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Article Cite This: J. Med. Chem. 2018, 61, 10793−10813

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Structure−Activity Relationships of Photoswitchable DiaryletheneBased β‑Hairpin Peptides as Membranolytic Antimicrobial and Anticancer Agents Oleg Babii,† Sergii Afonin,† Aleksandr Yu. Ishchenko,‡,§ Tim Schober,∥ Anatoliy O. Negelia,⊥ Ganna M. Tolstanova,⊥ Liudmyla V. Garmanchuk,⊥ Liudmyla I. Ostapchenko,⊥ Igor V. Komarov,*,‡,§,# and Anne S. Ulrich*,†,∥ †

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), POB 3640, 76021 Karlsruhe, Germany Institute of High Technologies, Taras Shevchenko National University of Kyiv, Vul. Volodymyrska 60, 01601 Kyiv, Ukraine § Enamine Ltd., Vul. Chervonotkatska 78, 02066 Kyiv, Ukraine ∥ Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany ⊥ Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Prosp. Hlushkova 2, 03022 Kyiv, Ukraine # Lumobiotics GmbH, Auerstraße 2, 76227 Karlsruhe, Germany

J. Med. Chem. 2018.61:10793-10813. Downloaded from pubs.acs.org by 95.181.176.155 on 01/01/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Five series (28 structures) of photoswitchable β-hairpin peptides were synthesized based on the cyclic scaffold of the natural antibiotic gramicidin S. Cell-type selectivity was compared for all activated (diarylethene “ringopen”) and deactivated (“ring-closed”) forms in terms of antibacterial activity (MIC against Escherichia coli and Bacillus subtilis), anticancer activity (IC50 against HeLa cell line), and hemolytic cytotoxicity (HC50 against human erythrocytes). Correlations between the conformational plasticity of the peptides, their hydrophobicity, and their bioactivity were also analyzed. Considerable improvements in selectivity were achieved compared to the reference compound. We found a dissociation of the anticancer activity from hemolysis. Phototherapeutic indices (PTI), HC50(closed)/MIC(open) and HC50(closed)/IC50(open), were introduced for the peptides as safety criteria. The highest PTI for HeLa-selective toxicity were observed among analogues containing hydroxyleucine on the hydrophobic face. For one compound, high PTIs were demonstrated across a range of different cancer cell lines, including a doxorubicin-resistant one.

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INTRODUCTION The scientific and commercial interest in bioactive peptides has been increasing over the recent years, as peptides provide a rich source of leads for the development of novel therapeutics.1,2 In particular, research and development of membrane-active peptides are gaining momentum3,4 after a series of setbacks in the late 1990s and at the beginning of the new millennium.5 There are several reasons behind this “renaissance”. First, problems caused by unfavorable pharmacological characteristics of peptides, such as low in vivo stability and poor oral availability have been solved, and rational design principles enabling optimization of the ADME parameters have also been developed.6−8 Second, advancements in synthetic methodology and the upscaling of synthetic procedures have allowed production of peptides in sufficient amounts at competitive prices, which has opened the door to cost-efficient clinical trials of peptide drug candidates and manufacturing of the drugs. Third, the rapid spread of © 2018 American Chemical Society

antibiotic-resistant bacterial infections, the gap in the commercial development of antibiotics, as well as chemotherapy-induced drug resistance of advanced tumors have prompted pharmacologists to revive efforts toward developing membrane-active peptides because they could potentially address all these issues. Considering the challenges posed by acquired drug resistance, the major regulatory authorities undertook measures to facilitate peptide drug approval.9,10 Marketing approval of peptide-based drugs has indeed increased in recent years.11 However, despite this progress, several unresolved problems remain, such as limited selectivity in the delivery of a peptide to its site of action and the high systemic toxicity of intrinsically cytotoxic peptides. Coping with these issues demands novel approaches and strategies for the design of peptide drugs.12,13 Received: September 13, 2018 Published: November 19, 2018 10793

DOI: 10.1021/acs.jmedchem.8b01428 J. Med. Chem. 2018, 61, 10793−10813

Journal of Medicinal Chemistry

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Figure 1. (A) Schematic structure of gramicidin S (GS) (left) and its 3D molecular model (right); residue numbering (Orn = ornithine) with conformational elements (left), and amphipathicity of the two faces of GS (right: yellow, hydrophobic; blue, cationic; green, proline residue). (B) Mechanism of GS pore formation in lipid bilayers.30 (C) Schematic structure of light-controllable analogue 2 and reversible interconversion between its two thermally stable photoforms. The DAE-containing photoswitching fragment is shown in red and framed.

selectivity of local therapeutic action and reduced overall systemic toxicity, leading to significant improvements in safety. The idea of using light for local activation of therapeutic agents dates back to the beginning of the last century.21 A treatment modality called photodynamic therapy (PDT) was introduced in clinical practice in the mid-1950s, mainly in oncology,22,23 and is also being explored for the treatment of localized infectious diseases.24 PDT is based on the generation of cytotoxic singlet oxygen by light with the aid of photosensitizers. As a more recent alternative, reversibly photoisomerizable (“photoswitchable”) bioactive compounds are being developed toward actual practical applications in medicine,25 although their pharmacological properties still need to be improved further. In this paper, we present the results from our comprehensive attempt to establish a rational basis for such an improvement, based on photoswitchable analogues of the natural antimicrobial peptide gramicidin S (GS, 1, Figure 1), by performing an extensive structure− activity relationship studies.

Membranolytic peptides offer unique opportunities to combat drug-resistant microbial infections3,4 as well as cancers,14−17 but their high toxicity has always been the major hurdle to the marketing approval. Several promising peptidic anticancer drug candidates, e.g., of marine origin,18,19 failed at late stages of development due to their excessive toxicity. Only a few membrane-active peptides are used in clinics to date, and these compounds are limited to topical applications due to the life-threatening cytotoxic side effects upon systemic administration. A novel way to cope with the problems caused by such unwanted systemic toxicity entails the conversion of a drug into a light-controllable analogue and its use as a photopharmacological20 agent. Light-controllable drugs can be administered in the inactive, less toxic form and then activated by light only when and where required for treatment. The activation by light can be achieved with very high spatiotemporal precision; therefore, light-controllable drugs may have favorable pharmacological properties, such as high 10794



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better photocontrol of its anticancer and antibacterial activity, and especially to lower the toxicity of the closed isomers, aiming toward the treatment of cancer and/or localized infectious diseases.

Gramicidin S has been known for more than 75 years; it was one of the first antibiotics discovered.26 Its structure was thoroughly analyzed by X-ray,27,28 NMR,29−31 and cold-ion spectroscopy in the gas phase.32 These studies revealed that the decameric sequence cyclo[VOLfP]2 (O, ornithine; f, Dphenylalanine) of GS forms asymmetric macrocycle, consisting of one short antiparallel β-pleated segment flanked by type-II′ β-turns. This arrangement provides the GS molecule with two distinct “sides,” a hydrophobic face and a cationic face (Figure 1A), which persist in solution and in the membrane-bound state. GS has demonstrated excellent potency against a wide range of Gram-positive microorganisms (including drugresistant strains and biofilms).33 In combination with other antibiotics, it revealed notable synergy of therapeutic action, expanding the potency to Gram-negative bacteria.34 A cytotoxic activity of GS against cancer cells was also shown, and its antitumor efficacy was demonstrated in vivo.35,36 Despite its use for many decades (e.g., in over-the-counter throat lozenges), no acquired resistance against GS has ever been reported. This can be attributed to its rather unspecific mechanism of action against multiple cellular targets: GS exerts its initial and main cytotoxicity by destabilizing plasma membranes via the formation of transient pores (Figure 1B). These pores cause membrane depolarization, disturb the cellular homeostasis, and can lead to cell lysis.37−39 Additional contributions of GS interactions with several other membraneassociated and intracellular targets to prokaryotic cell death have also been demonstrated.40,41 Therefore, the development of any resistance to GS should require multiple simultaneous nonlethal changes in the target cell, which is highly improbable. Despite this potential, GS is used as a drug only for topical treatment, as it turned out to be too toxic for systemic administration.26 Since its discovery, significant efforts have been made toward decreasing the side toxicity by structural modifications of the GS molecule. Several papers reported significant improvement in the therapeutic index for GS analogues as antibacterial agents (we refer the readers to an account and a recent review in this field42,43), yet these efforts still have not yielded suitable drug candidates for systemic use. In our previous work,44,45 we incorporated a photoisomerizable diarylethene (DAE) fragment into the cyclic backbone of the GS molecule and obtained a number of analogues that existed as two thermally stable isomers, reversibly convertible into each other by light of different wavelengths. By replacing two amino acid residues in one βturn of the GS scaffold with a DAE-derived fragment, we could prepare and interconvert the “ring-open” (referring to the inner DAE cycle) and the “ring-closed” photoforms (an example is shown in Figure 1C). Comparative studies of these two isomers demonstrated differences of 1 order of magnitude in their cytotoxic activity against bacteria (4−16 times difference of the two photoforms in their minimum inhibitory concentration, MIC), as well as against human erythrocytes (3−11 times difference in concentration causing 50% lysis, HC50). For example, the “ring-closed” isomer 2(closed) has 5− 8 times lower cytotoxicity (in terms of IC50) against several human cancer cell lines compared to the corresponding “ringopen” isomer 2(open) (Figure 1C). Importantly, the more active form 2(open) could be generated in vivo by red light (637−664 nm), which penetrate as deep as ∼1 cm into the tissue, showing an appreciable antitumor efficacy in a rodent cancer model.45 These encouraging results prompted us to optimize the light-controllable GS analogues further, to achieve

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RESULTS SAR Approach and Overall Strategy. In previous work,44,45 we varied the structure of the DAE fragment in the photoswitchable GS analogues to achieve optimal chemical and photophysical characteristics, namely an efficient photoconversion from the less active (closed) isomer into the more active (open) isomer, thermal and chemical stability of both isomers, and a bathochromic shift of the light that generates the active isomer (for better tissue penetration). We also screened different positions of the DAE fragment within the βturn of the GS scaffold to find the best architecture for a pronounced change in activity upon photoisomerization. The original, optimally performing (hit) compound 2 (Figure 1C) has been taken as a reference for the present study. For the systematic SAR analysis in this work, we synthesized 27 modified analogues of 2 and purified each of them in the thermally stable “ring-open” and “ring-closed” forms by HPLC. The resulting compounds (both photoforms) as well as the original hit (compound 2) were screened against two representative bacterial strains, Gram-positive (G+) Bacillus subtilis and Gram-negative (G−) Escherichia coli, and against the commonly used human epithelial cancer cell line HeLa (protection from light was maintained wherever possible to avoid uncontrolled photoisomerization). Standard colorimetric tests were used to obtain MIC values (for bacteria) and IC50 values (concentration to inhibit 50% of cell growth of HeLa culture) as measures of the respective biological activities of the tested compounds. To obtain a measure of the toxic side effects of these compounds against noncancerous human cells, we performed a standard hemolysis assay and determined the HC50 values (concentration causing 50% lysis of suspended human erythrocytes compared to the action of 0.1% Triton X100). This is only a crude approximation, as the toxicity of a chemical compound in vivo is caused by a multitude of different processes,46 but is a justified measure because hemolysis was reported to be the main cause of systemic toxicity of the parent compound, GS.47 Five series of GS analogues were synthesized; the corresponding structures are shown in Figures 2−6. For each isolated photoform, we determined not only the cytotoxic activity but also their retention times on a C18 reverse-phase HPLC column under identical conditions in order to monitor trends in the hydrophobicity−hydrophilicity balance as described earlier.48 Conformational plasticity was also monitored in both aqueous and membrane-mimicking isotropic environments (trifluoroethanol solutions) by recording far-UV circular dichroism spectra for each compound. The spectra of the purified “ring-closed” isomers were collected in darkness, and the same samples were remeasured after complete photoconversion to the “ring-open” forms. When designing derivatives of our initial hit, we advantageously used the results of numerous SAR studies that are available in the literature on the parent molecule GS.36,42,43,49−67 These studies had been performed in vitro using assays similar to the ones we chose for our study. All previous studies had been aimed at improving the antibacterial activity of GS and decreasing its hemolytic toxicity. No systematic studies comparing the in vitro anticancer activity of 10795



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Figure 2. Photoswitchable gramicidin S analogues with varying ring size of the β-sheet fragment compared to that in 2, with an inversion of the hydrophobic/hydrophilic faces and with point mutations (indicated in blue) leading to a variation in overall hydrophobicity. The photoswitching fragment (DAE; see Figure 1C) is schematically shown in red. N-Me = Nα-methyl.

sure that the molecule has a sufficiently high overall hydrophobicity. Structural considerations led to the conclusion that cyclic peptides would form stable β-hairpins only if they contain an even but not an odd number of amino acid residues.68 Residues with high β-conformational propensities (Val, Ile, Thr, Phe, Tyr, and Trp) should be preferably placed within the β-strands.69 It also turned out that delicate changes in the composition, leading to a modulation of amphipathicity and overall hydrophobicity of the parent molecule, generated more selective antibacterial GS analogues. With respect to ring size, it was shown that shortening the peptide backbone to less than eight amino acid residues abolished any cellular toxicity, while increasing the size to 12 or 14 residues resulted in a significant rise in nonselective toxicity.50 Dissociation of the antimicrobial activity on the one hand and the cytotoxicity against human erythrocytes on the other hand was achieved in these studies by further optimizing the overall hydrophobicity−hydrophilicity balance, using point mutations with D-amino acids within the β-sheet fragment of a 14-mer peptide.

GS, and its analogues, however, have been performed before our work. In the previous studies cited above, most of the GS structural variations resulted in decreased overall (and nonselective) cytolytic action, but some of the derivatives demonstrated equal or even higher antimicrobial potencies than the parent compound. More importantly, it was proven feasible to dissociate the bacteriolytic and hemolytic activities and thereby eventually to improve the antibacterial selectivity.42,51,52,56−58,61,63−66 These studies are important milestones toward clinical applications of GS-based antibacterial drugs, as the researchers identified key factors that had a positive influence on the antimicrobial action of the GS analogues and their cytotoxic activity against noncancerous human cells. It was shown that to maintain high membranolytic activity, it is important to (1) preserve the β-sheet-like structure within the cyclic molecule, at least when it is bound to cell membranes, (2) maintain the net positive charge and amphipathic “sidedness” of the GS molecule, and (3) make 10796



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Table 1. Biological Activities and Selectivity Indices (SI) for the First Series of GS Analogues (3−8) Compared with the Original Hit, Compound 2i

a

MIC(G−), MIC against Gram-negative E. coli. bMIC(G+), MIC against Gram-positive B. subtilis. cIC50, concentration causing 50% viability loss of HeLa cells. dHC50, concentration causing 50% lysis of human RBC. eSIG−, selectivity index for Gram-negative bacteria vs RBC, HC50/MIC(G−) ratio. fSIG+, selectivity index for Gram-positive bacteria vs RBC, HC50/MIC(G+) ratio. gSIC, selectivity index for cancer cells vs RBC, HC50/IC50 ratio. hMeasurements were performed using mg/mL concentrations, and toxicity values were recalculated to μM. 2-fold dilution method was used for the measurements; no curve fitting was performed, therefore, accuracy of the values is within a factor of 2. iThe SI values are highlighted in gray if equal to that of the corresponding photoform of 2, marked green if improved, and pink if inferior. For each parameter, the best values within the series, including the highest toxicities and lowest hemolysis, are shown in bold.

in the types of residues flanking the photoswitchable fragment: cationic Lys in the case of 3, and a nonpolar amino acid in the case of 4. The three 14-mer peptides (nonphotocontrolled variants had been described in the literature42,56) contained four Lys residues. Finally, one of the peptides in this series also contained an N-methylated Lys residue at the β-turn in place of proline.70 Within our series of 14-mers, we further varied the amphipathicity using point mutations, i.e., of L-Lys to D-Lys (compound 6), by swapping two neighboring Lys and Leu residues so that Lys is moved to the hydrophobic face of the scaffold while Leu entered the polar face (7) or by introducing an additional cationic amino acid within the β-turn region (8). Experimental cytotoxicity data for the first series of GS analogues and their corresponding selectivity indices (SI) toward Gram-positive and Gram-negative bacteria and cancer cells are shown in Table 1. Increasing the ring size seems to be beneficial in terms of selectivity: the 12-mer 4(open) and 14-mer 5(closed) demonstrated improvements in all three SI values (SIG−, SIG+, SIC). For the 12-mers (3 and 4), an overall decrease in the toxicity against eukaryotic cells was observed, whereas the 14-mers 5−8 generally possessed lower IC50 and HC50 values. The 14-mer 5 showed an extremely low HC50 of 0.1 μM in the open form. This was accompanied by a moderate to significant rise in antibacterial action. Its anticancer selectivity, however, is not better than that of the reference compound (in terms of SIC, only the single analogue 4(open) exceeds the selectivity index of the corresponding photoform of 2, all other peptides being inferior). In general, increasing the ring size of the GS analogues to 14 amino acid residues had a deteriorating effect

This approach yielded analogues that retained the high antimicrobial activity of GS while offering 7- to 38-fold higher antibacterial selectivity (measured as the concentration causing 100% hemolysis divided by MIC, HC100/MIC).42,56 This breakthrough was followed by a series of reports that achieved a moderate to high (up to 10-fold) improvement in the antibacterial therapeutic index (HC100/MIC or HC50/MIC) compared to that of GS by other structural changes. Namely, beneficial effects on the antibacterial selectivity were observed upon making alterations such as introducing positively charged moieties or aminoacylated residues at one57,64 or both of the Pro sites,65 by replacing D-Phe at the β-turn with parasubstituted D-Phe analogues59 or with pyridine-containing dehydroamino acids,58 by increasing the net charge in “inverted” GS analogues containing four positively charged and two β-branched bulky hydrophobic amino acid residues in the β-sheet fragment,61−63 by N-methylation of the backbone at the β-strand and β-turn regions,66 and by replacing the βturn with hydroxy- and oxygen-containing mimics.43,67 Selectivity in the Cytotoxic Action of the Photoswitchable GS Analogues. In the first series of our photoswitchable GS analogues (Figure 2), we varied the macrocycle ring size. According to the β-sheet periodicity rule,51 we synthesized 12- and 14-mer peptide analogues (counting the DAE-fragment as an equivalent of two amino acid residues). Analogues 3−8 were designed to be able to adopt the β-hairpin structure that is crucial for biological activity. The two 12-mer peptides (3 and 4) contain four cationic side chains (Lys), similar to the nonphotocontrollable GS analogues described in the literature.42,56 They differ only 10797



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Figure 3. Second series of gramicidin S analogues with point mutations affecting amphipathicity. Modifications, compared to 2, are highlighted and listed in blue, and the DAE-containing fragment is schematically shown in red.

Table 2. Biological Activities and Selectivity Indices for the Second Series of GS Analogues (9−12) Compared with the Original Hit, Compound 2a

a The SI values are highlighted in gray if equal to that of the corresponding photoform of 2, marked green if improved, and pink if inferior. For each parameter, the best values within the series, including the highest toxicities and lowest hemolysis, are shown in bold. MIC(G−), MIC against Gramnegative E. coli. MIC(G+), MIC against Gram-positive B. subtilis. IC50, concentration causing 50% viability loss of HeLa cells. HC50, concentration causing 50% lysis of human RBC. SIG−, selectivity index for Gram-negative bacteria vs RBC, HC50/MIC(G−) ratio. SIG+, selectivity index for Gram-positive bacteria vs RBC, HC50/MIC(G+) ratio. SIC, selectivity index for cancer cells vs RBC, HC50/IC50 ratio. Measurements were performed using mg/mL concentrations, and toxicity values were recalculated to μM. 2-fold dilution method was used for the measurements; no curve fitting was performed, therefore, accuracy of the values is within a factor of 2.

on the differences in activity between the corresponding “ringopen” and “ring-closed” isomers (i.e., on the efficiency of photoswitching the biological activity). For some analogues (5 and 8), we even observed a “reversal” in the antibacterial activity upon photoswitching (comparing to 2 and other analogues), where the ring-closed isomers were more active than the ring-open ones. However, for the eukaryotic cells, the ring-open isomers were always more toxic. It should be noted that the “reversal” is highly undesirable if one envisions application of the compounds for therapy because the more active isomer in this case is generated by UV light, which is toxic per se and has much lower tissue penetration depth.

The second series of analogues (Figure 3) was designed by introducing point mutations into the original hit 2 in order to investigate the influence of the hydrophobicity−hydrophilicity balance on the activity profile. In this series, the hydrophobicity was varied in compounds 9−11 without changing the “sidedness” of the molecules. Mutant 9 has Pro changed to LysN‑Me and contains two extra methylene units in both cationic side chains to compensate for the net charge increase. The aliphatic residues Leu and Val in the original hit were mutated to Ala, giving 10 and 11. As a control, a hydrophobic valine residue was replaced with charged Orn in 12. The 10798



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Figure 4. Third series of gramicidin S analogues with point mutations to affect the polarity and stability of the β-hairpin. Modifications, compared to 2, are highlighted and listed in blue, and the DAE fragment is schematically shown in red. AsniPr = Nγ-isopropyl-asparagine; DabiBu = Nγisobutyryl-diaminobutyric acid; and Adm = adamantylglycine.

results of the cytotoxicity measurements for these analogues are summarized in Table 2. The results clearly show that reducing the overall hydrophobicity leads to a decrease in both the hemolytic and Grampositive antibacterial activities (see the data for compounds 10 and 11), as was described for nonphotocontrollable GS analogues.43,51 Notably, alongside hemolysis, the anticancer activity also decreased proportionally. For peptides 9 and 12, a congruent increase in the nonselective antimicrobial activity

was observed (compare also to 8), suggesting that higher net charge plays an important role in this type of toxicity. With the exception of compound 11, the efficiency of photoswitching the biological activity in this series was less than that for the hit compound 2. Further mutations of the original hit were performed based on literature data, mainly paying attention to the polarity of the residues adjacent to the DAE-unit. In our third series (Figure 4), the two cationic ornithine residues on the polar face of the 10799



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Table 3. Biological Activities and Selectivity Indices for the Third Series of GS Analogues (13−20) Compared with the Original Hit, Compound 2a


for biological activity, D-Phe was replaced with a D-Pro to generate an “ideal” β-turn,70 and both valine residues were replaced by Ile. The Val/Ile exchange in this case was performed to compensate for the loss of hydrophobicity caused by the Phe/Pro mutation. Other analogues in this series were peptides 19 and 20 with inverted polar and nonpolar faces, each bearing two 1-adamantane residues and a high net positive charge. Similar “inverted” GS analogues were reported to have low hemolytic activity while maintaining a strong antimicrobial effect.61,63 However, their anticancer activity has not been studied so far. The results of screening this series are shown in Table 3. Compounds 13 and 14 maintained high activities compared to that of the original hit, but they demonstrated decreased photoswitching efficiency against eukaryotic cells, which depreciates all of the tested selectivity indices. In 13, the introduction of Arg instead of the original ornithine residues led to a strong increase in the hemolytic activity of the 13(open) isomer. In 14(closed), substitution of two Leu residues with Tyr had a clear deteriorating effect; an increase in hemolysis was accompanied by substantial loss of anticancer activity. Compounds 19 and 20 bearing the adamantane side chains also had inferior characteristics, contrary to the expectations based on the literature results.61,63 In addition, they demonstrated highly unwanted “inverted” antibacterial photoswitching, their closed forms being more active than their open forms, possibly due to the enormous steric bulk that was

scaffold were replaced by Arg, giving peptide 13. Arginine was selected because many natural β-structured anticancer peptides, such as tachyplesin and gomesin, have Arg-rich sequences.17 In 14, the two leucine residues were replaced with Tyr, while in 15 they were replaced with a Val and Thr each. These mutations aimed at screening the nonpolar face of the peptide by introducing slightly less hydrophobic amino acids than in the original hit compound. Again, the choice of Val and Thr was based on their abundance in natural peptides with reported anticancer activity.17 Analogues 16 and 17 were designed with the intention to stabilize the secondary structure. It is known that an extended β-strand conformation is stabilized by the presence of β-branched amino acids,69 and β-sheets can be further stabilized by intrastrand noncovalent interactions. These intrastrand interactions can be achieved by introducing amino acid residues containing hydrogen bond donors/acceptors (such as Ser, Thr, Asn, or Gln) or by installing an oppositely charged pair to form a salt bridge. On the basis of our data for the second series, however, we reasoned that the introduction of highly polar residues on the hydrophobic face of the peptide would reduce its hydrophobicity and cause a loss of overall activity. To compensate for this effect, we introduced Nγ-isopropyl-asparagine and Nγisobutyryl-diaminobutyric acid, both of which containing additional aliphatic substituents, affording the new GS analogues 16 and 17, respectively. In 18, to elucidate whether the presence of the original D-phenylalanine residue is critical 10800



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Figure 5. Fourth series of gramicidin S analogues with N-alkylation and hydroxylation mutations. Modifications, compared to 2, are highlighted and listed in blue, and the DAE fragment is schematically shown in red. LeuOH = hydroxyleucine; N-Bu = Na-butyl.

activity in the open photoforms. Hence, they have good photoswitching efficiency and are promising candidates for further development. Compound 18 showed slightly improved selectivity indices compared to those of original hit for both photoforms against all cell types, suggesting that the aromatic benzyl group is not essential for the biological activity of gramicidin S derivatives. The fourth series of peptides (Figure 5) was synthesized with the intention of probing the influence of backbone Nalkylation and proline replacements, which affect the β-hairpin stability, and hydrophobicity was subtly modulated by introducing hydroxy-containing residues into the original hit sequence. All these modifications frequently occur in natural

attached rigidly to the peptide backbone. This result corroborates our data for compounds 9 and 12 (second series). Taken together with the results described above (comparing the 12-mers 3 vs 4 and the 14-mers 5 vs 8), these findings strongly suggest that an increase in the net positive charge rather than the charge density leads to an overall increase in toxicity and to a reduction in any cell-type selectivity. In contrast, a subtle increase in the polarity of the residues on the hydrophilic face of the molecule, together with a stabilization of the β-sheet, resulted in a significant increase in the selectivity indices, especially for the antibacterial action. Compounds 15−17 showed very low hemolytic activity in the closed photoforms, yet, at the same time, high antibacterial 10801



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Table 4. Biological Activities and Selectivity Indices for the Fourth Series of GS Analogues (21−27) Compared with the Original Hit, Compound 2a

a

The SI values are highlighted in gray if equal to that of the corresponding photoform of 2, marked green if improved, and pink if inferior. For each parameter, the best values within the series, including the highest toxicities and lowest hemolysis, are shown in bold. MIC(G−), MIC against Gramnegative E. coli. MIC(G+), MIC against Gram-positive B. subtilis. IC50, concentration causing 50% viability loss of HeLa cells. HC50, concentration causing 50% lysis of human RBC. SIG−, selectivity index for Gram-negative bacteria vs RBC, HC50/MIC(G−) ratio. SIG+, selectivity index for Gram-positive bacteria vs RBC, HC50/MIC(G+) ratio. SIC, selectivity index for cancer cells vs RBC, HC50/IC50 ratio. Measurements were performed using mg/mL concentrations, and toxicity values were recalculated to μM. 2-fold dilution method was used for the measurements; no curve fitting was performed, therefore, accuracy of the values is within a factor of 2.

biologically active peptides, especially in those synthesized nonribosomally.71,72 Alkylation of exposed NH groups, apart from the influence on amphipathicity, was reported to enhance the passive permeability of cyclic peptides by abolishing backbone aggregation and by increasing conformational rigidity through limiting rotation of the ϕ and ψ angles.73,74 As demonstrated before, this might increase the cell permeability and enhance the therapeutic index of the GS analogues.66 The hydroxyleucine derivative was introduced with the intention of moderately decreasing the overall hydrophobicity, which had been shown to substantially lower the hemolytic activity in the previous series. In addition, the hydroxyl group could rigidify and stabilize the β-sheet in the open isomers by forming interstrand hydrogen bonds, which would not be stable in the closed isomers. Hypothetically, this might further increase the difference in activities between the two photoforms and thereby improve the corresponding therapeutic indices. The formation of interstrand H-bonds was recently reported for hydroxy-containing cyclic β-hairpin peptides.75 The first sign of a beneficial influence of hydroxysubstituted amino acid residues within the β-sheet was already mentioned above in the analysis of our third series of peptides (compound 15). Analysis of the toxicity data (Table 4) allowed us to make the following conclusions. Backbone N-methylation (compound 21) improved antibacterial selectivity mostly by decreasing the toxicity against eukaryotic cells. The selectivity indices of the peptidomimetics with β-structure stabilization and Pro-substitution (22,23) did not improve much. An

increase in their antieukaryotic activity of the closed photoforms was notable, but unfortunately this was observed for both cell types. At the same time, we were pleased to find that introduction of the hydroxy-containing side chains was beneficial, primarily for efficiently photoswitching the biological activities. A single hydroxyleucine residue in the amino acid sequence (compounds 24−26) seems to be better than two (compound 27). The best position of hydroxyleucine within the backbone is seen in compound 25. In these hydroxyleucine containing compounds, improvement in both the antibacterial and anticancer activity was observed, the efficiency of the photoswitching was high, and hemolysis low, in accordance with our hypothesis. It was of interest to find out whether the high cytotoxic activity and photoswitching efficiency of compound 25 would be retained across a broad range of cancer cell lines, including drug resistant ones. We thus determined IC50 values of 25 against the human hepatocellular carcinoma cell line HepG2, the human breast cancer cell line MCF-7, the human pancreatic cancer cell line MIA PaCa-2, against the doxorubicin-resistant human lung adenocarcinoma cell line MOR/0.2R, as well as the nonresistant parent cells MOR. The corresponding cytotoxicities of our initial hit, compound 2, were taken as the control. The obtained data are listed in Table 5. As can be seen from Table 5, compound 25 is highly toxic in its open form against all of the studied cancer cell lines, regardless whether they are drug-resistant or not, and its closed form is much less toxic than the open form. 10802



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positive bacteria, and we implemented mostly those design principles from the literature that had been developed using sensitive, i.e., Gram-positive strains. Assuming that the main mechanism of action of GS and its analogues is the destabilization of cellular membranes, a direct correlation between antibacterial activity and cytotoxic activity against HeLa cancer cells was expected. This expectation is based on the known fact that cancer cells expose a higher density of anionic lipids and other negatively charged molecules on their plasma membrane exterior compared to noncancerous eukaryotic cells.76,77 In this respect, they resemble bacteria, for which the highly negative ζ-potential has been recognized as a hallmark. Binding of our cationic GSderived molecules to model lipid membranes was shown to be mediated primarily by electrostatic interactions with negatively charged lipids.78 Hence, cancer cells, similar to prokaryotic cells, should be more sensitive to the membranolytic action of GS and its analogues than healthy eukaryotic cells, whose outer membranes are enriched in zwitterionic lipids. In our previous studies, we had explored this aspect experimentally by designing the original light-controllable analogue 2(open): its cytotoxicity against cancerous HeLa cells was indeed at least twice as high as that against mouse aortic endothelial cells.45 Interestingly, in the current comprehensive study, we could not confirm this expectation, as no direct correlation between antibacterial and anticancer activity was found. This result, together with the observed strong correlation between the anticancer and hemolytic activities, suggests either a stronger association of membranolytic activity with the eukaryotespecific lipid composition (e.g., presence of cholesterol), or the involvement of additional molecular mechanisms (other than

Table 5. Cytotoxicities against a Set of Cancer Cell Lines, Measured for Compounds 2 and 25 IC50 (μM) peptide (photostate) 2(open) 2(closed) 25(open) 25(closed)

MCFHepG2 7 5 75 5 100

5 50 6 150

MIA-PaCa-2 6 50 6 150

MOR MOR/0.2 3 50 5 150

3 50 5 100

HeLa 5 41 9 150

Finally, we prepared a fifth series of analogues, consisting of dimers 28 and 29 (Figure 6), to probe for cooperativity in the cytotoxic action. The two fragments of cyclic GS derivatives were connected by a flexible linker in two different alignments, using click-chemistry. Table 6 lists the experimental data for these dimers. One of these dimers, compound 28, demonstrated better characteristics than the reference hit 2, mainly due to lower hemolytic activity of the closed photoform. In contrast, the other mode of monomer connection (compound 29) led to a decrease in all SI values due to increased hemolysis. Analyzing the whole set of the experimental data, we noted several general trends in the cytotoxicity selectivity. First, the net charge, irrespective of the photostate of the analogues, has the strongest impact on modulating cell type selectivity: the overall toxicity and any difference between cell types decreases upon increasing the number of cationic substituents. Second, regarding the antibacterial activity, most of our analogues are good candidates only for the treatment of infections caused by Gram-positive bacteria. This is not surprising, as we modified the gramicidin S, which is intrinsically selective toward Gram-

Figure 6. Fifth series, consisting of homodimeric gramicidin S analogues. Modifications, compared to 2, are highlighted and listed in blue, and the DAE-containing fragment is schematically shown in red. 10803



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Table 6. Biological Activities and Selectivity Indices for the Fifth Series, Consisting of Dimeric Gramicidin S Analogues (28− 29), Compared with the Original Hit, Compound 2a


An ideal cytotoxic DAE-containing drug candidate for photopharmacology should have a low cytotoxicity in its ring-closed form and a high antibacterial or anticancer activity in the ring-open form. It would not be preferable, though still acceptable, if the hemolytic activity of the “ring-open” form is also high. As we have shown before,45 the less toxic (closed) form of the photoswitchable compound is activated only at the site of the lesion, hence the rest of the organism remains unaffected by the highly cytotoxic (open) isomer. To illustrate this effect, we define and introduce here two phototherapeutic indices (PTI): HC50(closed)/MIC(open), and HC50(closed)/ IC50(open). These indices should be maximized in the hit-tolead optimization in order to find the best antibacterial or anticancer drug candidates, respectively. The phototherapeutic indices calculated from the above data are listed in Table 7. Significant improvement compared to conventional selectivity indices (SI) is now observed, with clear photoswitchable leads: compound 24 is most suitable as an antibiotic against Grampositive bacteria (9-fold better than the initial hit and 30-fold better than the parent GS), and compound 25 promises the best anticancer activity in being selective toward HeLa cells (2and 7-fold better than 2 and GS, respectively). Notably, both of these compounds contain a hydroxyleucine residue, which appears to enhance the general efficiency of photoswitching any of the measured biological activities. The improvements in the therapeutic indices brought about by the photoswitching are clearly visible in the plots where the activities of the “ring-open” isomers are plotted against the hemolytic activities of the corresponding “ring-closed” isomers. Now, the points for almost all of the compounds fall below the diagonal, i.e., in the area where safety is enhanced. A clear improvement is seen for all activities (Figure 7B,D,F). The farther the distance of a compound to the diagonal in the lower right triangle, the better is its therapeutic window in terms of selectivity and safety. Conformational Plasticity of the Photoswitchable GS Analogues. As seen from the conformational analysis by CD (Figure 8, and Supporting Information, Figures S2−S6), significant conformational plasticity is evident for all of the studied GS analogues, both when moving from an aqueous to a membrane-mimicking environment and upon changing their

membranolytic effects) being more pronounced in the toxic action against prokaryotic cells. The above conclusions are in line with our finding that both of the compounds studied in more detail, 2 and 25, show a very similar toxicity in their open forms against a broad range of different cancer cell lines, including drug resistant ones, while their closed forms are much less cytotoxic (Table 5). Safety of Photoswitchable GS Analogues. Plotting MIC or IC50 values against HC50 allowed an easy visual assessment of the general trends in the safety of our peptides. As noted above, hemolysis was shown to be the main cause of the in vivo toxicity of GS and its analogues, so the safest candidates can be found in the lower-right corner of these plots, where the antibacterial or anticancer activity is high and hemolysis is low. Figure 7 (7A,C,E) summarizes all data measured in this study, as listed above in Tables 1−4 and 6. There is a clear difference in the activities of the GS analogues against Gram-positive and Gram-negative bacteria. For a representative of the former class, B. subtilis (Figure 7A), almost all data points are found below the diagonal, suggesting a relative safety of our compounds against Gram-positive species. Several analogues within the series [e.g., 4(open), 15(open), 16(open), 17(open), 27(open)] may be regarded as most promising. In contrast, the points in the MIC(G−)HC50 plot lie mostly above the diagonal, demonstrating that the GS-based compounds are not suitable for application against Gram-negative bacteria. Finally, in the IC50-HC50 plot (Figure 7E), the data points (especially for the closed photoforms) are grouped closer to the diagonal than in the case of both MIC-HC50 plots. Obviously, dissociation of the anticancer and hemolytic activity is a challenging task, and this is the very situation where photoswitching may improve the safety and reduce the side effects of a systemically administered drug.20 To illustrate the potential of photoswitching, we re-evaluated the data by taking into account the different activity and toxicity of the corresponding open and closed forms. Compared to previous SAR studies on GS analogues, our study thus addresses an additional parameter in the optimization, namely the ability to control the biological activity with light. 10804



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Figure 7. Toxicity of the GS analogues 2−28 correlated with their in vitro hemolysis (A,C,E) and their corresponding phototherapeutic potentials (B,D,F). Selective toxicity against Gram-positive (A) and Gram-negative (C) bacteria, and against HeLa (E) cells as determined in this study. Open circles represent (open) isomers, and filled circles represent (closed) isomers. Resultant phototherapeutic potentials for antibiotic activities against Gram-positive (B), Gram-negative (D) bacteria, and for anticancer cytotoxic action (F). For each peptide, the IC50 (open) values are plotted against HC50(closed). The compounds numbers are indicated next to the data points. Values for the parent GS (1) and the initial hit compound 2 are marked with diamonds, GS additionally highlighted with black.

photoforms. The spectral signatures of the closed forms resemble a random coil conformation with the most

pronounced negative CD band close to 200 nm, which decreases upon photoswitching. Thereupon, a negative CD 10805



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Table 7. Phototherapeutic Indices (PTI) for the Photoswitchable GS Analogues (2−29) Compared to the Natural Parent Peptide Gramicidin Se

a

PTIG−: phototherapeutic index against Gram-negative bacteria, HC50(closed)/MIC(G−)(open) ratio. bPTIG+: phototherapeutic index against Gram-negative bacteria, HC50(closed)/MIC(G+)(open) ratio. cPTIC: phototherapeutic index against cancer cells, HC50(closed)/IC50(open) ratio. d PTI values for GS were calculated from MIC(G−) of 28, MIC(G+) of 1.8, IC50 of 3.5, and HC50 of 10.5 μM, measured in control experiments under conditions identical to testing the open forms of the photoswitchable analogues. eThe PTI values are white if equal to GS, are marked green if improved, and are marked pink if inferior. For each parameter, the best values are shown in bold.

amphiphilic cyclic antibiotic gramicidin S. The experimental data allow us to draw the following conclusions: (i) photoswitching of the GS analogues has a beneficial effect on the safety of the compounds; (ii) increasing the ring size of the backbone from 10 to 14 amino acids (counting the diarylethene fragment as an equivalent of two residues) has a negative effect, as the hemolytic activity increases and the photoswitching efficiency decreases. The optimal ring size is 10 or 12 residues; (iii) significant alteration of the hydrophobicity−hydrophilicity balance impairs the therapeutic indices of the peptidomimetics; (iv) a subtle increase in the polarity on the hydrophilic face of the molecules with a concomitant insertion of hydrophobic residues on the same face is beneficial for the pharmacological characteristics of the peptidomimetics. For compounds containing hydroxyleucine as a side chain, hemolysis is particularly low, and the efficiency of photoswitching is high. (v) Dimerization of the cyclic peptides might be beneficial, as it lowers the hemolytic activity, but the mode of the ring connection significantly influences the cytotoxic activity; (vi) there is no correlation between antibacterial activity and cytotoxic activity against HeLa cancer cells within the studied GS analogues; different compounds were found as potential leads for anti-Gram-negative, antiGram-positive, and anticancer applications; (vii) some representative compounds were found to be active against a broad set of cancer cell lines, including a doxorubicin-resistant one.

signal between 215 and 225 nm becomes dominant, suggesting a conformational change of the peptidic backbone into a more β-pleated structure. The “structuring” toward the conformation of the parent compound GS is observed for all peptides upon photoswitching from the closed into the open photoform. This conformational change was expected to correlate well with the changes in cytotoxic activity, but this is only observed in the case of eukaryotic cells. For erythrocytes and HeLa cells, the open photoforms were indeed always more toxic than the closed ones. The change in antibacterial activity upon photoswitching, however, does not correlate with the changes in conformation. As noted above, we even observed an “inverted” toxicity change for some of the compounds (5, 8, 14, and 23 against B. subtilis; 8, 12, 19, and 20 against E. coli), where the more structured open forms demonstrated lower toxicity than the corresponding less structured closed forms. These facts confirm that the β-hairpin conformation of the amphiphilic GS analogues is a more dominant factor in the killing of eukaryotic cells compared to prokaryotic cells. Apparent Hydrophobicity and Activity of the Photoswitchable GS Analogues. We measured the retention time (RT) of all peptides on a reverse-phase HPLC column under identical conditions, as described in the literature,48 in order to assess their overall hydrophobicity and detect correlations with the biological activities. Such correlations had been reported for nonphotoswitchable GS analogues56 based on the notion that the stationary hydrocarbon C18 phase mimics biological membranes.79 The RT values would thus reflect the hydrophobicity-driven membrane affinity of the peptides and hence their potency in targeting lipid bilayers. Here, we observed a systematically lower hydrophobicity in every pair for the less structured closed than for the open photoisomers (Table S2 in the Supporting Information). Moreover, as seen in Figure S14 (see Supporting Information), a correlation between RT and both eukaryotic toxicity indices (IC50 and HC50) was observed, which was most pronounced for the closely related decamer variants (our second, third, and fourth series).

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EXPERIMENTAL SECTION

1. Chemical Synthesis, General. All chemicals and solvents were purchased from Sigma-Aldrich, Iris Biotech, ABCR, Fisher, Carl Roth, and Biosolve. Noncanonical amino acids Fmoc-Adm-OH, FmocLeuOH-OH, and Fmoc-OrnN3-OH were purchased at Iris Biotech. NFmoc protected diarylethene-based photoswitching building block (N-Fmoc-DAE, 4-(2-(5-((((9H-fluoren-9-yl)methoxy)carbonyl)-Lprolyl)-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-5-methylthiophene-2-carboxylic acid) was synthesized according to published procedures.45 Reversed-phase HPLC (RP-HPLC) for all new compounds was done on a Jasco system equipped with a diode array detector. The following columns and eluting conditions were employed for the peptides: Vydac (218TP) C18 (4.6 mm × 250 mm); column temperature, 40 °C; flow rate, 1.5 mL/min for

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CONCLUSION In summary, we have varied the structure of photocontrollable diarylethene-modified β-hairpin peptides, derived from the 10806



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Figure 8. (A) Illustration of the DAE photoisomerization and the corresponding conformational changes of the peptidic fragments in the photoswitchable GS analogues. (B) CD spectra of gramicidin S (left, water) and of the initial photoswitchable hit 2 [right: 2(closed) red, water; black, TFE; 2(open), blue, water; green, TFE]. (C) Selected (the five most intense ones) CD spectra in water of closed and open analogues, shown in the left and right panels, respectively. Characteristic bands (see text) are indicated by blue and red dotted lines.

Figure 9. Synthesis of Fmoc-AsniPr-OH building block. (a) HBTU/DIPEA in DMF, then isopropylamine; (b) TFA/DCM. analytical HPLC. Vydac (218TP) C18 (22 mm × 250 mm); column temperature, 40 °C; flow rate, 17 mL/min for preparative HPLC. Eluent A: 90% H2O, 10% acetonitrile, 5 mM hydrochloric acid. Eluent B: 10% H2O, 90% acetonitrile, 5 mM hydrochloric acid. Gradient slopes of 1% and 4% B/min for analytical and preparative HPLC were used, respectively. All the newly synthesized peptidic compounds have ≥95% purity according to analytical HPLC with UV detection (215 nm). Analytical 1H NMR spectra were recorded on a Bruker Avance 300 spectrometer and referenced to tetramethylsilane. Mass spectra for

peptide identification were recorded on a Bruker Autoflex III instrument, using MALDI-TOF. Analytic samples were cocrystallized on a Bruker stainless steel target with a matrix of 3,5-dihydroxybenzoic acid or α-cyano-4-hydroxycinnamic acid from acidic H2O/ acetonitrile solutions. For the synthesis of peptide 16, the required Fmoc protected amino acid Fmoc-AsniPr-OH was synthesized. The synthesis was performed in two steps (Figure 9) starting from commercially available Fmoc-Asp(OH)-OtBu as described below. 10807



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Table 8. Nomenclature, Sequences and Molecular Mass (Calculated and Determined by MALDI-TOF Mass Spectrometry) of the Synthesized Peptides (GS, 2−29) mol wt (cyclic sequence) [m/z] peptide

linear sequencea

calcd

measured

GS 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

fPVOLfPVOL DPro Sw-VOLfPVOL DPro Sw-KVKLfPVKLK DPro Sw-LKVKfPKLKV DPro Sw-VKLKVfPLKVKL DPro Sw-VKLKVfPLkVKV DPro Sw-VKLKVfPKLVKV DPro Sw-VKLKVf-LysN‑Me-LKVKL DPro Sw-VKLf-LysN‑Me-VKL DPro Sw-VOLfPVOA DPro Sw-AOLfPVOA DPro Sw-OOLfPVOL DPro Sw-VRLfPVRL DPro Sw-VOYfPVOY DPro Sw-TOVfPVOV DPro Sw-AsniPr-OVfPVOV DPro Sw-DabiBu-OVfPVOV DPro Sw-IOLpPIOL DPro Sw-O-Adm-OfPO-Adm-K DPro Sw-O-Adm-OfPO-Adm-T DPro Sw-VOV-PheN‑Me-PV-OrnN‑Me-V DPro Sw-IOIf-LeuN‑Me-LOI DPro Sw-IOLf-LeuN‑Bu-IOL DPro Sw-LeuOH-OVfPVOV DPro Sw-VOVfP-LeuOH-OV DPro Sw-VOVfPVO-LeuOH DPro Sw-LeuOH-OVfP-LeuOH-OV DPro Sw-LeuOH-OLf-OrnN3N‑Me-VOL (monomer) DPro Sw-OrnN3-OLfPVOL (monomer)

1141.447 1280.685 1565.083 1565.083 1777.371 1763.345 1763.345 1822.455 1353.822 1238.605 1210.552 1295.699 1364.765 1396.758 1254.604 1309.683 1323.710 1258.679 1480.964 1453.896 1280.685 1338.807 1380.887 1282.658 1282.658 1282.658 1312.684 2773.536 (dimer) 2681.399 (dimer)

1141.219 1280.128 1565.174 1565.185 1776.224 1762.205 1762.246 1822.245 1353.369 1238.097 1210.062 1295.124 1364.111 1396.633 1254.072 1309.322 1323.318 1258.514 1480.601 1453.592 1280.331 1338.375 1380.389 1282.341 1282.406 1282.405 1312.385 2773.057 2681.020

a Canonical amino acids are represented by one letter code. Lower case letters represent the amino acids with D-configuration. Noncanonical amino acids are presented by a three-letter code. Superscript indices N-Me and N-Bu, next to a three-letter abbreviation, indicate N-methylation and Nbutylation, respectively. LeuOH corresponds to (2S,3R)-β-hydroxyleucine; AsniPr, Nγ-isopropyl-asparagine; DabiBu, Nγ-isobutyryl-diaminobutyric acid; Adm, L-1-adamantyl-glycine; OrnN3, L-δ-azidoornithine; DProSw, DAE photoswitching fragment (4-(2-(5-((L-prolyl-2-methylthiophen-3yl)cyclopent-1-en-1-yl)-5-methylthiophene-2-carboxyl). Monomers were dimerized via propargyl ether by means of Cu(I)-catalyzed “click” reaction;

2. (S)-tert-Butyl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(isopropylamino)-4-oxobutanoate (Fmoc-AsniPrOtBu). Fmoc-Asp(OH)-OtBu ((S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid, 2 g, 4.85 mmol) was dissolved in 20 mL of DMF and combined under stirring with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 1.821 g, 1.05 equiv, 4.79 mmol) and DIPEA (N,N-diisopropylethylamine, 0.927 mL, 1.1 equiv, 5.34 mmol). The solution was stirred for 2 min, and then isopropylamine (0.412 mL, 1 equiv, 0.287 g, 4.85 mmol) was added. After 2 h, the solution was poured into water (100 mL) and extracted with ethyl acetate (3 × 50 mL). Organic extracts were dried over anhydrous Na2SO4. The solvent was then removed, and the crude material Fmoc-AsniPr-OtBu was crystallized from ethyl acetate/hexane mixture (1:1, v/v). Yield: 1.58 g (72%), white powder. 1H NMR (300 MHz, DMSO-d6) δ = 1.03 (t, 6H, 2 CH3), 1.37 (s, 9H, tBu), 2.41 (q, 1H, CH), 3.35 (qd, 2H, CH2), 3.82 (m, 1H, CH), 4.22−4.30 (m, 3H, CHCH2), 7.32 (t, 2H, aromatic), 7.42 (t, 2H, aromatic), 7.53−7.56 (d, 1H, NH), 7.69− 7.71 (d, 2H, aromatic), 7.74−7.76 (d, 1H, NH), 7.88−7.90 (d, 2H, aromatic). 3. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4(isopropylamino)-4-oxobutanoic Acid (Fmoc-AsniPr-OH). The O-tert-butyl protection in Fmoc-AsniPr-OtBu was removed with TFA/

DCM mixture (1:1, v/v, 10 mL, 30 min). The volatiles were removed on a rotary evaporator, and the obtained material was lyophilized from water/acetonitrile mixture (2:1, v/v, 100 mL), yielding 1.38 g of pure Fmoc-AsniPr-OH. 1H NMR (300 MHz, DMSO-d6) δ = 1.03 (t, 6H, 2 CH3), 2.43 (q, 1H, CH), 3.61 (broad, 2H, CH2), 3.80 (m, 1H, CH), 4.19−4.37 (m, 3H, CHCH2), 7.32 (t, 2H, aromatic), 7.41 (t, 2H, aromatic), 7.49−7.52 (d, 1H, NH), 7.69−7.71 (d, 2H, aromatic), 7.71−7.72 (d, 1H, NH), δ = 7.88−7.90 (d, 2H, aromatic). HRMS (ESI): calcd for C22H24N2O5 [M + H]+ 396.1685, found 396.1747. 4. Peptide Synthesis, Purification, and Characterization. Peptides were synthesized by automated solid phase peptide synthesis80 with an automatic peptide synthesizer BiotageSyro II. Double-coupling protocol (20 min/coupling step) with 4 equiv was used. Fmoc-protected amino acids were activated with HBTU and HOBt (1-hydroxybenzotriazole) using DIPEA in DMF. Fmoc deprotection in all cases was performed with 20% piperidine (20 min in DMF). The linear sequences were synthesized on a 2-chlorotrityl resin, preloaded with the first amino acid. Typical resin load was 0.5−0.8 mmol/g; the reaction scale was 0.2 mmol. For the synthesis of peptide 26, a LeuOH (β-hydroxyleucine)-preloaded 2-chlorotrityl resin was prepared according to the literature protocol.80 10808



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Coupling of the noncanonical amino acids was performed manually using 1.2 equiv of the Fmoc-protected amino acid, activated with 1.2 equiv of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), 1.2 equiv HOBt, and 2.4 equiv of DIPEA in DMF (0.5 mL DMF per 0.1 mmol of an Fmoc-protected amino acid). The N-methylation was performed on-resin following the published protocol.81 The free N-terminus was o-NBS (ortho-nitrobenzenesulfonyl-) protected using 5 equiv of o-NBS-Cl and 10 equiv of 2,4,6trimethylpyridine (sym-collidine) in NMP, rt, 15 min reaction time. Next, the N-terminus was methylated using either a Mitsunobu reaction-mediated method (5 equiv of triphenylphosphine, 10 equiv of MeOH, 5 equiv of diisopropylazodicarboxylate in THF, rt, 5 min reaction time) or a DBU-mediated method (3 equiv of DBU (1,8diazabicyclo[5.4.0]undec-7-ene) followed by 10 equiv of dimethyl sulfate in NMP, rt, 5 min reaction time). Cleavage of the o-NBS group was performed with 10 equiv of 2-mercaptoethanol and 5 equiv of DBU in NMP, rt, 15 min reaction time. The DBU-mediated method was used for the N-methylation of L-δ-azidoornithine. Introduction of the noncanonical amino acid N-γ-isobutyryldiaminobutyric acid (DabiBu) in 17 was done using the following procedure. Fmoc-Dab(Dde)-OH was coupled to the linear sequence at the appropriate place. Next, the Dde side chain protection was selectively removed by NH2OH·HCl/imidazole solution in NMP/ DCM following the published protocol.82 Then 1.25 g (1.80 mmol) of NH2OH·HCl and 0.918 g (1.35 mmol) of imidazole were suspended in 5 mL of NMP, and the mixture was sonicated until complete dissolution. This solution was diluted with 5 mL of DCM and used to treat the resin for 2 h. The resin was washed 5 times with NMP, and the free side chain NH2 group was capped with the isobutyric acid activated with PyBOP/HOBt/DIPEA as described above (5 equiv). The synthesis was continued according to a general protocol. After completion of a linear sequence, the resin was washed with DCM and dried under vacuum. The linear precursors were cleaved from the resin without side chain deprotection by a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol and DCM (1:3, v/v; 10 mL; 15 min). The solutions were filtered from the resin and dried using a rotary evaporator. The obtained oils were suspended in an acetonitrile/water mixture (1:1, v/v) and lyophilized, the crude linear precursors were used for the cyclization without further purification. The cyclization step was done in DCM (0.8 L per 0.2 mmol load) with the activating mixture of PyBOP (3 equiv) and HOBt (3 equiv) predissolved in DMF (2 mL), followed by addition of DIPEA (6 equiv). The reaction mixture was stirred for 16 h. Afterward, the solvent was evaporated on a rotary evaporator and residual material was lyophilized. The final deprotection of the cyclized peptides was done with a deprotecting cocktail, containing trifluoroacetic acid, triisopropylsilane, and water (92.5:2.5:5, v/v/v; 10 mL), incubating for 30 min at room temperature. The volatiles were removed on a rotary evaporator and the residual oils were lyophilized. The crude peptides were dissolved in 10 mL of water/acetonitrile mixture (2:1, v/v) and analyzed on analytical RP-HPLC. Individual peaks fractions from analytical RP-HPLC were collected and analyzed with MALDITOF mass spectrometry. In all cases, the major components in crude materials were confirmed to be target products. The peptides were purified on a preparative RP-HPLC. A typical method exploited a gradient of 30−50% B. Final yields of peptides in an open form varied between 30 and 60 mg with the purity ≥95% confirmed by analytical RP-HPLC and MALDI-TOF. Numerical mass data of the synthesized peptides are listed in Table 8 (see Supporting Information file for the spectra). 5. Preparation of Photoswitchable Peptides in the “RingClosed” Photoform. All peptides were prepared with the DAE fragment in its closed form in 100% purity by RP-HPLC separation of an equilibrium mixture of the two photoforms (photostationary state, as obtained after UV irradiation). The following protocol was used: 20 mg of the peptide in the open form (synthesized as described above) were dissolved in 10 mL of a freshly prepared saturated urea solution. The solution was degassed by alternating vacuum exposure/ argon refilling. Degassed solutions were exposed to UV light, using a LUMATEC light source (Superlite) in a 500 mL conical flask under

constant stirring (kept at rt in argon atmosphere). The irradiation conditions were as follows: the spectral range 320−400 nm, light power 2.1 W, power density approximately 10.5 mW/cm2, 15 cm distance from the tip of the light guide to the solution, 8 min exposure time. This procedure yielded in average 40% of the closed form (40− 95% for the entire range of peptides). After the UV irradiation, the urea solutions were diluted by water (20 mL), and the pure closed forms were obtained by preparative RP-HPLC. Chromatographically separated fractions were combined and lyophilized in the aluminum foil wrapped (light-protection) glass flasks and kept as dry powders at −20 °C in the dark. 6. Cell Viability Assay. All materials were from Sigma-Aldrich or Biopharma (Ukraine). The cell lines Hep G2 (human hepatocyte carcinoma, ECACC 85011430), MCF-7 (human breast adenocarcinoma, ECACC 86012803), MIA-PaCa-2 (human pancreatic carcinoma, ECACC 85062806), MOR (human lung adenocarcinoma, ECACC 84112312), MOR/0.2R (human lung adenocarcinoma, doxorubicin-resistant, ECACC 96042335), and HeLa (human cervix epitheloid carcinoma, ECACC 93021013) were obtained from European Collection of Authenticated Cell Cultures. The cytotoxicity of the peptides in both photoforms was evaluated with a standard colorimetric test using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Cell viability was characterized by conventional IC50 values83,84 using standard 96-well microtiter plates. As a culture medium, Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 40 μg/mL gentamicin was used. Cells (100 μL per well) were plated with a starting concentration of about 5 × 104 cells/mL. After a 24 h period of adaptation (5% CO2, 100% humidity, 37 °C), the tested compounds were added at different concentrations (in 100 μL of medium) to each well, except for the control, and the cells were incubated for an additional 24 h. Next, 10 μL of the 12 mM MTT solution were added to each well, and the plates were left for 4 h incubation. Afterward, the medium from each well was removed, and 100 μL of DMSO was added instead. The amount of formazan was colorimetrically assayed at 570 nm. Four individual repetitions and 10 different 2-fold dilution series were used for each tested peptide (256−0.5 and 512−1 μg/mL for open and closed forms, respectively). All experiments with the closed light sensitive form were performed under dim light to prevent uncontrolled photoisomerization of the peptidomimetics. The obtained cell viability in % was plotted against peptide concentration. IC50 values were extrapolated from the cell killing curves (see Supporting Information, Figures S11−S13). 7. Hemolytic Activity Assay. To test the hemolytic activities of peptides, human erythrocytes (RBC) were used. RBC concentrates in 10% citrate−phosphate−dextrose medium were obtained from Karlsruhe municipal hospital. Two 172 mM Tris-HCl buffers with the isotonic osmolarity of blood (310 mOs/mL) HB1 and HB2 were used in the assay. HB1, pH 7.6 at 4 °C; HB2, pH 7.6 at 37 °C. The erythrocytes were washed twice in HB1 by centrifugation at 300g, 4 °C. Peptides were dissolved in 40 μL of DMSO and then diluted to 400 μL by HB2. The starting peptide solutions were then used to construct a 2-fold dilution series of 200 μL aliquots in HB2 in 1.5 mL Eppendorf tubes. Two independent series of 10 dilutions were made (starting 256 or 192 μg/mL). The hemolysis reaction was initiated by the addition 200 μL of 0.5% RBC suspension in HB2. The samples were incubated under periodic agitation for 30 min at 37 °C, after which their content was centrifuged for 10 min at 13000g. The absorption of the supernatant at 540 nm gives the extent of hemolysis, with 0% taken from the peptide-free negative control, and 100% after treatment with 0.1% Triton X-100 (positive control). To ensure that the colorimetric readout does not interfere with the absorption of DAE fragment, the samples in the closed photoform were backconverted to their open colorless forms by exposure to visible light before the absorption measurement. HC50 values were extrapolated as peptide concentrations causing 50% hemolysis (see Supporting Information, Figures S9, S10). 8. Antimicrobial Activity Assay. Bacterial cultures (Bacillus subtilis, DSM 347, and Escherichia coli, DSM 1103) were obtained 10809



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from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The antimicrobial activities of GS and its analogues 2−28 were measured using a 2-fold microdilution assay according to standard protocols with some modifications.85,86 The peptides were dissolved in 50% ethanol to give stock solutions with the concentration 1024 μg/mL. Sterile flat-bottom polystyrene nontissue culture-treated 96-well Nunc plates were used for the assay. First, 50 μL of the double concentrated Mueller−Hinton (MH) medium (Mast Diagnostika, Germany) were filled to the initial row. Next, 50 μL of the nonconcentrated MH medium were applied to the remaining test wells for the positive (without peptides) and the negative (sterility) controls. Addition of the peptide stock solutions (50 μL) to the wells of the initial raw provided the starting peptide concentration for the gradient. For construction of the gradient, all subsequent rows were filled by transfer of 50 μL aliquots from the preceding rows. From the last row of wells, 50 μL aliquots were discarded. Bacterial suspensions were prepared from the culture in the exponential growth phase, which was diluted first to the OD550 = 0.2 and then 1:100 or 1:1000 for Gram-positive and Gram-negative strains, respectively, to inoculate each well with 5 × 105 CFU/mL, excluding the negative control. To examine bacterial growth, the plates were incubated for 22 h at 37 °C without agitation. Then, 20 μL of an aqueous solution (0.2 mg/mL) of the redox indicator resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) were added to each well. The plates were incubated for 2 h at 37 °C, and the redox status of resazurin was determined visually and spectrophotometrically using a microplate reader FLASHScan 550 (Jena Analytic, Germany). The difference in absorbance of resorufin at 570 nm and resazurin at 600 nm was used to assess bacterial growth and thereby to determine MIC, the lowest concentrations that inhibit bacterial growth. MIC measurements were done 1−6 times, each time at least in three replicates. 9. Circular Dichroism Spectroscopy. CD spectra were recorded on a Jasco J-815 spectropolarimeter. Measurements were performed using 200 μL aliquots with 50 μg/mL concentrations in quartz glass cells (Suprasil, Hellma) of 1 mm path length and kept in the measurement chamber under constant N2 flow. Spectra were recorded between 260 nm and 180 nm at 0.1 nm intervals at 25 °C (maintained using a water-thermostated house-built rectangular copper cell holder). Three consecutive scans at a scan rate of 20 nm/min, 8 s response time, and 1 nm bandwidth were averaged for each sample and for the (subtracted) baseline of the corresponding peptide-free sample. The peptides were first analyzed in their closed photoforms prepared in advance by preparative HPLC (see above). Two aliquots were taken per peptide, 17.5 μg each, and lyophilized prior to the CD measurements in 0.5 mL Eppendorf tubes. Each lyophilized sample was freshly dissolved in either deionized Milli-Q water or trifluoroethanol (1 min vortexing; 5 min ultrasonic bath, 40 °C), transferred into the CD measurement cell and immediately measured to record a closed photoform spectrum. Next, the same glass cell was light source by the LUMATECH illuminator (the spectral range 570 ± 10 nm, 5 min, photoisomerization was confirmed by discoloration of the solution), and the CD spectrum of an open photoform was then recorded. Care was taken to maintain the peptides in the closed photoform under minimal light exposure.

Article

AUTHOR INFORMATION

Corresponding Authors

*For I.V.K.: phone, (+38 044) 5213566; E-mail, Igor. [email protected]. *For A.S.U.: phone, (+49 721) 60843222; E-mail, Anne. [email protected]. ORCID

Igor V. Komarov: 0000-0002-7908-9145 Anne S. Ulrich: 0000-0001-5571-9483 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): I.V.K., S.A., O.B., and A.S.U. are inventors on the issued patent family: Peptidomimetics possessing photocontrolled biological activity; (WO2014127919 (A1), EP2958934 (B1), US9481712 (B2), UA113685 (C2)).

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ACKNOWLEDGMENTS We acknowledge EU funding by the EU H2020-MSCA-RISE2015 through the PELICO project (grant 690973). This work was also supported by the DFG-GRK 2039 (S.A., T.S, A.S.U.) and by the BMBFVIP+ (O.B., A.S.U.). I.V.K. acknowledges the Alexander von Humboldt Foundation for financial support as a recipient of the Georg Forster Research Prize in 2016. We thank Dr. Wadhwani (KIT, Karlsruhe) for access to the peptide synthesis facility.

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S Supporting Information *

ABBREVIATIONS USED GS, gramicidin S; PDT, photodynamic therapy; DAE, diarylethene; CD, circular dichroism; MIC(G−), MIC determined against Gram-negative E. coli; MIC(G+), MIC determined against Gram-positive B. subtilis; HC50, concentration causing 50% lysis of RBC; SIG−, selectivity index for Gram-negative bacteria vs RBC, HC50/MIC(G−)ratio; SIG+, selectivity index for Gram-positive bacteria vs RBC, HC50/ MIC(G+) ratio; SIC, selectivity index for cancer cells vs RBC, HC50/IC50 ratio; RT, retention time; DIPEA, N,N-diisopropylethylamine; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; PyBOP, benzotriazol-1yl-oxytripyrrolidinophosphonium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole); LeuOH, (2S,3R)-β-hydroxyleucine; AsniPr, Nγ-isopropyl-L-asparagine; DabiBu, Nγ-isobutyryl-L-diaminobutyric acid; Adm, L-1-adamantylglycine; OrnN3, L-δazidoornithine; DProSw, DAE photoswitching fragment (4-(2(5-((L-prolyl-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-5methylthiophene-2-carboxyl); MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; MH, Mueller−Hinton medium; CFU, colony-forming units; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; o-NBS, ortho-nitrobenzenesulfonyl-

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01428. Experimental data file including graphs demonstrating phototherapeutic potential of the photoswitchable GS analogues, circular dichroism spectra, RP-HPLC analysis, hemolysis and HeLa cytotoxicity data, MALDITOF spectra (PDF) Molecular formula strings (CSV)

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