50 6.70 6.71 1.93 0.09

>20 >20 >20 5 0.25

>20 >20 >20 10 0.50

>10 >10 >10 >10

>1.49 >1.49 >5.18

a

IC50, the concentration (μg/mL) that affords 50% inhibition of growth; MIC, minimum inhibitory concentration is the lowest test concentration (μg/mL) that allows no detectable growth; MBC, minimum bactericidal concentration is the lowest test concentration (μg/mL) that kills the organism; CTX, cytotoxicity, SI, selectivity index (CTX/IC50).

DIC and HONB in DMF under MW heating at 50 °C for 30 min to produce dipeptides 10a−f, which were deprotected under acidolysis conditions and subsequently neutralized in situ (in the presence of DIEA) and subjected to next coupling step immediately. The deprotected dipeptides upon reaction with Boc-His(2aryl)-OH (4a−f) in the presence of DIC and HONB in DMF under MW heating afforded Boc group protected tripeptides 11a−f, which under acidolysis conditions produced free

protected tripeptides (7a−f) upon acidolysis reaction in the presence of 6 N HCl in MeOH for 15 min at ambient temperature produced the second set of peptides (8a−f) (Scheme 2). For the peptides of series 2, 2-aryl-L-histidines (3a−f) upon esterification with MeOH in the presence of HCl gas afforded 2-aryl-L-histidine methyl esters (9a−f) in high yields (Scheme 3). The latter compounds 9a−f were neutralized using DIEA and subsequently reacted with Boc-Trp-OH in the presence of 6610

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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MIC = MFC = 0.63 μg/mL or 0.72 μM) and emerged as the most active peptide across both series. The in vitro activity data show that in addition to the substituents pattern at the C-2 position of the imidazoles ring, the nature of the group at both termini also greatly influenced the antifungal potency. For the Boc-protected peptides, the incorporation of a methyl ester group (peptides 11a−f) offers optimal tuning of lipophilicity compared to a NHBzl group (peptides 7a−f) at the Cterminus. For Boc-deprotected free peptides (8a−f and 12a−f), increase in lipophilicity by the introduction of C2-Ar group in histidine residue results in enhanced activity. In general, peptides with free N-terminus (8a−f and 12a−f) demonstrated higher potency compared to Boc protected peptides (7a−f and 11a−f). The peptides 7b, 8e, 12e, and 12f also displayed antibacterial activity against S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) (Table 2). The peptide 12e exhibited IC50 value of 4.33 μg/mL and MIC of 10 μg/mL against S. aureus, while 12f showed promising IC50, MIC, and MBC values of 1.93, 5, and 10 μg/mL, respectively, against MRSA. Cytotoxicity Evaluation. The absence of cytotoxic effects is one of the most essential criteria to determine the potential of membrane targeted antimicrobial peptides. The selectivity toward antimicrobial activity by synthetic peptides is a prerequisite as both humans and microbes belong to sister clades in the tree of eukaryotes. To determine the safety profile, synthesized peptides were evaluated for cytotoxic study in a panel of cell lines. The in vitro cytotoxicity of the synthetic peptides was determined against four human cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two noncancerous mammalian cells (VERO and LLCPK1) by neutral red uptake assay.24 The results show that all synthetic peptides (series 1 and 2) were nontoxic up to the highest tested concentration of 10.0 μg/mL, which indicates a higher selectivity index (>1- to >28-fold) for anticryptococcal activity and a selective index (>1.3 to >5.8-fold) for antibacterial activity. Therefore, it could be concluded that activity exhibited by peptides is not due to a general cytotoxic effect of synthetic peptides. Upon the basis of in vitro activity and cytotoxicity results, we selected peptide 12f for detailed evaluation, including hemolytic effect, time kill kinetics, proteolytic stability, mechanism of action investigations, and synergistic action with known drugs. Hemolytic Study. The hemolytic activity of the antifungal peptides is considered as a marker of selectivity toward fungal cells and determined upon the basis of amount of hemoglobin released from human red blood cells (hRBCs). We re-examined peptide 12f against a new C. neoformans strain (ATCC350), wherein it exhibited MIC value of 0.39 μg/mL or 0.44 μM. All subsequent studies were conducted on C. neoformans strain (ATCC350). Varied concentrations of peptide 12f were incubated with hRBCs, and the release of hemoglobin was measured. Triton X-100 was used as a positive control for this study. The peptide 12f was tested up to the highest test concentration of 100 μg/mL. The peptide 12f did not exhibit any hemolysis at its MIC (Figure 2). At the highest test concentration of 100 μg/mL, peptide 12f exhibited only 17.74% hemolysis. It can be concluded that 12f has a nonhemolytic nature at its MIC value and high selectivity to C. neoformans cells. Several earlier reports have established a link between increases in hydrophobicity and increase in hemolytic effects, resulting in assumption that the presence of

peptides 12a−f. All dipeptides and tripeptides were purified on neutral alumina using a fully automated flash purification system. In Vitro Antimicrobial Activity. For a better understanding of the structure−activity profiles of the synthetic peptides, both Boc protected peptides (7a−f and 11a−f) and free peptides (8a−f and 12a−f) were evaluated for in vitro activity against a panel of fungal (C. albicans, C. glabrata, C. krusei, Aspergillus f umigatus, and C. neoformans) and bacterial (E. coli, S. aureus, and MRSA) strains. The results are summarized for C. neoformans (Table 1), S. aureus, and MRSA (Table 2). The peptides were found to be inactive against Candida, Aspergillus, and E. coli (data not shown). The MIC (minimum inhibitory concentration) values of evaluated peptides were determined by using a protocol suggested by the Clinical and Laboratory Standard Institute (CLSI) guidelines.22 The marketed drug amphotericin B, which is used for the treatment of C. neoformans infections but has toxicity toward mammalian cells, was used as a positive control in antifungal evaluation, while ciprofloxacin was used as a standard drug for antibacterial activity evaluation.23 A clear pattern confirming the role of lipophilicity in antifungal activity was observed for peptides 7a−f that contained Boc and NHBzl groups at the N- and C-terminus, respectively. We noted that an incremental increase in the overall lipophilicity in the designed peptides is proportional to decrease in activity. Peptide 7a (R = H) and peptide 7b (R = C6H5) exhibited IC50 values of 0.80 μg/mL and 1.40 μg/mL, respectively. At the same time, peptides 7c−f containing bulkier aryl groups produced IC50 values between 12.61 and 14.0 μg/ mL or no inhibition of C. neoformans at the highest tested concentration of 20 μg/mL. This trend indicates that a combination of NHBzl and Boc group at both termini and the presence of bulkier aryl group at the C-2 position of the imidazole ring results in dramatic reduction in antifungal activity of this class of peptides. In contrast, peptides 8a−f obtained directly from 7a−f by removal of Boc group at the Nterminus exhibited a reverse trend. In these cases, the absence of Boc group at the N-terminus and an increase in lipophilicity affected by the introduction of bulkier aryl group at the C-2 position of imidazole ring result in enhanced antifungal activity. For example, peptide 8f (R = 2-biphenyl) exhibited inhibition of C. neoformans at IC50 = 1.40 μg/mL, MIC = MFC = 2.50 μg/ mL, and was the most promising of series 1. These observations confirm that a delicate balance of bulk to charge ratio is essential to retain and enhance antifungal activity (Table 1). In complete reversal of the results obtained for Boc protected peptides 7a−f (series 1), peptides 11a−f (series 2) containing Boc and OMe groups at the N- and C-terminus exhibited a linear increase in activity with the increased lipophilicity induced by the aryl substitution on both histidine residues with the exception of peptide 11f. The activity profiles of Bocdeprotected peptides 12a−f follow the trajectory of 8a−f, wherein an increase in lipophilicity results in gradual increment in activity. Peptide 12a (R = H) produced IC50 of 9.81 μg/mL, but peptide 12e containing a bulky 4-tert-butylphenyl group at the C-2 position of the imidazole ring was found to be a promising inhibitor of C. neoformans with IC50 value of 0.83 μg/ mL compared to 0.69 μg/mL for amphotericin B. The MIC and MFC values of 12e were 1.25 μg/mL similar to that for amphotericin B. The peptide 12f containing a biphenyl group at the C-2 position of the imidazole ring exhibited even better inhibition of C. neoformans (IC50 = 0.35 μg/mL or 0.40 μM, 6611

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Proteolytic Stability. Proteolytic stability is one of the most crucial parameters in the discovery of peptide-based therapeutics.26 In general, natural peptides due to long sequences are highly susceptible to proteolytic degradation as compared synthetic peptide containing modified amino acids and modified termini. In order to examine the proteolytic stability, peptide 12f was digested with proteolytic enzyme trypsin in a ratio of 100:0.5 mol/mol and analyzed by RPHPLC after 2 h (Figure 4). There was no observed change in the retention time and peak intensity of the trypsin treated 12f (Figure 4a) and untreated 12f (Figure 4b). Hence it may be assumed that incorporation of modified amino acids and modified terminus resulted in the stability of 12f against proteolysis. Mechanism of Action Investigations. High fungicidal potency, no apparent cytotoxicity, proteolytic stability, and rapid killing of C. neoformans cells by 12f prompted us to examine its mechanism of action. The amphipathic peptides are presumed to exhibit activity due to their microbial membranes disruptive properties, and thus mechanism of action was investigated by using various microscopic visualization techniques. We conducted the confocal laser scanning microscopy (CLSM) investigation to know the effect of 12f on the membrane permeability and intracellular localization in fungal cells. The scanning electron microscopy (SEM) study was performed to examine the effect of 12f on the C. neoformans cells morphology, and the high resolution transmission electron microscopy (HRTEM) study allowed us to visualize the effects of 12f on both cell wall and cell membrane of C. neoformans cells. Confocal Laser Scanning Microscopy. Propidium iodide (PI) is a red fluorescent DNA binding dye when excited at 480 nm. In the presence of membrane active molecules, it permeabilizes cell wall of microbial cells resulting in compromised membrane permeability. Under confocal microscopic examination, uptake of PI after 1 h by C. neoformans cells in the presence of 12f at its MIC revealed red fluorescence due to permiabilization of cell membrane when excited by the PI filter at 488 nm laser (Figure 5B), whereas the cells without 12f treatment did not show any fluorescence due to intact membranes (Figure 5A). This confirms that PI enters the fungal cells treated with 12f possibly because C. neoformans cells were not able to control the chemical traffic from the surface. In order to understand the localization of 12f inside the C. neoformans cells, we synthesized FITC-labeled (fluorescein isothiocyanate; green fluorescent dye, excitation at 480 nm) 12f and control peptide 8b. The examination of control peptide 8b suggests that it was not able to internalize inside the cells as seen from the intact cell surface (Figure 5C). FITC-labeled 12f upon incubation with C. neoformans cells translocated inside Cryptococcus cells, after cell membranes permiabilization and disruption as evident from the increased green fluorescence (Figure 5D). It could be presumed that 12f interacts with the intracellular targets and the killing of C. neoformans might be the result of multiple effects. DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent dye commonly used to detect the nuclear fragmentation of genetic materials in microbial cells under confocal microscopic examination. The DAPI dye binds selectively to the DNA (minor groove of adenine- and thymine-rich sequence) so that any changes in fluorescence intensity reflect the extent of damage to DNA caused by the membrane disruptive peptides.

Figure 2. Hemolytic activity of 12f against human RBCs. The 12f peptide was incubated with 0.4% hRBCs in PBS. The standard deviation from three independent experiments is plotted.

amphiphilic histidine residues and tryptophan in the case of 12f results in no hemolytic effects.25 Time Kill Kinetics. There is always a need to discover rapid fungicidal agents to combat antifungal resistance. Peptide 12f showed potent fungicidal activity against C. neoformans, and time kill kinetics is an important method to judge the efficacy of investigational antimicrobials. For better understanding of fungicidal nature, time kill kinetics experiments with 12f, amphotericin B (Amp B), and control peptide 11a (inactive peptide) were performed. In the time kill assay, the colony forming units (CFUs) of the C. neoformans were rapidly reduced after treatment with 12f at the concentration of 0.39 μg/mL (MIC). The complete killing of C. neoformans cells in the presence of 12f was observed within 4 h. The known antifungal drug Amp B at the concentration of 0.89 μg/mL (MIC) also completely killed C. neoformans cells after 4 h of incubation (Figure 3). However, the negative control peptide 11a did not show any growth inhibition and exhibits a growth trajectory similar to the untreated control. Therefore, it could be interpreted that peptide 12f successfully inhibited the growth of C. neoformans cells.

Figure 3. Time kill kinetics of the 12f, Amp B, and negative control peptide 11a. The standard deviation from three independent experiments is plotted. 6612

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Figure 4. RP-HPLC chromatograms of 12f untreated (a) and after 2 h of incubation with trypsin (b).

Figure 5. Confocal micrographs of C. neoformans cells to assess the effect of 12f at 0.39 μg/mL (MIC) inside the treated cells: (i) membrane permeabilization assay using propidium iodide (PI) after 1 h; (ii) intracellular localization of FITC-labeled 12f after 1 h and FITC-labeled 8b (control peptide); (iii) nuclear fragmentation effect of DAPI after 1 h. Images in (A) and (E) are fungal cells incubated alone, and image in (C) is obtained with FITC-labeled 8b. Images in (B), (D), and (F) correspond to 12f peptides treated cells after 1 h of incubation.

DAPI staining of C. neoformans cells treated with 12f showed a high split blue fluorescence intensity (Figure 5F) within the cells, as compared to untreated cells (Figure 5E). Therefore, these results indicated that 12f interacts with genetic material and causes nuclear fragmentation resulting in the fast killing of C. neoformans cells after membrane damage. Electron Microscopy. Scanning electron microscopy (SEM) study was performed to understand the cell surface morphological changes in the C. neoformans upon treatment with 12f at its MIC (0.39 μg/mL). The untreated cryptococcal cells have clean smooth surface (Figure 6A,B) as compared to cells treatment with 12f (Figure 6C,D). Peptide 12f induced cell wall breakage was visible with an increase in the coarseness on the cell surface (Figure 6C,D). It is assumed that a positively charged peptide binds with the negatively charged cell surface

of C. neoformans cells by electrostatic interactions. The other possibility is that amphiphilic peptide may aggregate over cell surface, resulting in its distortion. It is concluded that 12f treated C. neoformans cells were unable to sustain under the collective effects induced via nonspecific electrostatic interactions and amphiphilicity, resulting in severe disruption of cell surface integrity and membrane permeabilization. In the TEM examinations, 12f treatment resulted in severe effect on the C. neoformans cell wall and membrane as compared to untreated cells showing intact cell structure. Untreated fungal cells show intact cell membranes under TEM examination (Figure 7A−C). Figure 7A showed intact cell morphology of many cells. On the other hand, Figure 7B,C focused on single cell visualization and showed defined and intact cell wall and plasma membrane. The peptide 12f treated 6613

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Figure 6. Surface morphological study of C. neoformans cells with 12f and untreated fungal cells by SEM. Images A and B represent morphology of untreated cells (7000×, 25 mm; 5000×, 24 mm). Images C and D represent fungal cells (8000×, 24 mm; 3700×, 24 mm) treated with 12f. The peptide was used at its MIC.

Figure 7. Morphological study of C. neoformans cells treated with 12f and untreated fungal cells by high resolution transmission electron microscopy. Images A, B, and C represent untreated cells. Images D, E, and F represent cells treated with 12f. The peptide was used at its MIC. The scale bar for images A and D is 0.5 μm, and for other images it is 0.2 μm.

TEM images show severe distruption in cell wall and cytoplasmic membrane resulting in fungal cell lysis (Figure 7D−F). The confocal microscopic study using PI revealed that the peptide 12f permeabilizes cell membranes of C. neoformans cells. Cellular uptake of FITC-labeled 12f peptide inside the fungal cells indicated that peptide binds with the multiple intracellular sites. The DAPI experiment demonstrates that 12f after permiabilization inside C. neoformans cells binds with the fragmented genetic material cells, resulting in inhibition. The

SEM results indicate that peptide 12f interacts with negatively charged cell surface of C. neoformans cells via electrostatic interactions, resulting in the disruption of their surface integrity. Finally, the TEM results show that 12f acts by severe disruption of both cell wall and plasma membrane, which results in permeabilization inside cells and interaction with intracellular sites, responsible for its rapid fungicidal nature. These mechanistic studies reveal that the high anticryptococcal effect of potent peptide 12f is probably a multistep process involving initial membrane permeabilization, cell wall and plasma 6614

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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Table 3. Evaluation of Synergistic Effect of 12f Peptide with Fluconazole (FLC) and Amphotericin B (Amp B) S. no.

antifungal drug alone, MIC (μg/mL)

peptide 12f alone, MIC (μg/mL)

concn of antifungal drug and concn of 12f in combination (μg/mL)

fraction inhibitory concn (FICI)

effect

1 2

9.57 (FLC) 0.89 (Amp B)

0.39 0.39

2.37 (FLC) + 0.024 (12f) 0.055 (Amp B) + 0.012 (12f)

0.308 0.092

synergistic synergistic

neoformans cells. Combinations of 12f with marketed antifungal first line drugs such as Amp B and fluconazole were found be remarkably synergistic in nature. It can be concluded that peptide 12f has great potential in peptide-based antifungal drug development. The discovery of histidine-rich peptide-based antimicrobials also provided insights that rationally modified histidines as building blocks offer a better choice in search of rapidly acting and potent ultrashort membrane disruptive antifungal agents.

membrane disruption, interaction with genetic materials and other intracellular components of the targeted fungi cells, ultimately leading to rapid cell death. Synergistic Effect Study of 12f with Fluconazole and Amphotericin B. The emergence of resistance against marketed antifungal agents has renewed interest in the evaluation of the combination therapy of antifungals. The use of new structural class of antifungals with existing antifungal drugs acting via different mode of actions is a significant step in developing more efficacious synergistic antifungal combinations and offers ways to combat drug resistance.27 For the synergistic study, we selected fluconazole and Amp B by considering their antifungal potency, toxicity, and different mode of actions when compared with 12f. The study was performed against C. neoformans (ATCC350), and as shown earlier, 12f produced MIC of 0.39 μg/mL while fluconazole and Amp B exhibited MIC values of 9.57 and 0.89 μg/mL, respectively. The in vitro synergy was determined using checkerboard dilution assay. The fraction inhibitory concentration index (FICI) was determined for combination of 12f with fluconazole and Amp B in inhibiting C. neoformans cell growth that showed synergy at sub-MIC values (≤0.5 is considered to be synergistic). The results listed in Table 3 indicate that 12f showed significant synergism with both fluconazole and Amp B. Peptide 12f at noncidal concentration resulted in potentiation of fluconazole by 4-fold (MIC from 9.57 μg/mL to 2.37 μg/mL). At the same time, MIC value of peptide 12f was lowered by 16-fold in this combination (MIC from 0.39 μg/mL to 0.024 μg/mL). The MIC for Amp B was lowered by 16-fold in combination with 12f (MIC from 0.89 μg/mL to 0.055 μg/mL), while the MIC of peptide 12f was lowered by 32-fold in this combination (MIC from 0.39 μg/mL to 0.012 μg/mL). FICI values of 12f in combinations with fluconazole and Amp B were found to be 0.308 and 0.092, respectively. These results indicate that the synergistic combinations of 12f peptide with clinically used drugs fluconazole and Amp B represent a dynamic strategy to improve antifungal potency. This also opens up a highly efficacious approach to combat antifungal drug resistance in a cost-effective manner using less toxic therapeutics.

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EXPERIMENTAL SECTION

General Chemistry. All starting material chemicals were purchased from Sigma-Aldrich, Chem-Impex, and Alfa-Aesar and used further without any additional purification. Thin-layer chromatography (TLC) was performed using aluminum plates precoated with silica gel (0.25 mm, 60 Å pore size) impregnated with a fluorescent indicator (254 nm). Visualization on TLC was achieved by the use of UV light (254 nm) and also staining with molecular iodine. Automated flash column chromatography was undertaken on silica gel (230−400 mesh size). 1H and 13C NMR spectra were recorded on Bruker AVANCE III 400 MHz instrument using CD3OD as solvent and TMS as internal standard. Proton and carbon chemical shifts are expressed in parts per million (ppm, δ scale) and were referenced to NMR solvent CD3OD, δ 3.31, 49.0 ppm. The following abbreviations were used to describe peak patterns when appropriate: br = broad, s = singlet, d= doublet, t = triplet, q = quadruplet, m = multiplet. Coupling constants (J) were reported in hertz unit (Hz). High resolution mass spectra (HRMS) were obtained with MAXIS-Bruker using ESI-TOF method. HPLC analysis was performed on the SHIMADZUprominence using Supelcosil LC-18 column (25 cm × 4.6 mm) run for 40 min with a flow of 1 mL/min, using a gradient run of 95−5% (A/B), where buffers A and B were 0.1% TFA in H2O and CH3CN, and detection at 220 or 254 nm; all peptides exhibited ≥95% purity. All MW-irradiation mediated peptide coupling reactions were carried out on a CEM Discover microwave syntheszier. General Procedure for the Synthesis of 2-Aryl-L-histidines (3a−e). N-α-Trifluoroacetyl-L-histidine methyl ester (1, 3.76 mmol, 1.0 equiv) was dissolved in CH2Cl2 (10 mL) and CF3CO2H (1.5 equiv). A solution of AgNO3 (0.2 equiv) in water (10 mL) was added, followed by ArB(OH)2 (1.5 equiv) and (NH4)2S2O8 (2.0 equiv). The reaction mixture was stirred at ambient conditions for 12−14 h, and progress of the reaction was monitored by TLC. After the completion of reaction, the pH of the reaction mixture was adjusted to 10−12 by the addition of aqueous ammonia solution. The extraction of the reaction mixture was carried out with ethyl acetate (3 × 50 mL), and the organic layer was washed with brine solution (2 × 10 mL). The extract was concentrated under reduced pressure and purified on an automated flash column chromatography system to provide 2a−e. The protected amino acid (2a−e, 1 mmol) was dissolved in 6 N HCl (5 mL), and reaction mixture was heated at 100 °C for 12 h. The removal of solvent afforded 2-aryl-L-histidines (3a−e). General Procedure for the Synthesis of N-α-Boc-2-aryl-Lhistidines (4a−f). 2-Aryl-L-histidine (3a−e, 1 equiv) was dissolved in a mixture of H2O−dioxane (2:3). The pH of the reaction mixture was adjusted to 12 by addition of aqueous solution of 4 N NaOH followed by the addition of di-tert-butyl dicarbonate (2 equiv). Reaction mixture was allowed to stir at ambient conditions for 12 h. Reaction mixture was concentrated under reduced pressure, methanol (20 mL) was added, and stirring continued for additional 12 h at ambient temperature. The reaction mixture containing N-α-Boc-His(2-aryl)O-Na+ was concentrated and treated with saturated aqueous solution

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CONCLUSIONS We have designed and synthesized two series of His(2-Ar)-TrpHis(2-Ar) classes of amphipathic peptides. Several synthetic peptides of this scaffold have displayed high selectivity and potency against C. neoformans, leading to the identification of the most potent peptide 12f. Peptide 12f did not display hemolytic effects at MIC and exhibited 2-fold higher potency compared to clinical used drug Amp B. Peptide 12f acts rapidly in the time kill kinetics study, and results are comparable with Amp B. Peptide 12f was also found to be proteostable against proteolytic enzyme trypsin. The mechanism of action studies by confocal microscopy revealed that the presence of 12f permeabilizes cell surface, translocates intracellularly, and causes nuclear fragmentation of genetic material resulting in killing of C. neoformans cells. The TEM results indicate that 12f acts by cell wall and plasma membrane disruption of C. 6615

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

Journal of Medicinal Chemistry

Article

4H), 7.48 (d, J = 6.3 Hz, 1H), 7.28−7.08 (m, 12H), 6.96 (s, 1H), 6.76 (s, 2H), 4.63 (br s, 2H), 4.22 (br s, 3H), 3.21 (d, J = 12.1 Hz, 2H), 3.03−2.89 (m, 3H), 2.78 (d, J = 8.2 Hz, 1H), 2.33 (s, 3H), 2.30 (s, 3H), 1.31 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 174.3, 173.9, 172.6, 157.8, 147.9, 140.0, 139.9, 139.5, 137.9, 135.5, 134.7, 130.5, 129.4, 128.7, 128.5, 128.4, 128.3, 128.1, 126.4, 124.7, 122.6, 120.3, 120.1, 119.2, 112.4, 110.3, 80.9, 56.1, 54.8, 52.2, 43.8, 30.3, 29.9 28.6, 28.4, 21.3. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 848.4248, found 848.4226. HPLC: tR = 27.9 min, 97.8% purity. Boc-His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]-NHBzl (7d). Yield = 63%. 1H NMR (400 MHz, CD3OD): δ 7.76−7.73 (m, 4H), 7.50 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.22−7.16 (m, 3H), 7.11−7.05 (m, 4H), 6.98−6.92 (m, 5H), 6.75 (d, J = 6.4 Hz, 2H), 4.64 (t, J = 6.6 Hz, 2H), 4.28−4.24 (m, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.22 (d, J = 6.3 Hz, 2H), 3.02−2.89 (m, 3H), 2.79− 2.74 (m, 1H), 1.33 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 173.0, 172.4, 171.2, 160.2, 160.1, 156.4, 146.5, 138.0, 136.5, 128.0,127.3, 126.9, 126.7, 126.4, 123.3, 122.7, 122.6, 121.2, 118.7, 117.8, 113.9, 113.8, 111.0, 108.8, 79.5, 54.7, 54.4, 54.4, 53.5, 42.5, 31.3, 28.6, 27.2, 26.9. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 880.4146, found 880.4093. HPLC: tR = 27.4 min, 97.2% purity. Boc-His[2-(4-tert-butylphenyl)]-Trp-His[2-(4-tert-butylphenyl)]-NHBzl (7e). Yield = 57%. 1H NMR (400 MHz, CD3OD): δ 7.75 (t, J = 9.0 Hz, 4H), 7.52−7.42 (m, 5H), 7.30 (d, J = 8.0 Hz, 1H), 7.20−7.05 (m, 7H), 6.98 (t, J = 7.2 Hz, 1H), 6.79 (s, 2H), 4.66 (t, J = 6.0 Hz, 2H), 4.25 (br s, 3H), 3.27−3.21 (m, 2H), 3.04−2.90 (m, 3H), 2.82−2.76 (m, 1H), 1.34 (s, 18H), 1.31 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 174.4, 173.9, 172.7, 157.9, 153.1, 153.0, 147.9, 139.5, 137.9, 135.5, 129.4, 128.7, 128.6, 128.5, 128.3, 128.1, 126.8, 126.2, 124.7, 122.6, 120.1, 119.2, 112.5, 110.3, 80.9, 56.1, 54.9, 52.2, 43.8, 35.5, 31.6, 30.3, 30.0, 28.6, 28.4. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 932.5187, found 932.5117. HPLC: tR = 32.3 min, 98.0% purity. Boc-His(2-biphenyl)-Trp-His(2-biphenyl)-NHBzl (7f). Yield = 53%. 1H NMR (400 MHz, CD3OD): δ 7.86 (s, 4H), 7.65−7.56 (m, 9H), 7.45−7.29 (m, 7H), 7.21−6.99 (m, 8H), 6.82 (d, J = 7.0 Hz, 2H), 4.68 (s, 2H), 4.31−4.21 (m, 3H), 3.23 (br s, 1H), 3.01−2.77 (m, 5H), 1.34 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 174.4, 173.9, 172.6, 157.8, 147.5, 142.5, 141.5, 139.5, 138.0, 135.5, 130.2, 130.1, 129.9, 129.9, 129.4, 128.7, 128.6, 128.6, 128.3, 128.1, 127.8, 126.8, 124.7, 122.6, 120.6, 120.1, 119.3, 112.5, 110.3, 80.9, 56.0, 54.8, 52.2, 43.8, 30.4, 30.0, 28.6, 28.4. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 972.4561, found 972.4128. HPLC: tR = 31.3 min, 97.5% purity. His-Trp-His-NHBzl (8a). Yield = 92%. 1H NMR (400 MHz, CD3OD): δ7.57−7.49 (m, 6H), 7.39−7.24 (m, 4H), 7.16−7.12 (m, 3H), 7.05 (t, J = 7.4 Hz, 1H), 4.67 (t, J = 7.5 Hz 1H), 4.58 (t, J = 6.9 Hz, 1H), 4.49 (t, J = 6.9 Hz, 1H), 4.37−4.33 (m, 2H), 3.50−3.47 (m, 1H), 3.40−3.37 (m, 1H), 3.27−3.16 (m, 3H), 3.08 (dd, J = 15.4, 7.4 Hz, 1H). 13C NMR (100 MHz, CD3OD): δ 173.0, 169.7, 167.7, 137.8, 136.2, 134.3, 133.2, 128.4, 127.2, 126.9, 126.3, 126.0, 124.1, 121.6, 118.7, 118.5, 117.9, 117.2, 111.5, 108.2, 55.3, 53.5, 51.8, 27.0, 26.4, 26.1. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 568.2784, found 568.2782. HPLC: tR = 17.8 min, 95.8% purity. His(2-phenyl)-Trp-His(2-phenyl)-NHBzl (8b). Yield = 97%. 1H NMR (400 MHz, CD3OD): δ 8.01 (d, J = 8.4 Hz, 2H), 7.81−7.74 (m, 4H), 7.61−7.56 (m, 4H), 7.45−7.38 (m, 4H), 7.30−7.24 (m, 2H), 7.20−7.09 (m, 3H), 7.06−6.93 (m, 3H), 4.70 (t, J = 7.5 Hz, 1H), 4.55 (t, J = 7.0 Hz, 1H), 4.40 (t, J = 6.6 Hz, 1H), 4.14 (d, J = 14.8 Hz, 1H), 3.99 (d, J = 9.1 Hz, 1H), 3.48 (dd, J = 16.1, 5.7 Hz, 1H), 3.28−3.22 (m, 2H), 3.18−3.06 (m, 2H), 2.88 (dd, J = 12.8, 7.5 Hz, 1H). 13C NMR (100 MHz, CD3OD): δ 173.0, 169.5, 167.6, 144.8, 144.5, 143.7, 138.4, 137.7, 136.2, 132.3, 129.6, 129.4, 129.0, 128.5, 128.3, 127.7, 127.3, 127.1, 127.0, 126.9, 126.6, 126.1, 124.0, 122.2, 121.4, 121.2, 119.5, 118.9, 117.7, 111.4, 108.1, 55.3, 54.4, 52.7, 42.5, 27.1, 26.7, 26.6. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 720.3410, found 720.3404. HPLC: tR = 23.4 min, 95.5% purity. His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]-NHBzl (8c). Yield = 96%. 1H NMR (400 MHz, CD3OD): δ 7.90−7.83 (m, 4H), 7.55−7.42 (m, 6H), 7.30−7.02 (m, 10H), 4.72 (br s, 2H), 4.37−

of KHSO4 to pH 4 to generate N-α-Boc-2-aryl-L-histidine. The solvent was removed under reduced pressure, and the resulting residue was extracted with tert-butanol (3 times) to afford 4a−e. General Synthetic Procedure for Tripeptides 8a−f. N-α-Boc2-aryl-His-OH (4a−f, 1 equiv) was dissolved in DMF (2 mL) in a microwave (MW) reaction vessel. Benzylamine (1.2 equiv), DIC (1.2 equiv), and HONB (1.2 equiv) was added to the vessel, and reaction mixture was heated under MW irradiation at 60 °C for 30 min. The solvent was removed and residue was purified using a solvent combination of hexane/EtOAc (3:2) on a fully automated flash column chromatographic system to afford Boc-His(2-aryl)-NHBzl (5a−f). The latter compounds 5a−f were dissolved in a solution of 6 N HCl in MeOH (5 mL), and reaction mixture was stirred at 25 °C for 15 min. The removal of solvent afforded the Boc-deprotected amino acids. The latter compounds were neutralized with DIEA (3 equiv) in DMF (2 mL) followed by the addition of Boc-Trp-OH (1.2 equiv), DIC (1.2 equiv), and HONB (1.2 equiv). The reaction mixture was subjected to MW heating at 60 °C for 30 min. The solvent was removed and crude product was purified using a solvent combination of CHCl2/MeOH (98:2) on neutral alumina to afford dipeptides 6a−f. The Boc-deprotections were carried out as described above, and to the residue were added DIEA (3 equiv), N-α-Boc-His(2-aryl)-OH (4a−f, 1.2 equiv), DIC (1.2 equiv), and HONB (1.2 equiv) in DMF (2 mL). The reaction mixture was subjected to MW heating at 60 °C for 30 min. The solvent was removed and crude product was purified using a combination of CHCl2/MeOH (97.5:2.5) on neutral alumina to afford 7a−f. The latter compounds 7a−f upon acidolysis with 6 N HCl for 15 min at ambient temperature cleanly afforded final tripeptides 8a−f. General Synthetic Procedure for Tripeptides 12a−f. 2-Aryl-Lhistidine (4a−f, 1 equiv) was dissolved in dry methanol, and HCl gas was passed through the solution for 2 h followed by stirring at ambient temperature for 12 h. Reaction mixture was concentrated under reduced pressure to afford 9a−f. Dipeptides (10a−f) were synthesized by coupling 9a−f (1 equiv) with Boc-Trp-OH (1.2 equiv) in the presence of DIEA (3 equiv), DIC (1.2 equiv), and HONB (1.2 equiv) in DMF (2 mL) under MW heating at 60 °C for 30 min. The peptides were purified using a solvent combination of CHCl2/MeOH (98:2) on neutral alumina to afford 10a−f. Dipeptides 10a−f upon Bocdeprotection in the presence of 6 N HCl in MeOH (5 mL) at 25 °C for 15 min produced the free dipeptides. The latter dipeptides upon coupling with N-α-Boc-His(2-aryl)-OH (4a−f, 1 equiv) in the presence of DIEA (3 equiv), DIC (1.2 equiv), and HONB (1.2 equiv) in DMF (2 mL) under MW heating at 60 °C for 30 min produced crude tripeptides. The tripeptides were purified using a solvent combination of CHCl2/MeOH (97:3) on neutral alumina and afforded Boc-protected tripeptides 11a−f. The Boc latter peptides (1 mmol) in 6 N HCl (5 mL) were stirred for 15 min at ambient temperature to produce peptides 12a−f. Characterization Data of Synthesized Peptides. Boc-His-TrpHis-NHBzl (7a). Yield = 60%. 1H NMR (400 MHz, CD3OD): δ 7.66 (s, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.30−7.24 (m, 3H), 7.16−7.04 (m, 5H), 6.83 (s, 1H), 6.71 (s, 1H), 4.61−4.54 (m, 2H), 4.27−4.15 (m, 3H), 3.20−3.17 (m, 2H), 2.95−2.71 (m, 4H), 1.31 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 173.3, 172.6, 171.5, 156.7, 137.7, 136.3, 135.2, 134.0, 132.6, 128.3, 127.1, 127.0, 123.8, 121.6, 119.1, 117.8, 116.8, 111.5, 108.4, 80.6, 54.8, 54.6, 53.6, 42.7, 28.7, 28.5, 27.3, 26.7. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 668.3309, found 668.3309 . HPLC: tR = 21.9 min, 97.4% purity. Boc-His(2-phenyl)-Trp-His(2-phenyl)-NHBzl (7b). Yield = 61%. 1 H NMR (400 MHz, CD3OD): δ 7.93−7.82 (m, 3H), 7.75−7.52 (m, 5H), 7.48−7.29 (m, 6H), 7.24−6.98 (m, 8H), 4.69−4.65 (m, 2H), 4.39 (br s, 1H), 4.22 (br s, 2H), 3.19−3.07 (m,3H), 3.02−2.91 (m, 3H), 1.37 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 174.2,173.6, 171.9, 157.8, 146.7, 144.2, 140.9, 139.4, 137.9, 133.4, 131.4,130.3, 130.1, 129.5, 129.1, 128.7, 128.4, 128.2, 127.9, 127.4, 127.0, 124.9, 122.6, 120.1, 119.2, 112.5, 110.2, 81.1, 56.3, 55.8, 54.3, 43.8, 29.8, 29.1, 28.6, 28.4. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 820.3935, found 820.3927. HPLC: tR = 26.3 min, 95.7% purity. Boc-His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]NHBzl (7c). Yield = 66%. 1H NMR (400 MHz, CD3OD): δ 7.69 (s, 6616

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

Journal of Medicinal Chemistry

Article

128.6, 126.3, 124.7, 122.4, 119.8, 119.2, 112.3, 110.4, 80.8, 55.6, 54.8, 54.1, 52.7, 30.5, 29.9, 28.6, 28.3, 21.3. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 773.3775, found 773.3779. HPLC: tR = 26.1 min, 96.7% purity. Boc-His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]-OMe (11d). Yield = 61%. 1H NMR (400 MHz, CD3OD): δ 7.76 (d, J = 8.4 Hz, 4H), 7.54 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.15 (s, 1H), 7.09 (t, J = 7.2 Hz, 1H), 7.03−6.94 (m, 6H), 6.91 (s, 1H), 4.71−4.63 (m,2H), 4.36−4.32 (m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.66 (s, 3H), 3.24−3.10 (m, 3H), 3.05−2.98 (m, 2H), 2.91−2.88 (m, 1H), 1.36 (9H). 13C NMR (100 MHz, CD3OD): δ 174.5, 174.0, 172.2, 162.5, 157.7, 147.3, 143.3, 138.0, 133.4, 128.8, 128.5, 124.8, 122.9, 122.4, 119.9, 119.2, 115.5, 112.3, 110.3, 81.0, 55.9, 55.9, 55.5, 53.7, 52.9, 30.7, 30.1, 28.6, 27.9. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 805.3673, found 805.3650. HPLC: tR = 24.7 min, 95.9% purity. Boc-His[2-(4-tert-butylphenyl)]-Trp-His[(4-tert-butylphenyl)]-OMe (11e). Yield = 56%. 1H NMR (400 MHz, CD3OD): δ 7.62 (t, J = 9.5 Hz, 4H), 7.49−7.41 (m, 3H), 7.35−7.19 (m, 3H), 7.01− 6.87 (m, 3H), 6.69 (d, J = 9.7 Hz, 2H), 4.57 (br s, 2H), 4.20 (br s, 1H), 3.53 (s, 3H), 3.10−3.04 (m, 2H), 2.98−2.70 (m, 4H), 1.21 (s, 18H), 1.17 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 173.8, 172.7, 171.4, 157.6, 152.9, 152.8, 148.0, 138.0, 133.4, 128.8, 128.7, 128.6, 126.8, 126.7, 126.2, 124.7, 122.4, 119.9, 119.3, 112.3, 110.4, 80.8, 55.9, 55.6, 54.1, 52.8, 35.5, 31.6, 30.7, 29.8, 28.6, 27.7. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 857.4714, found 857.4711. HPLC: tR = 31.1 min, 97.8% purity. Boc-His(2-biphenyl)-Trp-His(2-biphenyl)-OMe (11f). Yield = 54%. 1H NMR (400 MHz, CD3OD): δ 7.83 (d, J = 8.2 Hz, 4H), 7.64− 7.50 (m, 9H), 7.45 (t, J = 7.5 Hz, 2H), 7.40−7.30 (m, 5H), 7.15 (s, 1H), 7.12−7.08 (m, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.86 (d, J = 11.2 Hz, 2H), 4.66−4.57 (m, 2H), 4.32 (br s, 1H), 3.65 (s, 3H), 3.22−3.07 (m, 3H), 3.01−2.93 (m, 2H), 2.87−2.80 (m, 1H), 1.31 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 172.3,171.3, 168.8, 157.2, 147.8, 145.3, 143.2, 139.9, 138.1, 136.6, 135.3, 133.8, 131.3, 129.9, 129.2, 128.7, 128.6, 127.9, 127.4, 127.1, 127.0, 126.9, 126.4, 126. 0,125.5, 125.4, 124.7, 123.4, 121.3, 118.6, 117.9, 116.5, 111.1, 80.0, 54.8, 53.1, 52.7, 52.0, 30.2, 28.9, 27.2, 27.0. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 897.4088, found 897.4053. HPLC: tR = 29.6 min, 98.2% purity. His-Trp-His-OMe (12a). Yield = 93%. 1H NMR (400 MHz, CD3OD): δ 7.70 (d, J = 12.1 Hz, 2H), 7.58 (t, J = 9.5 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.21−7.10 (m, 3H), 6.83 (t, J = 13.2 Hz, 2H), 4.68−4.53 (m, 2H), 3.90−3.86 (m, 1H), 3.60 (s, 3H), 3.24−2.89 (m, 6H). 13C NMR (100 MHz, CD3OD): δ 171.1, 170.7, 170.5, 134.6, 133.7, 130.5, 125.4, 122.7, 120.2, 117.6, 116.5, 115.3, 110.1, 107.0, 52.9, 52.1, 51.1, 28.5, 26.6, 25.6. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 493.2312, found 493.2308. HPLC: tR = 17.3 min, 98.2% purity. His(2-phenyl)-Trp-His(2-phenyl)-OMe (12b). Yield = 96%. 1H NMR (400 MHz, CD3OD): δ 7.91−7.83 (m, 4H), 7.56−7.46 (m, 7H), 7.33−7.22 (m, 2H), 7.18−7.11 (m, 3H), 7.02 (t, J = 8.2 Hz, 1H), 4.69−4.64 (m, 1H), 4.61−4.57 (m, 1H), 4.35 (s, 1H), 3.67 (s, 3H), 3.45−3.35 (m, 2H), 3.26−3.14 (m, 3H), 3.07−3.01 (m, 1H). 13C NMR (100 MHz, CD3OD): δ 174.5, 171.5, 169.2, 146.3, 138.3, 132.1,128.9, 127.4, 127.0, 124.8, 124.2, 122.8, 122.1, 119.3, 119.2, 115.3, 112.9, 109.8, 56.7, 55.9, 54.3, 51.89, 28.4, 28.0, 27.8. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 645.2938, found 645.2938. HPLC: tR = 20.6 min, 97.8% purity. His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]-OMe (12c). Yield = 95%. 1H NMR (400 MHz, CD3OD): δ 7.86 (t, J = 9.4 Hz, 4H), 7.56 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 4.4 Hz, 5H), 7.33 (d, J = 8.1, 1H), 7.26 (s, 1H), 7.09 (t, J = 7.4 Hz, 1H),7.02−6.98 (m, 1H), 4.84−4.81 (m, 1H), 4.69−4.66 (m, 1H), 4.40−4.37 (m, 1H), 3.69 (s, 3H), 3.51−3.40 (m, 2H), 3.28−3.22 (m, 3H), 3.13−3.07 (m, 1H), 2.45 (s, 3H), 2.41 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 174.8, 171.3, 169.0, 146.7, 145.2, 144.9, 144.7, 138.0, 132.3, 131.5, 131.4, 130.7, 128.5, 128.1, 128.0, 127.6, 125.1, 122.5, 121.1, 120.9, 119.8, 119.2, 119.1, 112.4, 109.8, 56.8, 53.4, 53.1, 52.9, 28.6, 28.0, 27.8, 21.6,

4.19 (m, 3H), 3.68−3.36 (m, 3H), 3.22−3.08 (m, 3H), 2.43 (s, 6H). 13 C NMR (100 MHz, CD3OD): δ 174.7, 171.1, 169.0, 146.7, 145.2, 144.9, 144.6, 139.5, 137.9, 131.5, 131.5, 131.1, 129.5, 128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.6, 125.1, 122.7, 121.2,121.1, 120.7, 120.1, 119.2, 118.9, 112.5, 109.8, 56.8, 53.9, 53.1, 44.2, 28.6, 28.1, 28.0, 21.6. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 748.3723, found 748.3724. HPLC: tR = 24.7 min, 97.5% purity. His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]NHBzl (8d). Yield = 91%. 1H NMR (400 MHz, CD3OD): δ 7.85 (d, J = 8.9 Hz, 2H), 7.75 (d, J = 6.9 Hz, 2H), 7.41 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.24−7.17 (m, 4H), 7.14−7.01 (m, 10H), 4.69−4.65 (m, 1H), 4.50 (t, J = 7.0 Hz, 1H), 4.32−4.29 (m, 1H), 4.15−4.01 (m, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 3.39−3.35 (m, 1H), 3.27−3.02 (m, 4H), 2.88 (dd, J = 15.6, 7.1 Hz, 1H), 13C NMR (100 MHz, CD3OD): δ 173.0, 169.5, 167.8, 162.6, 145.4, 144.0, 137.7, 136.2, 128.8, 128.5, 128.3, 128.0, 127.2, 127.0, 126.9, 126.5, 124.0, 119.0, 118.7, 117.7, 117.2, 115.1, 115.0, 114.8, 111.5, 108.1, 55.4, 55.3, 55.2, 52.6, 51.8, 42.8, 29.9, 27.1, 26.7. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 780.3622, found 780.3603. HPLC: tR = 24.0 min, 98.4% purity. His[2-(4-tert-butylphenyl)]-Trp-His[2-(4-tert-butylphenyl)]NHBzl (8e). Yield = 94%. 1H NMR (400 MHz, CD3OD): δ 7.84 (d, J = 8.5 Hz, 2H), 7.72−7.60 (m, 6H), 7.44 (t, J = 6.7 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.22−7.16 (m, 4H), 7.12−7.04 (m, 5H), 4.67−4.63 (m, 1H), 4.52 (t, J = 7.1 Hz, 1H), 4.31−4.28 (m, 1H), 4.14−3.98 (m, 2H), 3.42 (d, J = 5.0 Hz, 1H), 3.26−3.07 (m, 4H), 2.97−2.91 (m, 1H), 1.30 (s, 9H), 1.28 (9H). 13C NMR (100 MHz, CD3OD): δ 175.9, 173.2, 169.4, 145.4, 144.0, 137.6, 136.1, 134.3, 129.2, 128.4, 127.2, 127.0, 126.9, 126.7, 126.4, 125.9, 124.1, 121.8, 119.8, 119.1, 117.7, 111.6, 108.1, 52.8, 51.8, 51.1, 42.6, 34.6, 31.4, 30.9, 30.1. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 832.4662, found 832.4632. HPLC: tR = 30.6 min, 95.5% purity. His(2-biphenyl)-Trp-His(2-biphenyl)-NHBzl (8f). Yield = 92%. 1 H NMR (400 MHz, CD3OD): δ 7.99 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 3H), 7.63 (t, J = 9.0 Hz, 4H), 7.52− 7.40 (m, 9H), 7.32 (d, J = 8.1 Hz, 1H), 7.25−7.11 (m, 7H), 7.06 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.3 Hz, 1H), 4.73−4.66 (m, 2H), 4.36 (t, J = 6.5 Hz, 1H), 4.21−4.08 (m, 2H), 3.45−3.40 (m, 1H), 3.29−3.16 (m, 4H), 3.20−2.97 (m, 1H). 13C NMR (100 MHz, CD3OD): δ 174.9, 169.6, 167.6, 144.9, 144.6, 143.5, 138.6, 137.9, 136.3, 134.2 129.7, 128.9, 128.4, 128.2, 127.7, 127.1, 127.1, 127.0, 126.7, 126.6, 123.8, 121.4, 121.1, 119.3, 118.9, 117.9, 117.7, 111.3, 108.3, 55.3, 52.5, 51.8, 42.5, 27.2, 26.7, 26.6. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 872.4036, found 872.4026. HPLC: tR = 28.9 min, 97.3% purity. Boc-His-Trp-His-OMe (11a). Yield = 65%. 1H NMR (400 MHz, CD3OD): δ 7.91−7.83 (m, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.18−7.05 (m, 4H), 6.97−6.85 (m, 2H), 4.66−4.57 (m, 2H), 4.26 (s, 1H), 3.63 (s, 3H), 3.24−3.15 (m, 2H), 3.05−2.94 (m, 3H), 2.86−2.78 (m, 1H), 1.30 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 175.4, 172.6, 171.9, 156.7, 136.3, 134.8, 133.4, 131.6, 130.3, 127.1, 123.9, 121.5, 119.0, 117.9, 116.8, 111.5, 108.4, 80.8, 54.5, 54.3, 52.6, 52.4, 28.3, 27.8, 27.3, 27.0. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 593.2836, found 593.2838. HPLC: tR = 19.3 min, 97.6% purity. Boc-His(2-phenyl)-Trp-His(2-phenyl)-OMe (11b). Yield = 61%. 1 H NMR (400 MHz, CD3OD): δ 7.85−7.80 (m, 4H), 7.53−7.49 (m, 7H), 7.31 (d, J = 8.1 Hz, 1H), 7.15−7.05 (m, 4H), 6.98 (t, J = 7.5 Hz, 1H), 4.75−4.69 (m, 1H), 4.58 (t, J = 7.1 Hz, 1H), 4.36 (br s, 1H), 3.65 (s, 3H), 3.22−3.03 (m, 5H), 2.95−2.90 (m, 1H), 1.35 (s, 9H). 13 C NMR (100 MHz, CD3OD): δ 173.9, 173.8, 172.8, 157.7, 148.0, 137.9, 132.4,128.8, 127.9, 127.5, 124.8, 124.2, 124.1, 122.8, 122.4, 119.9, 119.2, 115.3, 115.2, 112.3, 110.3, 80.9, 56.0, 55.8, 54.1, 52.8, 30.7, 29.9, 28.6, 27.9. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 745.3462, found 745.3450. HPLC: tR = 24.9 min, 95.6% purity. Boc-His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]OMe (11c). Yield = 58%. 1H NMR (400 MHz, CD3OD): δ 7.68−7.66 (m, 4H), 7.51 (d, J = 7.7 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.22−7.16 (m, 4H), 7.10−7.04 (m, 2H), 6.96 (t, J = 7.3 Hz, 1H), 6.98 (d, J = 6.8 Hz, 2H), 4.66 (t, J = 6.9 Hz, 2H), 4.31−4.28 (m, 1H), 3.62 (s, 3H), 3.30−3.09 (m, 3H), 3.05−2.84 (m, 3H), 2.34 (s, 3H), 2.30 (s, 3H), 1.33 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 173.9, 173.8, 170.4, 157.3, 148.1, 147.2, 139.8, 139.7, 138.0, 130.5, 130.4, 128.8, 128.7, 6617

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

Journal of Medicinal Chemistry

Article

21.5. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 673.3251, found 673.3251. HPLC: tR = 21.6 min, 98.7% purity. His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]OMe (12d). Yield = 92%. 1H NMR (400 MHz, CD3OD): δ 7.92− 7.87 (m, 4H), 7.55−7.47 (m, 2H), 7.35−7.31 (m, 2H), 7.24 (s, 1H), 7.12−7.06 (m, 5H), 7.01−6.97 (m, 1H), 4.83−4.80 (m, 1H), 4.66 (t, J = 7.3 Hz, 1H), 4.35 (br s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.67 (s, 3H), 3.49−3.38 (m, 2H), 3.23−3.18 (m, 3H), 3.09−3.03 (m, 1H). 13C NMR (100 MHz, CD3OD): δ 174.8, 171.3, 169.0, 164.5, 164.4, 146.8, 145.3, 139.3, 138.0, 130.4, 130.0, 129.6, 128.5, 127.6, 125.1, 122.5, 120.6, 119.9, 119.1, 118.8, 116.2, 116.1, 112.4, 109.8, 56.7, 56.3, 56.2, 53.3, 53.1, 52.9, 28.6, 28.0, 27.8. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 705.3149, found 705.3130. HPLC: tR = 20.8 min, 96.4% purity. His[2-(4-tert-butylphenyl)]-Trp-His[2-(4-tert-butylphenyl)]OMe (12e). Yield = 91%. 1H NMR (400 MHz, CD3OD): δ 7.76 (d, J = 8.6 Hz, 2H), 7.66 (t, J = 9.1 Hz, 4H), 7.52 (d, J = 8.6 Hz, 2H), 7.44−7.33 (m, 3H), 7.25−7.14 (m, 3H), 7.07 (t, J = 7.5 Hz, 1H), 4.58−4.50 (m, 2H), 4.22 (q, J = 3.8 Hz, 1H), 3.54 (s, 3H), 3.41 (d, J = 4.5 Hz, 1H), 3.26 (dd, J = 14.2, 5.6 Hz, 1H), 3.13−2.88 (m, 4H), 1.32 (s, 9H), 1.25 (s, 9H). 13C NMR (100 MHz, CD3OD): δ 172.0, 168.6, 166.2, 155.4, 155.2, 147.4, 144.0, 142.4, 137.9, 134.6, 134.5, 130.7, 127.5, 125.5, 125.2, 125.1, 124.5, 124.0, 122.8, 120.0, 117.7, 117.6, 117.5, 110.1, 106.2, 54.3, 51.4, 50.5, 50.1, 33.1, 33.0, 28.6, 28.5, 25.2. HRMS (ESI-TOF): m/z [(M + H)+] calculated for 757.4190, found 757.4189. HPLC: tR = 28.2 min, 96.3% purity. His(2-biphenyl)-Trp-His(2-biphenyl)-OMe (12f). Yield = 95%. 1 H NMR (400 MHz, CD3OD): δ 7.40 (d, J = 7.4 Hz, 2H), 7.33−7.22 (m, 10H), 7.17−7.07 (m, 10H), 6.94−6.84 (m, 3H), 4.47 (br s, 1H), 4.28 (br.s, 1H), 4.12 (br.s, 1H), 3.40 (s, 3H), 3.12−3.05 (m, 2H), 2.93−2.73 (m, 3H), 2.59−2.51 (m, 1H). 13C NMR (100 MHz, CD3OD): δ 173.0, 170.3, 167.7, 144.4, 143.6, 142.3, 138.6, 136.4, 133.7, 133.1, 131.7, 130.7, 129.5, 129.0, 128.5, 127.7, 127.5, 126.9, 126.7, 126.6, 126.5, 126.5, 125.9, 124.0, 121.8, 121.5, 118.8, 117.6, 115.9, 111.5, 108.2, 55.3, 52.6, 51.8, 29.2, 27.2, 26.6. HRMS (ESITOF): m/z [(M + H)+] calculated for 797.3564, found 797.3549. HPLC: tR = 27.9 min, 98.2% purity. Synthesis of FITC-Labeled Peptides. To a mixture of pyridine− DMF−CH2Cl2 (12:7:5, 1 mL), peptide 12f (0.01 mmol, 1 equiv) or 8b (0.01 mmol, 1 equiv) was added followed by the addition of FITC (1.2 equiv). The reaction mixture was allowed to stir for 12 h at ambient temperature. The purification involved using a solvent combination of CH2Cl2/MeOH (97−95:3−5) on neutral alumina to afford orange colored FITC-labeled peptides. Antimicrobial Activity Determination. The antibacterial activities of synthesized peptides were evaluated against Staphylococcus aureus (ATCC 29213), methicillin-resistant S. aureus (ATCC 33591, MRSA), Escherichia coli (ATCC 35218), Mycobacterium intracellulare (ATCC 23068), and Pseudomonas aeruginosa (ATCC 27853). S. aureus, MRSA, P. aeruginosa, and E. coli susceptibility to test peptides was determined using CLSI methods,28 and M. intracellulare susceptibility to the synthetic peptides was tested by using the modified Alamar blue procedure of Franzblau et al.29,30 The antifungal activities of the peptides against Cryptococcus neoformans (ATCC90113) were determined according to modified CLSI methods.22 Test Procedure. All pathogenic strains were obtained from ATCC, Manassas, VA. The test synthetic peptides were serially diluted in 20% DMSO/saline and transferred in duplicate to 96-well flat-bottomed microplates, and inocula were prepared by correcting the OD630 of microbe suspensions inincubation broth [Sabouraud Dextrose (Difco) for C. neoformans, cation adjusted Mueller−Hinton (Difco) at pH 7.3 for nonmycobacterial bacteria, and 5% Alamar blue (BioSource International, Camarillo, CA) in Middlebrook 7H9 broth with OADC enrichment, pH 7.3, for M. intracellulare] to afford C. neoformans 1.5 × 103 CFU/mL, nonmycobaterial bacteria 5 × 105 CFU/mL, and M. intracellulare 2.0 × 106 CFU/mL. The drug controls [ciprofloxacin (ICN Biomedicals, Ohio) for bacteria, and amphotericin B (ICN Biomedicals, Ohio) for C. neoformans] were used in each assay of tested peptides. All organisms were read at either 530 nm using the

Biotek Power wave XS plate reader (Bio-Tek Instruments, Vermont) or 544ex/590em (M. intracellulare) using the Polarstar Galaxy plate reader (BMG Lab Technologies, Germany) prior to and after incubation: MRSA/E. coli/P. aeruginosa at 35 °C for 16−20 h, C. neoformans at 35 °C for 70−74 h, and M. intracellulare at 37 °C and 10% CO2 for 70−74 h. The MIC value was defined as the lowest test concentration that allows no detectable growth (for M. intracellulare no color changes from blue to pink). For all the peptides, minimum fungicidal or bactericidal concentrations were determined by removing 5 DL from each clear (or blue) well, transferring to agar and incubating as mentioned previously. The MFC and MBC for tested peptides were defined as the lowest test concentration that kills the organism (exhibited no growth on agar plates). The C. neoformans strain (ATCC350) used in the time kill, hemolysis, microscopic examination, and synergistic combination studies was obtained from Microbial Type Culture Collection, Institute of Microbial Technology (MTCC-IMTECH), Chandigarh and National Collection of Pathogenic Fungi (NCPF), Post-Graduate Institute of Medical Education and Research (PGIMER), Chandigarh. The C. neoformans was cultured in yeast extract−peptone−dextrose (YEPD broth, HiMedia, India) and RPMI-1640 media (HiMedia, India) and stored with 15% glycerol at −80 °C as frozen stocks. The fungal cells were freshly revived on respective agar plates from the stock before each experiment. For agar plates, 2.5% (w/v) bacteriological agar (HiMedia, India) was added to the medium.31 Cytotoxicity Assay. All synthetic peptides were evaluated for cytotoxicity in a panel of mammalian cell lines to determine their safety profile. The in vitro cytotoxicity studies were performed against four human cancer cell lines (SK-OV-3, SK-MEL, KB, and BT-549) and two noncancerous mammalian kidney cell lines (LLC-PK1 and VERO).24 All cell lines were obtained from ATCC(American Type Culture Collection). The assay was performed in 96-well tissue culture-treated microplates, and targeted peptides were tested up to a highest concentration of 10 μg/mL. The cells (25 000 cells/well) were seeded to the wells of the plate and incubated for 24 h for confluence. The peptide samples were added and plates again incubated for 48 h. The number of viable cells was calculated through a modified version of neutral red uptake assay. The marketed drug doxorubicin was used in study as a positive control, while DMSO was used as the negative control. The tested peptides (7a−f, 8a−f, 11a−f, and 12a−f) were found to be noncytotoxic up to a concentration of 10.0 μg/mL. Hemolytic Assay. For the hemolytic activity of 12f, RBCs (human red blood cells) containing 10% citrate phosphate dextrose were harvested by spinning (1000g; 5 min; at 25 °C) and washed three to five times with phosphate buffer saline (PBS). The packed cell volume was used to make a 0.8% (v/v) suspension in PBS. The RBC suspension (100 μL) was transferred to each well of a 96-well microtiter plate and mixed with 12f peptide solution at varied test concentrations. The microtiter plate was incubated at 37 °C for 1 h, and the samples were centrifuged (1000g; 5 min; at 25 °C). The supernatant (100 μL) was transferred to new wells, and the absorbance was measured with a microtiter plate reader (BioRad model 680) at 414 nm to monitor RBC lysis. The cells with PBS alone were used as the negative control, and RBCs lysed using 0.1% Triton X-100 were taken as positive control (100% lysis).32 Time Kill Assay. In order to understand the antifungal nature of peptide 12f against C. neoformans cells, time kill kinetic was performed. C. neoformans cells (∼1 × 104 CFU/mL) were inoculated in RPMI1640 medium containing 12f and amphotericin B separately. The tubes containing treated and untreated samples were incubated (30 °C; 200 rpm), and 100 μL aliquots were removed at predetermined time intervals (4, 8, 12, 16, and 24 h). The 10 μL aliquots were serially diluted (10-fold) in saline water and plated on YEPD agar plates. C. neoformans cells were counted after incubating the plates at 30 °C for 48 h.33 Proteolytic Stability. To study the proteolytic stability, peptide 12f and trypsin were taken at ratio of 100:0.5 (mol/mol) in 0.1 M NH4CO3, 0.1 mM CaCl2. At pH 8.3, tested solution was incubated in a rotary shaker (37 °C, 200 rpm, for 1 h). An aliquot was injected into a 6618

DOI: 10.1021/acs.jmedchem.7b00481 J. Med. Chem. 2017, 60, 6607−6621

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RP-HPLC analytical C18 column using the water−acetonitrile liner gradient of 95−5% as acetonitrile (0.1% TFA)−water (0.1% TFA) at a flow rate of 1 mL/min over 40 min.34 Propium Iodide (PI) Uptake. The permeabilization effect of peptide 12f on C. neoformans cell wall was checked by using a membrane impermeant dye PI.35 For study, overnight grown C. neoformans (ATCC350) cells (∼1 × 104 CFU/mL) were suspended in a RPMI-1640 medium containing 12f at the MIC and PI (1.49 μM). After incubation for 1 h at 30 °C with constant shaking (200 rpm), cells were harvested by centrifugation and suspended in phosphate buffer saline (PBS, pH 7.4). Then cells were examined by confocal microscopy (Olympus Fluoview FV1000 SPD; Olympus, Tokyo, Japan) with a wavelength of >560 nm for PI, and C. neoformans cells without treatment of 12f served as control. FITC-Labeled Peptide Uptake. To examine the localization of 12f inside C. neoformans cells, the peptide was labeled with FITC. C. neoformans cells (∼1 × 104 CFU/mL) were suspended in a RPMI1640 medium containing FITC-labeled 12f at the MIC concentration. After incubation for 1 h cells were harvested and suspended in PBS, then visualized by confocal microscopy with excitation and emission wavelengths of 488 and 515 nm, respectively. Examination of C. neoformans cells with 8b treatment served as control. Detection of Nuclear Fragmentation. DAPI staining was used for the analysis of nuclear fragmentation of C. neoformans cells.36 C. neoformans cells (∼1 × 104 CFU/mL) were suspended in RPMI-1640 medium containing 12f peptide at MIC value and incubated at 30 °C at 200 rpm for 1 h in water bath. After incubation, C. neoformans cells were harvested by centrifugation and suspended in PBS and incubated in the dark with DAPI at 3.01 μM for 10 min. The untreated C. neoformans cells with DAPI served as a control, and treated cells were examined by confocal microscopy with excitation at 350 nm and emission at 470 nm. Scanning and Transmission Electron Microscopy. The C. neoformans cells (∼104) suspension from overnight cultures was prepared in RPMI 1640 at pH 7, and then 12f at MIC concentration was added to the cells (∼104 cell) and incubated at 30 °C for 1 h. The C. neoformans cells were fixed with 2% glutaraldehyde in 0.1% phosphate buffer for 1 h at room temperature. The treated with 12f and untreated samples were washed with 0.1 M phosphate buffer (pH 7.2) and postfixed 1% OsO4 in 0.1 M phosphate buffer for 1 h at 4 °C. For SEM, the samples were dehydrated in acetone and dropped on round glass coverslip with hexamethyldisilizane (HMDS). Further, the samples were dried at room temperature and then sputter-coated with gold and observed under the SEM (JEOL 6100, Japan). For ultrastructure study, samples were dehydrated with graded acetone, clearing with toluene and infiltrated with toluene and Araldite mixture at room temperature and then finally in pure Araldite at 50 °C and imbedded in tube (1.5 mL) with pure Araldite mixture at 60 °C overnight. Semithin and ultrathin section cutting was done with ultramicrotome (Ultramicotome Lecia EM UC6). The sections of samples were taken on the 3.05 mm diameter and 200 mess copper grid, stained with uranyl acetate and lead acetate before observing under the HRTEM (FEI Technai G2 F20, The Netharlands) and analyzed at 120 kV.37 Checkerboard Assay. The interaction of peptide 12f with fluconazole and amphotericin B, well-known marketed antifungal drugs, was evaluated by the checkerboard method as described previously and expressed as the fractional inhibitory concentration index (FICI), i.e., sum of the FIC for each agent. FIC value of the most effective combination with 12f was used in calculating the FICI. FICI = FIC of A + FIC of B = CAcomb/MICAalone + CBcomb/MICBalone, where MICAalone and MICBalone are the MICs of drugs A and B when acting alone and CAcomb and CBcomb are concentrations of drugs A and B at the isoeffective combinations, respectively. The FICI of evaluated combinations of 12f and drugs was interpreted as synergistic when it was ≤0.5. When it was >4.0, it was interpreted as antagonist, and any value in between was interpreted as indifferent.38

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00481. Representative peptide analogues including HPLC chromatograms and 1H and 13C NMR and HRMS spectra (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 91+172-2292024. ORCID

Rahul Jain: 0000-0002-9180-2812 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.K.S. thanks University Grant Comission (UGC), India, for the award of senior research fellowship. Marsh Wright and John Trott are acknowledged for excellent technical support in biological testing at NCNPR. Antimicrobial testing was supported by the NIH, NIAID, Division of AIDS, Grant AI 27094 (antifungal) and the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-1-603 (antibacterial).

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ABBREVIATIONS CAMP, cationic antimicrobial peptide; DIEA, N,N-diisopropylethylamine; DIC, N,N′-diisopropylcarbodiimide; HONB, Nhydroxy-5-norbornene-2,3-dicarboxylimide; CTX, cytotoxicity; SI, selectivity index; hRBC, human red blood cell; CLSM, confocal laser scanning microscopy; PI, propidium iodide; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2phenylindole; Amp B, amphotericin B; FLC, fluconazole; SEM, scanning electron microscopy; HRTEM, high resolution transmission electron microscopy; RP-HPLC, reverse-phase high pressure liquid chromatography

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REFERENCES

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