Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

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Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes Nuria Rodríguez-Vázquez, H. Lionel Ozores, Arcadio Guerra, Eva González-Freire, Alberto Fuertes, Michele Panciera, Juan M. Priegue, Juan Outeiral, Javier Montenegro, Rebeca García-Fandiño, Manuel Amorín and Juan R. Granja* Departamento de Química Orgánica, Centro Singular de Investigación en Química Biológica y Materiales Moleculares, (CIQUS), Campus Vida, Universidad de Santiago, 15782 Santiago de Compostela, Spain Abstract: Peptide nanotubes are novel supramolecular nanobiomaterials that have a tubular structure. The stacking of cyclic components is one of the most promising strategies amongst the methods described in recent years for the preparation of nanotubes. This strategy allows precise control of the nanotube surface properties and the dimensions of the tube diameter. In addition, the incorporation of 3aminocycloalkanecarboxylic acid residues in the nanotube-forming peptides allows control of the internal properties of the supramolecular tube. The research aimed at the application of membrane-interacting self-assembled cyclic peptide nanotubes (SCPNs) is summarized in this review. The cyclic peptides are designed to interact with phospholipid bilayers to induce nanotube formation. The properties and orientation of the nanotube can be tuned by tailoring the peptide sequence. Hydrophobic peptides form transmembrane pores with a hydrophilic orifice, the nature of which has been exploited to transport ions and small molecules efficiently. These synthetic ion channels are selective for alkali metal ions (Na+, K+ or Cs+) over divalent cations (Ca2+) or anions (Cl–). Unfortunately, selectivity was not achieved within the series of alkali metal ions, for which ion transport rates followed the diffusion rates in water. Amphipathic peptides form nanotubes that lie parallel to the membrane. Interestingly, nanotube formation takes place preferentially on the surface of bacterial membranes, thus making these materials suitable for the development of new antimicrobial agents.

Keywords: Antimicrobials, Ion channels, Membrane, Nanotubes, peptide, Self-assembling.

Compartmentalization, i.e. the separation of components into distinct parts, is an important process for life and nanotechnology [1]. This type of spatial confinement can lead to qualitative and quantitative differences between the inner contents and the wider environment and provides a large surface area for contact with surrounding media to implement the appropriate conditions required to carry out certain functions. In this sense, living organisms are based on the cell as their basic component. The cells are also compartmentalized as they are comprised of closed parts, e.g. the organelles such as the cell nucleus, Golgi apparatus, peroxisomes and so on, within the cytosol. In living organisms a membrane, which is a single or double lipid layer, usually surrounds these compartments. Nature has selected phospholipids as one of key components of the biological membrane. They consist of a polar head that confers some affinity for the water and two hydrocarbon chains that provide hydrophobic character. In aqueous media these molecules, because of their structure, are programmed to spontaneously form bilayers that generate vesicles [2]. The resulting lipid bilayers are insulator materials that are almost impermeable to ions and polar molecules. However, membranes in living

cells carry out a variety of functions that include control of nutrient entry, exit of residues, generation and maintenance of differences between contents and the environment and they also allow communication with the surrounding media and other cells (transport, signal detection, and regulation). In order to carry out such functions the selective transport of ions and molecules through the membrane is a particularly relevant process. In general, membrane proteins, such as ion channels or pumps, are responsible for carrying out most of these functions [3]. The membrane proteins are large complexes that are characterized by the presence of a central channel that spans the membrane, with access to the channel region regulated by additional areas on one or both sides of the membrane. These proteins are functional holes in the phospholipid membrane that minimizes the inherent repulsion between hydrophilic substrates and the lipidic environment but at the same time creates selective filters to differenciate diferent types of substrates. To achieve this task Nature uses different features such as size exclusion, ion hopping, charge repulsion, ion pairing, hydrophobic interactions, and so on. The selectivity and efficiency of this natural process has stimulated the creativity to design synthetic mimics that further can develop artificial functions and properties [4].

*Address correspondence to this author at the Departamento de Química Orgánica, Centro Singular de Investigación en Química Biológica y Materiales Moleculares, Campus Vida, Universidad de Santiago, 15782 Santiago de Compostela, Spain; Tel: +34 881 815 746; Fax: +34 881 815 704; E-mail: [email protected]

The importance of membrane compartmentalization, it is not surprising that during the millions of years of evolution living organisms have developed a variety of mechanisms that target the membranes for protection or to attack other cells [5]. In general, these mechanisms are fast cell-killing

1. INTRODUCTION

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methods based on the perturbation of the cell membrane. In this way the required cell-survival chemical balance generated by the compartmentalization is destroyed [6]. For example, a range of toxins (e.g. Leukocidins, hemolysins), which have been developed by a variety of organisms, form orifices in the cell membrane and these allow the passage of small molecules and ions [7]. Additionally, a variety of pluricellular organisms have developed a first protection barrier, innate immunity, based on the interaction of small peptides with the microbial membrane that destabilizes cell membranes to cause rapid cell death [5,8]. These antimicrobial peptides are synthesized by a variety of living beings such as humans, plants, and frogs, amongst others. Although these peptides adopt a wide variety of structural motifs, ranging from helical to -sheet, extended conformation, loops, and so on, most of them have in common a cationic character due to the presence of basic residues (Lys and Arg) combined with a variable number of lipophilic residues [9]. The proposed mechanisms are based on two basic models, the barrel pore and the carpet-like mechanism (Fig. 1) [10]. In the barrel pore mechanism the peptides are transmembranally oriented to form small bundles that generate a hydrophilic hole, whereas in the carpet-like mechanism the peptides lie parallel to the membrane surface, thus modifying the membrane properties. Some additional models have also been proposed, such as toroidal pore or disordered toroidal pore, but these can be considered as intermediate models of the two basic mechanisms outlined above. Regardless of the perturbation model, it has also been suggested that an implicit concentration threshold for membrane disruption is required. In addition to this activity on the microbial cell membrane and the physical damage to the structure, which may include thinning, pore formation, altered curvature, modified electrostatics and localized per-

Rodríguez-Vázquez et al.

turbations, other biological responses of the host have also been proposed [10b]. Although the active forms are structurally very different, they have in common the adoption of amphiphilic 3-D conformations (Fig. 1). In the presence of the targeted membrane the random coiled structures fold to form an amphipathic structure in which the non-polar residues are in contact with the hydrocarbon chains of the phospholipid while the cationic and hydrophilic residues are in contact with the polar heads and the aqueous medium. Bacterial resistance to antimicrobial agents is becoming one of the major medical problems today, especially with the appearance of multi-drug resistant pathogens such as vancomycin-resistant enterococcus (VRE) or methicillinresistant staphylococcus aureus (MRSA) [11]. Indeed, there are warnings about everyday infections by these superbugs that may have reached levels that outstrip our ability to fight them with existing drugs. This problem is exacerbated by the difficulty and cost associated with the discovery of new antimicrobials with novel modes of action. In this respect, only four new classes of antimicrobials have been introduced in the last 40 years. Therefore, the search for new broadspectrum antimicrobial agents for the treatment of multidrug-resistant infections with negligible or almost nontoxic side effects has intensified. One of the approaches is based on natural antimicrobial peptides because they might offer an alternative strategy in the battle against multidrugresistant pathogens and they may also reduce the opportunities for emerging drug-resistant bacteria due to their mechanism of action [12]. Unfortunately, among the variety of antimicrobial peptides studied to date, only a few have gone on to clinical applications and the most promising results are those for topical applications. These limitations in the most

Fig. (1). Mode of action of antimicrobial peptides [6, 10]. These peptides fold in the presence of biological membranes to form amphipathic structures (helices, sheets, turns and so on). The aggregation of the folded peptides on the membrane destabilizes it through a variety of mechanisms such as barrel pore, toroidal pore or carpet-like.

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

relevant practical applications are probably due to the inherent nonspecific toxicity observed for these systems towards mammalian cells in systemic treatments [9]. The interest in this type of structure has encouraged scientists to develop simple and more efficient models that can mimic natural systems with the aim of developing more efficient antimicrobial agents. In addition, there is a great deal of interest in developing synthetic ion channels that can replace, in terms of efficiency and selectivity, the non-functional natural proteins in channelopathies [13]. In general, the proposed strategies are based, in a similar way to natural systems, on supramolecular approaches in which small molecules self-assemble under appropriate conditions to form well-ordered aggregates [4]. The final structure is the thermodynamically most stable and it is obtained in a reversible way through the formation of a variety of noncovalent interactions. 2. PEPTIDE NANOTUBES In this context, some years ago we started a program directed towards the formation of hollow rod-shaped structures (Self-assembling Cyclic Peptide Nanotubes, SCPNs) from cyclic peptides (Fig. 2) [14]. The original nanotubes used cyclic peptides (CPs) with an even number of -amino acids of alternating chirality (D,L--CPs) and these adopted a flat conformation in which the amide groups (NH and CO) lie perpendicular to the plane of the ring (Fig. 2) [15]. In this conformation, antiparallel -sheet-type interactions can be established between cyclic peptides (CPs) in such a way that they stack one on top of the other [16]. All of the amino acid side chains diverge outwards from the cyclic component. Thus, upon cyclic peptide stacking all of the side chains are exposed on the external surface of the cylindrical structure. Because of this side chain arrangement, the interior of the assembly remains empty creating the internal orifice in the peptide cylinder. In addition, the number of amino acids used in the cyclic peptide controls precisely the diameter of the nanotube [17]. Such properties allowed the design of a variety of peptide nanotubes with diverse properties and functions [18]. For example, in the original nanotubes, the cyclic peptide c-[(A-E-A-Q-)2] (entry 1, Table 1) [19] was soluble in basic aqueous solutions and nanotube formation took place upon protonation of the carboxylate groups of glutamic acid residues at acidic pH [15a]. On the other hand, peptide nanotubes were obtained from c-[(A-Q-)4] (entry 3, Table 1) and these were soluble in acidic media (trifluoroacetic acid) with nanotube formation triggered by slow water absorption [15b]. Interestingly, this design allowed the study of hydrophobic interactions in the packing of the nanotubes by changing the alanine residue for Leu, Phe or Val. SCPN formation is not only restricted to D,L-CPs as other platforms, such as -peptides (-CPs), peptides (-CPs) or hybrids such as ,- or ,-peptides and even ureas (CU), have also been shown to form nanotubes through a similar strategy (Fig. 2) [14,20]. In the last few years we have been working on cyclic peptides (,-CPs) that contain -aminocycloalkanecarboxylic acids (-Acas) [21]. The main characteristics of these peptides are their improved predisposition to adopt the flat conformation induced by the cis-configuration of the -amino acids and the projection of the methylene moiety of this amino acid (C2) towards

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the lumen of the cavity (entries 5 and 6, Table 1). The former characteristic gives the cavity its hydrophobic properties. Most of our studies were carried out with dimeric models (Fig. 2, where R3 is a methyl group) that permitted to identify the structural and thermodynamic parameters that govern the self-assembly process [22]. The ease of external surface modification and the use of dimeric models allowed the preparation of heterodimeric structures that were used to achieve electron and energy transfer processes [23]. 3. ION CHANNEL MODELS FROM PEPTIDE NANOTUBES One of the main advantages of these rod-shaped peptide materials is the simplicity with which their external surface properties can be modulated by simply selecting the appropriate amino acid sequence in the cyclic peptide. In this sense, the design of SCPNs that interact with the hydrophobic part of biomembranes was carried out through the appropriate selection of amino acids in the basic peptide subunit (Fig. 3). Thus, an eight-residue cyclic peptide formed mainly by Trp and Leu, c-[Q-L-(W-L-)3], (entry 8, Table 1) was prepared and shown to self-assemble in the membrane bilayers [24]. The nanotube formation was confirmed by FTIR experiments in which the typical bands due to antiparallel-type hydrogen bonding interactions characteristic of peptide nanotubes were observed [15,25]. In addition, the transmembrane tube efficiently transported alkali metal ions such as sodium or potassium through the channel. The planar lipid bilayer showed astonishing transport properties of more than 10 million ions per second, which is comparable to the levels found in natural systems. In general, most of these peptide nanotubes exhibit pronounced selectivity for alkali metal ions and the transport of divalent ions, such as Ca2+, or anions like Cl– was not detected. The rates of transport for the alkali metal ions followed the lyotropic series, meaning that the ions with larger diffusion rates in water (Cs+>K +>Na+>Li+) provided larger conductivities. Conductance experiments in planar bilayer membranes showed the characteristic gated events with multiple open and closed states (Fig. 4). The ionchannel transport rates were about three times faster than that of the natural ion transport antimicrobial peptide gramicidin A, for which the maximum conductance values observed (g max) indicated that transport of alkali metal cations followed their diffusion rates. Interestingly, transport experiment on liposomes showed that SCPNs had proton efflux rates similar if not higher than the natural linear peptide gramicidin A [24a]. These results do not provide information about ion-transport rates, which are on a much faster time scale, but reports about the rate limiting steps of the method, peptide diffusion and assembling. So, nanotube formation under appropriate conditions, at least six rings stacking, is a very fast process, competing in efficiency with the folding and dimerization of a small linear peptide such as gramicidin A. In general, most of the peptide nanotube recordings showed several conductance levels [24]. In Fig. (4) it is shown a especific recording corresponding to an ,-CP (entry 10, Table 1) in which three different levels are observed. These levels were assigned to nanotubes with different lengths. The length of the nanotube ensembles is limited by

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Fig. (2). Self-Assembling Cyclic Peptide Nanotube (SCPN) formation by the stacking of cyclic components in a flat conformation. Structures of some of the cyclic peptides (and ureas, CU) used in nanotube formation, such as D,L--CP in which the amino acids of opposite chirality are alternated, -CP and -CP (made of - or -amino acids), and hybrid derivatives like ,-CP, formed by alternation of - and -amino acids, ,-CP, in which - and -amino acids are combined and also hybrids of -amino acids and ureas (CUh). Model of parallel and antiparallel (illustrated for ,-CP) -sheet type interactions required in nanotube formation from the - or -derivatives (n = 1, 2) or D,L--CP and ,-CP, respectively. The former type of interaction can be studied with dimeric models, in which R3 would be an alkyl group.

the membrane thickness and by the inter-ring distance (4.85 Å) [26]. The structural features of the peptide nanotubes in which peptide subunits are stacked on top of each other restrict the length of the nanotubes to multiples of the interring distance. The use of the Nernst–Planck electrodifussion equation [27] allowed a correlation to be established between the conductance and the channel length. The most commonly observed conductance events were assigned to channels composed of five/six-stacked subunits of cyclic peptides. This observation fits quite well with the thickness of the hydrophobic part of the lipid bilayer. Non-ohmic current rectification was observed on the formation of heteromeric nanotubes (inset Fig. 4) [28]. The formation of these nanotubes

was achieved by the combination of two different CPs; firstly the channel-forming nanotubes, c-[(W-L-)4], and secondly a hydrophobic CP that contains two hydrophilic substituents at the amide group of its peptide skeleton c-[(W-LW-(CH2)nXN-L-)2] (entry 12, Table 1), where X denotes an ammonium or a carboxylate group. The former CPs blocked the growth of the SCPN due to the presence of the alkyl groups on the peptide backbone. Such structures confined these CPs to the membrane/water interface, thus facilitating their location at the edge of the nanotube as a molecular cap. The resulting stoppers therefore acted as the channel mouth and regulated the entry of ions into the nanotube lumen. Transport experiments in planar lipid bilayers showed that

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

Table 1.

a

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Examples of most relevant cyclic peptides used in nanotube formation described in this article along with an outline of the most relevant properties.

Entry

Peptidesa

1

c-[(A-E-A-Q-) 2]

Nanotube bundles. Triggered SCPN formation by acidification of a basic solution.

15a

2

c-[(A-E-A-Q-) 3]

Nanotube bundles. Triggered SCPN formation by acidification of a basic solution. Proof of diameter control by changing the ring size of the CP.

17a

3

c-[(X-Q-)4] X = A, L, V, F

Nanotube bundles. Triggered SCPN formation by dilution or basification of CP solution. Study of nanotube packing by hydrophobic effects.

15b

4

c-[(MeN-Ala-F-)4] c-[(MeN-Ala-F-)4]

Dimer-forming CPs. Evaluation of parallel versus antiparallel -sheet.

16

5

c-[(MeN-Acp-X-)n] X = F, L n = 2, 3, 4,….8

Dimer-forming cyclic peptides that contain -Acas. The -sheet in which the interactions take place is the -face. Control of pore diameter was demonstrated with a variety of ring sizes.

17c, 17d

6

c-[(Acp- MeN-A-) n] n = 2, 3, 4,

Dimer-forming cyclic peptides that contain -Acas. -Sheet interactions take place through its -face. Control of pore diameter was demonstrated with a variety of ring sizes.

17c, 17d

7

c-[Q-L-(W-L-)3]

First peptide nanotube that formed membrane channels and selectively transported alkali metal ions.

24a

8

c-[(-HTrp-)4]

-Peptide transmembrane nanotube with ion transport properties.

29a

9

c-[Q-L-(W-L-)4]

10

c-[Ach-Q-(Ach-W-)3]

Ion channel-forming CP that contains cyclic -amino acids.

32

11

c-[Ach-Q-(Ach-W-)2]

CP containing cyclic -amino acids that forms nanotubes on membranes and showed proton transport properties but did not transport ions.

32

12

c-[(W-L-W-(CH2)nXN-L-)2] X, COOH (n=1), NH2 (n=2)

Non-ohmic current rectifier cyclic peptide. These peptides are incorporated at the end of the membrane nanotube to form heteromeric tubes.

28

13

c-[(R-R-K-W-(L-W-)2]

Cyclic octapeptide with antimicrobial properties that was shown to have in vivo antibacterial efficacy.

40

14

c-[R-R-L-W-L-W-]

Potent antimicrobial hexapeptide against Gram-negative bacteria (E. coli).

40

15

c-[S-H-K-R-K-W-L-W-]

This peptide showed in vitro inhibition of Adenovirus (Ad-5) infections with an IC50 of 5 M.

44

16

c-[MeN-Acp-L- Ahf-T-]

Dimer-forming cyclic peptide that provided dimers with a hydroxylated cavity.

34b

17

c-[(MeN-Acp-Ach- MeN-Acp-Aga-)2]

Dimer-forming cyclic peptide made only by -Acas that incorporates sugar amino acids (SAAs) such as Aga.

50

18

c-[L-Nle-W-H-S-K-]

This peptide interacts strongly with A protein and inhibits its aggregation.

53

19

c-[A-Q-A-E-A-Q-A-C-]

This peptide was used to facilitate Doxorubicin delivery to overcome drug-resistant breast cancer cells.

55

Properties

Ion- and molecule-transporting peptide nanotube. Glucose and glutamic acid were proven to pass through the channel. More recently this peptide was also used for the transportation of the antitumor drug 5-Fluorouracil.

Ref.

30,54

Peptide sequences are written using the conventional one-letter code for amino acids, but residues having a D stereochemistry are underlined.

the nanotube conductance values either decreased or increased depending on the properties of the added CPs bearing ammonium or carboxylate groups, respectively. For the cationic CP (X = NH3) the reverse potentials were less negative than those for the homomeric nanotube made exclusively by c-[(W-L-)4], a finding that suggests some loss of the charge selectivity observed for this channel. -Peptides (-CP), oligoureas (CU) and oligourea/amide macrocycles (CUh) decorated with hydrophobic side chains

also showed ion transport properties [29]. -CPs, such as (c[(-HTrp-)4] (entry 8, Table 1), showed similar transport rates to the original nanotubes and current rectification was not observed despite the permanent macrodipole character of this nanotube as a consequence of the parallel-type interaction characteristics of the -nanotube. Guichard´s nanotubes, which are formed by the stacking of oligourea/amide macrocycles, represent an exception in these tubular transporter systems because they are anion-selective as a consequence of the anion-macrodipole

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Fig. (3). Nanotube organization on the lipid bilayer depends on the cyclic peptide structure. Hydrophilic regions are shown in blue and hydrophobic regions in gray. The hydrophobic CPs (gray) form single transmembrane nanotubes while amphipathic CPs can form nanotube bundles in which the hydrophilic groups tend to remain out of the alkyl groups or lie parallel to the membrane surface in a carpet-like structure.

Fig. (4). The peptide nanotube channels, because of the differences in length, generally presented different conducting states [24], as it is shown (top) in the 12 seconds of recording of c-[Ach-Q-(Ach-W-)3] (0.5 M KCl,10 mM MOPS, DPPC) in which it can be observed three levels of 0.6 pA, 1.3 pA and 2.9 pA (+80 mV) that correspond to three different ion channels with different number of peptide subunits [32]. Bottom, representation of the proposed mechanisms for the gating and variable conductance events observed with SCPNs. Inset, mode of interaction of the CP caps with modified ion conductance properties by the formation of heteromeric transmembrane channels [28].

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

interactions observed in these assemblies [29c]. In any case, the anion diameter was too large, when compared to the nanotube pore size, and this made it almost impossible for the transport to take place through the nanotube pore. Interestingly, hydrophilic molecules such as glucose or glutamic acid were also transported through the nanotube orifice [30]. In this case the nanotube-forming cyclic peptide was a decamer (entry 9, Table 1) and the octamer was unable to allow the passage of these biologically relevant molecules. This experiment confirmed that the transport was carried out by the supramolecular nanotube and showed that the octamer pore is too small to allow these molecules to pass through its cavity. The ion transport properties of the ten-residue peptide (c-[Q-L-(W-L-)4]) were also studied and the results confirmed nanotube formation and ion transport properties similar to those of the octapeptide homolog but with short mean open times. The increased conformational freedom of the decamer must be responsible for the faster open and closed states observed for these larger channels. Nanotubes derived from ,-CPs have one methylene moiety per -residue projected into the nanotube cavity to provide the hydrophobic properties. Computational studies carried out on both hexameric and octameric ,-CPs showed that in the presence of aqueous media the nanotube cavity is filled with partially ordered water molecules [31,32]. The number of water molecules varies with the internal diameter, ranging from 13–16 in the six-CP nanotube constituted by cyclic hexapeptides, to 23–28 for the nanotube (stack of six cyclic peptides) derived from octamers. The observation of the small nanotube cavity filled with water molecules in the hexapeptide nanotube led us to study their transport properties. Furthermore, the reduced number of water molecules per peptide subunit (2–3) also encouraged us to believe that the pores could have increased ion selectivity. Transport studies on fluorogenic micelles showed that the hexameric peptide (entry 11, Table 1) carried proton transport and this confirmed the transmembrane nanotube formation. Unfortunately, the cavity diameter of this nanotube is small thus ion transport was not observed. Metal ion transport was finally confirmed on working with the octamer c-[Ach-Q-(Ach-W-)3 ] (entry 10, Table 1). Ion conductance studies using patch clamp techniques showed K+ and Na+ transport rates for this peptide in the order of 107 ions s–1, around three times lower than the D,L--CP nanotubes (entry 7, Table 1) or two times lower than in the -tetrapeptide (entry 8, Table 1) [29a], which has a smaller diameter. The mean open time was also slightly shorter than that of the original nanotubes (D,L--CPs). The ion transport rates followed the order Cs+>K +>Na+, as one would expect from their relative mobilities in solution, although sodium flux was 30–40% greater than for the heavier alkali metal ions (K and Cs) on the basis of the measured conductances and the relative bulk diffusion coefficients. This minimal preference for sodium must be a consequence of the effective size of the conveyed ion and it highlights the influence that the dehydration penalty has on the transport rate. One of the advantages of the ,-peptide nanotubes is the projection of one methylene group (C2) of the -cyclic amino acid into the nanotube cavity. This carbon moiety can

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be modified to create nanotubes with new properties [33]. In this respect, our subsequent efforts to improve transport properties were focused on lumen functionalization and topological adjustment of the nanotube assembly. In the last few years we have turned our attention to the synthesis of different amino acids containing a variety of functional groups at this position, such as 2-fluoro- or 2-hydroxy-3aminocyclopentanecarboxylic acids (Fig. 5) [34]. The functional group, in order to allow peptide self-assembly and cavity modification, must be trans-oriented with respect to amino and carboxy groups. 4-Amino-3hydroxytetrahydrofuran-2-carboxylic acid (-Ahf-OH) was also synthesized, in this case starting form D-xylose, and it was used in the first proof of concept of cavity functionalization in peptide assemblies [34b]. This amino acid was implemented in a dimer-forming tetrapeptide (entry 16, Table 1, Fig. 5) and it was confirmed that the resulting CP adopted the required planar conformation. In this flat structure the hydroxyl group was in a pseudo-equatorial disposition pointing inwards in the ensemble. Interestingly, the presence of the hydroxyl groups projected into the cavity not only changed their internal properties but also increased the cyclic peptide stacking propensity and restricted the equilibrium towards the ensemble in which the two internal hydroxy groups were hydrogen bonded. We envisage that the access to peptide nanotubes with this type of modification will provide a broad range of new nanometric devices, such as selectivity filters, catalytic pores, receptors, or molecule containers. Computational studies, especially molecular dynamics calculations, are a powerful tool to study and understand the macromolecular and supramolecular behavior of a variety of chemical and biological systems. In this respect, membrane proteins, ion channels and transmembrane transport have benefitted from such studies [35]. MD simulations can act as a virtual microscope with high spatial and temporal resolution to provide detailed information about the fine structure of the resulting nanotubes. Membrane nanotubes have been quite extensively studied by these methods [36]. Our group has carried out a variety of these studies and the results confirmed the stability of the hydrogen-bonded tubular assembly of nanotubes derived from ,-cyclic peptides throughout the simulation time as well as the rapid entry of both water and cations into the nanotube [31,37]. We were also interested in the effect that lumen functionalization has on the ion transport upon insertion into a lipid bilayer. In this way, we simulated membrane nanotubes made of octapeptides containing two or four hydroxy-functionalized -amino acids [38]. These nanotubes showed more pronounced hydrophilic properties that decreased the crossing energy barriers for all alkali metal ions studied. These metal ions have a greater tendency to enter the nanotube cavity and, as a result, more than three potassium or caesium ions can be included in the nanotube. In addition, it was possible to observe for first time, to our knowledge, ion crossing on the nanosecond scale without the need to apply a transmembrane voltage or concentration gradient. Unfortunately, an improvement in ion selectivity was not observed, perhaps because the large channel diameter allowed the passage of most of the ions with their water coordination sphere intact.

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Fig. (5). Top, equilibrium model of cyclic peptides with a functionalized cavity, two different dimers can be formed, the Syn- and the Antidimers depending on the relative orientation of the functional group. Middle, different -amino acids prepared with modification at the C2 position [34a]. Bottom, Cyclic peptides prepared with some of these -Acas. The dimer forming properties of these systems were studied in solution. Bottom right, dimer structure of -CP obtained by X-ray diffraction.

4. ANTIMICROBIAL AGENTS BASED ON PEPTIDE NANOTUBES The nanotube orientation on the membrane depends on the cyclic peptide structure, as one would anticipate given that this factor governs the resulting nanotube structure. Thus all-hydrophobic cyclic peptides will try to maximize van der Waals contacts with lipid layers by forming nanotubes oriented perpendicular to the membrane. On the other hand, amphipathic cyclic peptides would lie parallel to the membrane or form barrel-stave pores in which the hydrophilic part of the nanotubes would be oriented towards the interior of the nanotube bundles (Fig. 3). This was confirmed by membrane studies on a variety of cyclic peptides that were deposited on liposome membranes and studied using different infrared techniques [39]. The hydrogen-bonded tubular structure can be confirmed by FT-IR, in which the hydrogen-bonded amide I and amide II bands (1620 cm–1, 1530 cm–1) and N–H stretching band (3300 cm–1) confirm the expected antiparallel -sheet-type structure that is characteristic of peptide nanotubes [15,25]. The parallelpolarized grazing angle IR spectrum of channel-forming nanotubes, i.e. those derived from hydrophobic peptides,

revealed a high amide I () to amide II band ratio, which confirmed the alignment of the nanotube almost perpendicular to the membrane surface. Polarized ATR-IR spectroscopy indicated that the nanotube is tilted with an angle of 7 ± 1° relative to the membrane plane. On the other hand, amphipathic cyclic peptides (cationic/hydrophobic) showed a parallel orientation with respect to the membrane layer. In this case, the calculated tilt angle of the resulting peptide nanotubes was 70 ± 5°. Such orientation provided the opportunity to disrupt the phospholipid bilayers by the carpet-like mechanism proposed for antibacterial peptides [8-10]. A series of cationic amphipathic cyclic peptides were tested against a variety of bacteria and it was found that both octapeptides with three hydrophilic residues in which at least two of them were cationic (R or K) and the hexapeptide with two hydrophilic amino acids were active at M concentration [40]. Interestingly, eight-residue cyclic peptides showed a preference for Gram-positive bacteria such as methicillin-resistant S. aureus (MRSA) while hexamers preferred Gram-negative bacteria (E. coli). In addition, these peptides had a low activity against mammalian cells (red blood cell), thus demonstrating some therapeutic index protection [41]. Several ex-

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

periments were carried out to elucidate the membrane interacting mechanism. These included infrared spectroscopy, which confirmed the nanotube peptide orientation with respect to the phospholipid bilayers, and fluorescence-based cell depolarization studies, in which fast and complete membrane depolarization was achieved at a concentration close to the MIC. Finally, a mode of action based on receptor/ligandmediated process was ruled out because enantiomeric CPs (entries 1 and 2, Table 2) have similar in vitro activities. In addition, the time-killing observed for these peptides is more consistent with a membrane permeation mechanism than a receptor/ligand-mediated process, which typically requires several hours. The broad spectrum of activity and membrane selectivity observed with the peptides discussed above highlights the effects that single amino acid substitutions have on membrane activity and selectivity. Indeed, it is interesting to note that a single mutation in the hydrophilic region produces a remarkable change in antimicrobial activity. For example, the peptide with the K-S-K sequence (entry 3, Table 2) showed strong activity against MRSA and low hemolytic activity, while exchanging the serine residue for a glutamine (K-Q-K, entry 5) reduced the antibacterial activity by almost a factor of ten while increasing the MIC against blood red cells. In addition, peptide c-[R-R-(L-W-)2] (entry 10, Table 2), displays a high potency and selectivity against E. coli with low levels of hemolysis, while other hexamers with modifications in the hydrophilic region (entry 9, Table 2) showed lower activity. All of these results demonstrate the importance of the interaction of these peptides and their resulting supramolecular tube with the membrane/aqueous media interface. In this process the hydrophobic groups are responsible for allowing the peptide to interact with the membrane alkyl chains, but surface recognition might derive mainly from the hydrophilic contacts with the membrane interface. It must be highlighted that one special characteristic of the nanotube strategy compared with other antimicrobial peptides is the possibility of each cyclic peptide rotating in the nanotube generated from one single cyclic peptide to give rise to several different nanotube assemblies with different surface presentations (Fig. 6). However, such diversity is limited, as shown in the inset in Fig. (6), by the antiparallel-type arrangement and the required amino acid pairwise arrangement, in which only D-residues can be hydrogen bonded to amino acids of the same chirality and L-residues can only interact with L-amino acids. The intravenous efficacy of cyclic peptides, which are obtained by different combinatorial syntheses, with MICs of less than 12 g/mL was later tested using two different infection methods, peritonitis and neutropenic-mouse thigh [42]. These antimicrobial CPs had a quite different sequence with respect to those used in previous studies since they had a reduced number of hydrophobic residues (2–3) and 3 to 4 lysines (entries 11–13, Table 2). These peptides showed prolonged systemic antimicrobial activity in infection models when the molecules were administered intravenously to treat an infection localized beyond the blood compartment. Pharmacokinetics studies were also carried and it is worth highlighting that the compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties.

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

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Six-residue cyclic peptides obtained from a combinatorial library were also tested against eukaryotic marine algae Ulva linza and Navicula perminuta (entries 1–5, Table 3) [43]. The resulting cyclic peptides, also derived from a combinatorial library, showed again very variable activity ranging from broad-spectrum activity to species with selective potencies coupled with low hemolytic profiles. Of particular interest was phenylalanine-rich peptide c-[K-W-F-F-F-H-] (entry 1, Table 3), which displayed a remarkable 100-fold selectivity towards U. linza. Once again, this result showed that this approach might help to engineer compounds à la carte towards specific microbial agents in conjunction with very low toxicity to other organisms. Finally, antivirus peptides were also developed using similar types of peptides and strategies [44]. The cyclic peptides selectively targeted and inhibited viral infections through the prevention of low pH development in endocytic vesicles. As a consequence of this effect, the virions were prevented from leaving the endosome. A recombinant adenovirus was used as a suitable viral infection model and this was inhibited without an apparent adverse effect on cell viability. The discovery in 2002 of a new class of cationic antibiotics named mannopeptimycins, which are six-residue cyclic glycopeptides made of alternating D and L amino acids [45] that resemble the structure of nanotube-forming cyclic peptides, provided the opportunity to explore a new nanotubeforming antimicrobial strategy. The antimicrobial potency was improved up to submicromolar concentrations through the introduction of hydrophobic aromatic groups near to the terminal mannosyl moiety. In addition, in some cases activation of the antimicrobial potency of natural peptides was proposed to take place by peptide-selective glycosylation [46]. Cyclic D,L--glycopeptides bearing side chains modified with simple saccharides, such as D-glucosamine, Dgalactose, or D-mannose, were explored as new antimicrobial agents. The proposed strategy involved the incorporation of a saccharide moiety in one of the hydrophilic residues of c-[W-L-W-K-S-K-S-K-] (entries 14–17, Table 2) [47]. The in vitro antibacterial activities of glycopeptides against Gram-positive species were tested and it was found that glycosylation did not adversely influence cell membrane uptake nor diminish the bactericidal activity, although depending on the position and the type of the glycosylated residue employed, the toxicity towards mammalian cells was modified significantly. The strategy of selective glycosylation in different positions of the CPs clearly showed reduced mammalian cell toxicity (up to approximately five times in some cases) even after prolonged exposure of tissue culture cells to CPs. We recently also designed cationic amphiphilic cyclic peptides containing cyclic -amino acids (-Aca) in order to explore their antimicrobial activity (entries 18-22, Table 2) [48]. In our design 4-Aminoproline (Apr) was used as a polar -Aca to be incorporated in the hydrophilic part of the cyclic peptide. Some of the examples showed in vitro antibacterial activities against Gram-positive species and low toxicity toward mammalian cells. The most potent derivatives were 3,-CPs in which only every fourth amino acid was a -Aca [49]. This structural motif was shown in previous studies with models to adopt the required flat conformation and to self-assemble into the corresponding dimers in

2656 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

Table 2.

Rodríguez-Vázquez et al.

Examples of antimicrobial cyclic peptides.

Peptidesa

Hemolysis

MRSA

VRE

B. Cereus

E. coli

Ref. HD50 (g/mL)

MIC (g/mL)

1

c-[K-Q-R-W-(L-W-)2]

45

6

2

c-[K-Q-R-W-(L-W-) 2]

40

3

c-[K-S-K-W-(L-W-)2]

100

mg/kg 2

80

40

8

80

40

5

40

4

c-[K-K-K-W-(L-W-)2]

50

8

90

5

c-[K-Q-K-W-(L-W-)2]

50

45

100

6

c-[K-R-K-W-(L-W-)2]

50

10

40

7

c-[R-R-K-W-(L-W-)2]

In vivof

50

6

15

40 6

g

40 40 40

5

g

8

c-[K-K-(L-W-)2]

80

10

17

9

c-[K-S-(L-W-)2 ]

90

75

100

40

10

c-[R-R-(L-W-)2]

90

35

5

40

11

c-[S-K-BY-K-N-K-S-K-]e

209

2

4.0h

42

8

6.7

h

42

4.7

h

42

12

c-[S-W-F-K-T-K-S-K-]

>400

10

40

g

40

13

c-[S-W-F-K-T-K-S-K-]

>400

8

14

c-[W-L-W-K-S-K-S-K-]

120

5

10

5

44

c

15

c-[W-L-W-K-S -K-S-K-]

105

2.5

5

5

47

16

c-[W-L-W-K-S-K-Sc-K-]

120

1.5

10

10

47

17

c-[W-L-W-K-Sd-K-S-K-]

190

5

10

5

47

18

c-[Ach-W-L-W-Ach-K-Q-K-]

230

16

16

8

>100

48

19

c-[Ach-W-L-W-Ach-K-Q-R-]

180

16

8

8

>100

48

20

c-[W-Acp-W-K-H-K-H-K-]

560

16

64

32

48

21

c-[Acp-W-Acp-K-Apr-K-]

530

64

64

64

48

22

c-[W-Acp-W-Apr-K-Apr-]

560

64

64

64

48

a

Peptide sequences are written using the conventional one-letter code for amino acids, but residues having D stereochemistry are underlined. b Inhibitor of A protein aggregation. c Ser(Man). d Z = Ser(Gal). e BY denoted O-benzyltyrosine. f PD50, 50% protective dose. g In vivo antibacterial efficacy of peptide nanotubes determined by survival rates of treated versus untreated mice challenged with lethal doses of methicillin-resistant S. Aureus by bolus intraperitoneal (right side) or sub-cutaneous dose of the peptide 45 min after MRSA injection. h In vivo efficacy of cyclic peptides in the peritonitis infection models.

which the hydrogen-bonding pattern was similar to that expected in the tubular structure. Interestingly, these types of peptides were unable to transport ions through a transmembrane nanotube [32]. The hydrophilic residues were two cationic forms and one glutamine and the least potent was the derivative that had five hydrophilic amino acids, three of which were Lys. The main novelty of this cyclic peptide is the formation of only one type of nanotube on the membrane because the relative rotation of the cyclic subunits of the nanotube is not possible because the -Acp must be hydrogen bonded to -Acp of the two neighbor CPs (Fig. 6). Inspired by the potency of mannopeptimycins we also started a program aimed at preparing nanotube-forming glycopeptides that contained -Aca in their sequences [50]. In particular, we decided to incorporate sugar amino acids (SAAs) such as -Aga that could be prepared in three synthetic steps from glucuronic acids (Fig. 5). Although this amino acid was previously used in the design of -turns [51],

we envisaged that its incorporation into cyclic peptides of alternating chirality would facilitate the adoption of the flat conformation required for nanotube formation. Once again preliminary studies were carried out on dimeric models but in this case we prepared an all -cyclic peptide (entry 17, Table 1). This model again allowed us to confirm the adoption of the flat conformation and the formation of the antiparallel-type interaction between the two-peptide rings. This interaction is not responsible for nanotube formation with small cyclic peptides such as the four-residue derivatives, which form nanotubes through a parallel-type interaction [52]. Cytotoxicity studies of CPs that incorporate these recently prepared -Acas are undergoing. Recently it has been reported that amphipathic cationic six-residue D,L--CPs such as c-[L-Nle-W-H-S-K] (entry 18, Table 1) interact strongly with A protein and inhibit its aggregation [53]. The results were explained on the basis of the structural similarities between the amyloids and the

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

2657

Fig. (6). Model of the nanotube diversity generated by one single CP of C2-symmetry by the relative rotation of each cyclic peptide in the nanotube. This diversity is limited by the antiparallel-type arrangement and the required amino acid pairwise arrangement, because Dresidues can only be hydrogen bonded to D-amino acids and L to L-residues. This limitation is showed in the inset. Table 3.

Examples of biocidal cyclic peptides against biofouling algae.

Peptidesa

Hemolysis

U. Linza

HD50 (g/mL)

a

N. permituta

E. coli

MRSA

LD66 (M)

MIC (g/mL)

1

c-[K-W-L-F-F-K-]

50

40

20

7.5

40

2

c-[K-W-W-F-W-K-]

>100

10

40

30

100

3

c-[K-W-F-F-L-H-]

>100

10

>100

10

>100

4

c-[K-W-F-W-W-K-]

>100

30

20

100

>100

5

c-[K-W-F-F-F-H-]

>100

1

100

100

>100

Peptide sequences are written using the conventional one-letter code for amino acids, but residues having D stereochemistry are underlined.

peptide nanotube architecture. These nanotubes prevented A protein aggregation into toxic forms through preferential binding and stabilized its non-toxic form. Another interesting application of this peptide architecture is for use as a drug delivery system. In this respect, the transport properties of hydrophobic cyclic decapeptide c-[QL-(W-L-)4] were tested for the transport of the antitumor drug 5-fluorouracil (5-FU) and an enhancement in its antitumor potency was observed [54]. The transport mechanism was studied by computational methods, the results of which suggest that the drug was transported by hopping through different potential energy minima distributed along the peptide nanotube. The hopping mechanism was driven by switching from hydrophobic interactions between 5-FU and the interior wall of the nanotube to hydrogen bonding interactions with the backbone amide groups (NH and CO). More recently, breast cancer cell treatment using doxorubicinloaded cyclic peptides was also addressed. In this case, instead of using the hydrophobic CPs the authors exploited bundles of peptide nanotubes obtained by an induced selfassembly process of hydrophilic CPs that contained a cysteine residue (c-[A-Q-A-E-A-Q-A-C-], entry 19, Table 1). These bundles were loaded with the anti-tumoral drug and the external surface was then pegylated to reduce nanotube toxicity and slowdown their clearance. These DOX-loaded supramolecular aggregates demonstrated improved drug properties (cytotoxicity, uptake and intracellular distribution)

[55]. In addition, the DOX-loaded SCPNs inhibited the activity of permeable glycoprotein (P-gp) in the cancer cell lines studied (MCF-7/ADR), thus showing their potential to overcome the multidrug resistance in tumor therapy. A number of these peptides were also tested against a variety of cancer cell lines and the different cytotoxicities with respect to red blood cells also suggest the opportunity to design combinatorial libraries to search for CPs with selective toxicity against a specific cancer type expanding the biological applications of this technology [40]. CONCLUSION Cyclic peptide nanotubes are biocompatible supramolecular materials that might have potential applications in the development of new therapies. The main characteristic of these materials is their simple preparation as they are self-formed from small, readily available components (CPs) under appropriate conditions. In addition, the most important structural characteristics of the peptide nanotubes, i.e. diameter, external surface properties and nature of the internal cavity, can be adjusted simply by selecting the appropriate cyclic peptide properties. The only nanotube parameter for which control remains elusive is the length, because precise control of the number of peptide rings that are stacked in the nanotube requires additional structural information to be encoded in the CP. This limita-

2658 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

Rodríguez-Vázquez et al.

tion can be overcome in the transmembrane nanotube, in which the membrane length restricts the number of peptide subunits in the nanotube. In addition, the different environments in which the nanotubes are grown allows the preparation of heteromeric structures and this further broadens the properties of SCPNs. In this respect, the fact that nanotube growth is derived mainly from the backbone interactions (hydrogen bonds formation) means that the combination of several peptides with different properties would allow the formation of nanotubes with very different properties and whose formation is induced by the conditions in the medium. Furthermore, the tubular external surface properties can be tuned according to the environmental conditions through the simple inter-strand rotation between two consecutive CPs, a process that leads to the formation of non-equivalent interactions for each -sheet. Appropriate unit design and optimization of the conditions for selfassembly allows the nanotube properties to be tailored for specific applications. This possibility was exploited by the modification of a few residues to produce nanotubes whose orientation on the membrane is precisely controlled, i.e. perpendicular or parallel to the bilayer. Such orientations provided nanotubes with different properties, for instance mimics of ion channels with remarkable transport properties of a variety of molecular entities. The design of drugs based on a supramolecular approach carried out according to the principles found for these membrane-active peptides may provide the drugs of the future, which would allow a personal prescription depending on the patient and the type of disease, type of cancer, infection and so on. In addition, this approach might help in the important battle against drug-resistance. The molecular approach to drug discovery involving the precise design of lead compounds on the basis of the perfect fit of the pharmacophore in the target (enzymes, receptors or nucleic acids) facilitates inactivation through a few structural changes on the biological target. The supramolecular approach would have a large sequence space and these systems could maintain their mode of action despite gross structural changes. Indeed, the experiments carried out with S. aureus showed that resistance did not develop easily even after prolonged exposure to the peptides at sublethal concentrations, a finding in agreement with the proposed interaction with multiple components of the bacterial membrane.

-Ach

= 3-aminocyclohexanecarboxylic Acid

-Acp

= 3-aminocyclopentanecarboxylic Acid

LIST OF ABBREVIATIONS

[5]

Apr

= 4-aminoproline

BY

= O-benzyltyrosine

CP

= Cyclic Peptide

DOX

= Doxorubicin

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Economy and Competitivity (MEC) and the ERDF [CTQ201015725 and CTQ2013-43264-R] and by the Xunta de Galicia (GPC2013-039). M.A., N.R.-V. and H.L.O. thank the MEC for R&C (MA) and FPU contracts. J.M. and R.G-F. received Juan de la Cierva contracts from the MEC. M.P. Thanks the Foreign Affairs and Cooperation Ministry for a MAE fellowship. REFERENCES [1]

[2]

[3]

[4]

[6]

DPPC = 1,2-Dipalmitoylphosphatidylcholine MOPS = 3-(N-morpholino)propanesulfonic Acid MRSA = Methicillin-resistant Staphylococcus aureus SAA

= Sugar Amino Acids

SCPN = Self-assembling Cyclic Peptide Nanotubes VRE

= Vancomycin-resistant Enterococcus

-Aca

= 3-aminocycloalkanecarboxylic Acid

[7]

a) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Eds. The Compartmentalization of Cells in Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002; b) Kang, S.; Douglas, T. Some enzymes just need a space of their own. Science, 2009, 327, 42-43; c) Zhu, Y.; Power, B. E. Lab-on-achip in vitro Compartmentalization Technologies for Protein Studies. In Protein-Protein interactions Advances in Biochemical Engineering/Biotechnology; Werther, M.; Seitz, H. Eds. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008, Vol. 11, pp. 81; d) Leamon, J.H.; Link, D.R.; Egholm, M.; Rothberg, J.M. Overview: methods and applications for droplet compartmentalization of biology. Nature Methods, 2006, 3, 541–543. a) Gaber, B.P.; Schnur J.M.; Chapman, D. eds. Biotechnological Applications of Lipid Microstructures, Plenum, 1988, New York; b) Lasic, D.D. Liposomes: From Physics to Applications Elsevier, 1993, Amsterdam; c) Bangham, A.D.; Hill, M.W.; Miller, N.G.A. Preparation and use of liposomes as models of biological membranes. Methods Membr. Biol., 1973, 1, 1–68. a) Gouaux, E.; MacKinnon, R. Principles of selective ion transport in channels and pumps. Science 2005, 310, 1461-1465; b) Hucho, F.; Weise, C. Ligand-gated ion channels. Angew. Chem. Int. Ed., 2001, 40, 3100-3116. a) For some of the latest studies see special number in Acc. Chem. Res., 2013, 46, 2741–3008; b) Chui, J.K.W.; Fyles, T.M. Ionic conductance of synthetic channels: analysis, lessons, and recommendations. Chem. Soc. Rev., 2011, 41, 148–175; c) Fyles, T.M. Synthetic ion channels in bilayer membranes. Chem. Soc. Rev., 2007, 36, 335–347; d) Sisson, A.L.; Shah, M.R.; Bhosale, S.; Matile, S. Synthetic ion channels and pores. Chem. Soc. Rev., 2006, 35, 1269–1286. a) Hancock, R.E.; Lehrer, R. Cationic peptides: a new source of antibiotics. Trends Biotechnol., 1998, 16, 82-88; b) Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature, 2002, 415, 389-395; c) Brogden, K.A.; Ackermann, M.; McCray, P.B. Jr; Tack, B.F. Antimicrobial peptides in animals and their role in host defences. Int. J. Antimicrob. Agents, 2003, 22, 465-478. a) van’t Hof, W.; Veerman, E.C.; Helmerhorst, E.J.; Amerongen, A.V. Antimicrobial peptides: properties and applicability. Biol. Chem., 2001, 382, 597-619; b) Shai, Y. Mode of action of membrane active antimicrobial peptides. Biopol. (Peptide Sci.) 2002, 66, 236-248. a) Kandel, E.R.; Schwartz, J.; Jessell, T.; Siegelbaum, S.; Hudspeth, A.J. Principles of Neural Science, 5th ed.; McGraw-Hill Professional: New York, 2012; b) Olson, S.; De, R. Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins. Proc. Natl. Acad. Sci. USA, 2011, 108, 7385-7390; c) Mueller, M.; Grauschopf, U.; Maier, T.; Glockshuber, R.; Ban, N. The structure of a cytolytic -helical toxin pore reveals its assembly mechanism. Nature, 2009, 459, 726-730; d) Guillet, V.; Roblin, P.; Werner, S.; Coraiola, M.; Menestrina, G.; Monteil, H.; Prévost, G.; Mourey, L.

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes

[8]

[9]

[10]

[11]

[12]

[13]

[14]

Crystal Structure of Leucotoxin S Component: New insight into the staphylococcal.barrel pore-forming toxins. J. Biol. Chem., 2004, 279, 41028-41037; e) Song, L.; Hobaugh, M.R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J.E. Structure of Staphylococcal Hemolysin, a heptameric transmembrane pore. Science, 1996, 274, 1859-1865; f) Parker, M.W.; Buckley, J.T.; Postma, J.P.M.; Tucker, A.D.; Leonard, K.; Pattus, F.; Tsernoglou, D. Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature, 1994, 367, 292-295; g) Bayley H. Toxin structure: Part of a hole? Curr. Biol., 1997, 7, R763-R767. a) Hancock, R.E.W.; Sahl, H.-G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol., 2006, 24, 1551-1557; b) Melo, M.N.; Ferre, R.; Castanho, M.A.R.B. Antimicrobial peptides: Linking partition, activity and high membrane-bound concentrations. Nat. Rev. Microbiol., 2009, 7, 245-250. a) Fjell, C.D.; Hiss, J.A.; Hancock, R.E.W.; Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug Discov., 2012, 11, 37-51; b) Powers, J.P.; Hancock, R.E. The relationship between peptide structure and antibacterial activity. Peptides, 2003, 24, 1681-1691. a) Matsuzaki, K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta., 1999, 1462, 1–10; b) Brogden, K.A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol., 2005, 3, 238-250. a) O'Connell, K.M.G.; Hodgkinson, J.T.; Sore, H.F.; Welch, M.; Salmond, G.P.C.; Spring, D.R. Combating multidrug-resistant bacteria: Current strategies for the discovery of novel antibacterials. Angew. Chem. Int. Ed., 2013, 52, 10706-10733; b) Kennedy, D. Time to deal with antibiotics. Science, 2013, 342, 777-777; c) Engler, A.C.; Wiradharma, N.; Ong, Z.Y.; Coady, D.J.; Hedrick, J.L.; Yang, Y.-Y. Emerging trends in macromolecular antimicrobials to fight multi-drug-resistant infections. Nano. Today, 2012, 7, 201-222; d) Cooper, M.A.; Shlaes, D. Fix the antibiotics pipeline. Nature, 2011, 472, 32-32; e) Lewis, K. Recover the lost art of drug discovery. Nature, 2012, 485, 439-440; f) Bush, K.; Courvalin, P.; Dantas, G.; Davies, J.; Eisenstein, B.; Huovinen, P.; Jacoby, G.A.; Kishony, R.; Kreiswirth, B.N.; Kutter E.; Lerner, S.A.; Levy, S.; Lewis, K.; Lomovskaya, O.; Miller, J.H.; Mobashery, S.; Piddock, L.J.; Projan, S.; Thomas, C.M.; Tomasz, A.; Tulkens, P.M.; Walsh, T.R.; Watson, J.D.; Witkowski, J.; Witte, W.; Wright, G.; Yeh, P.; Zgurskaya, H.I.. Tackling antibiotic resistance. Nat. Rev. Microbiol., 2011, 9, 894-896; g) Nikaido, H. Multidrug resistance in bacteria. Annu. Rev. Biochem., 2009, 78, 119-146; h) Chambers, H.F.; Deleo, F.R. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat. Rev. Microbiol., 2009, 7, 629-641; i) Payne, D.J.; Gwynn, M.N.; Holmes, D.J.; Pompliano, D.L. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov., 2007, 6, 29-40; j) Lowy, F.D. Antimicrobial resistance: the example of Staphylococcus aureus. J. Clin. Invest., 2003, 111, 1265-1273. a) Som, A.; Vemparala, S.; Ivanov, I.; Tew, G.N. Synthetic mimics of antimicrobial peptides. Biopol. (Peptide Sci.) 2008, 90, 83-93; b) Wiradharma, N.; Khoe, U.; Hauser, C.A.; Seow, S.V.; Zhang, S.; Yang, Y.Y. Synthetic cationic amphiphilic -helical peptides as antimicrobial agents. Biomaterials, 2011, 32, 2204-2212; c) Young, A.W.; Liu, Z.; Zhou, C.; Totsingan, F.; Jiwrajka, N.; Shi, Z.; Kallenbach, N.R. Structure and antimicrobial properties of multivalent short peptides. Med. Chem. Com., 2011, 2, 308-314; d) Afacan, N.J.; Yeung, A.T.; Pena, O.M.; Hancock, R.E. Therapeutic potential of host defense peptides in antibiotic-resistant infections. Curr. Pharm. Des., 2012, 18, 807-819. a) Kass, R.S. The channelopathies: novel insights into molecular and genetic mechanisms of human disease. J. Clin. Inves. 2005, 115, 1986-1989; b) Gilman, S. Ed. Neurobiol. dis., Academic Press. 2010, pp. 319. a) Garcia-Fandiño, R.; Amorín, M.; Granja, J.R. Synthesis of Supramolecular Nanotubes (In Supramolecular Chemistry: From Molecules to Nanomaterials) (Gale, P. A; Steed, J. W. Eds.) John Wiley & Sons Ltd: New York, 2012, vol. 5, pp. 2149-2182; b) Chapman, R.; Danial, M.; Koh, M.L.; Jolliffe, K.A.; Perrier, S. Design and properties of functional nanotubes from the selfassembly of cyclic peptide templates. Chem. Soc. Rev., 2012, 41, 6023-6041; c) Brea, R.J.; Reiriz, C.; Granja, J.R. Towards

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

[15]

[16]

[17]

[18]

[19]

[20]

2659

functional bionanomaterials based on self-assembling cyclic peptide nanotubes. Chem. Soc. Rev., 2010, 39, 1448-1456; d) Bong, D.T.; Clark, T.D.; Granja, J.R.; Ghadiri, M.R. Self-assembling organic nanotubes. Angew. Chem. Int. Ed., 2001, 40, 988-1011. a) Ghadiri, M.R.; Granja, J.R.; Milligan, R.A.; Mcree, D.E.; Khazanovich, N. Self-assembling organic nanotubes based on a cyclic peptide architecture. Nature, 1993, 366, 324-327; b) Hartgerink, J.D.; Granja, J.R.; Milligan, R.A.; Ghadiri, M.R. Selfassembling peptide nanotubes. J. Am. Chem. Soc., 1996, 118, 4350; c) Rosenthal, K.; Svensson, G.; Undén, A. Self-assembling peptide nanotubes from enantiomeric pairs of cyclic peptides with alternating D and L-amino acid residues. J. Am. Chem. Soc., 2004, 126, 3372-3373. a) Ghadiri, M.R.; Kobayashi, K.; Granja, J.R.; Chadha, R.K.; McRee, D.E. The Structural and Thermodynamic Basis for the Formation of Self-Assembled Peptide Nanotubes. Angew. Chem., Int. Ed. Engl., 1995, 34, 93-95; b) Kobayashi, K.; Granja, J.R.; Ghadiri, M.R. -Sheet Peptide Architecture: Measuring the Relative Stability of Parallel vs. Antiparallel -Sheets. Angew. Chem., Int. Ed. Engl., 1995, 34, 95-98. a) Khazanovich, N.; Granja, J.R.; Mcree, D.E.; Milligan, R.A.; Ghadiri, M.R. Nanoscale Tubular Ensembles with Specified Internal Diameters. Design of a Self-Assembled Nanotube with a 13-Å Pore. J. Am. Chem. Soc., 1994, 116, 6011-6012; b) Sun, X.C.; Lorenzi, G. P. On the Stacking of -Rings: The solution selfassociation behavior of two partially N-methylated cyclo(hexaleucines). Helv. Chim. Acta, 1994, 77, 1520-1526; c) Amorin, M.; Brea, R.J.; Castedo, L.; Granja, J.R. The smallest ,peptide nanotubulet segments: Cyclic ,-tetrapeptide dimers. Org. Lett., 2005, 7, 4681-4684; d) Brea, R.J.; Castedo, L.; Granja, J.R. Large-diameter self-assembled dimers of ,-cyclic peptides, with the nanotubular solid-state structure of cyclo-[(L-Leu-D-MeN-Acp)4-].4CHCl2 COOH. Chem. Commun., 2007, 31, 3267-3269. For some of the most important applications of peptide nanotubes, see for example: a) Motesharei, K.; Ghadiri, M.R. Diffusionlimited size-selective ion sensing based on SAM-supported peptide nanotubes. J. Am. Chem. Soc., 1997, 119, 11306-11312; b) Vollmer, M.S.; Clark, T.D.; Steinem, C.; Ghadiri, M.R. Photoswitchable Hydrogen- Bonding in Self-Organized Cylindrical Peptide Systems. Angew. Chem. Int. Ed., 1999, 38, 1598-1601; c) Sanchez-Quesada, J.; Ghadiri, M.R.; Bayley, H.; Braha, O. Cyclic peptides as molecular adapters for a pore-forming protein. J. Am. Chem. Soc., 2000, 122, 11757-1766; d) Horne, W.S.; Ashkenasy, N.; Ghadiri, M.R. Modulating charge transfer through cyclic D,L-peptide self-assembly. Chem.–Eur. J., 2005, 11, 1137-1144; e) Ortiz-Acevedo, A.; Xie, H.; Zorbas, V.; Sampson, W.M.; Dalton, A.B.; Baughman, R.H.; Draper, R.K.; Musselman, I.H.; Dieckmann, G.R. Diameter-selective solubilization of single-walled carbon nanotubes by reversible cyclic peptides. J. Am. Chem. Soc., 2005, 127, 9512-9517; f) Couet, J.; Samuel, J.D.J.S.; Kopyshev, A.; Santer, S.; Biesalski, M. Peptide-polymer hybrid nanotubes. Angew. Chem. Int. Ed., 2005, 44, 3297-3301; g) Ashkenasy, N.; Horne, W.S.; Ghadiri, M.R. Design of self-assembling peptide nanotubes with delocalized electronic states. Small, 2006, 2, 99102; h) de la Rica, R.; Pejoux, C.; Matsui, H. Assemblies of Functional Peptides and Their Applications in Building Blocks for Biosensors. Adv. Funct. Mater., 2011, 21, 1018-1026; i) Xu, T.; Zhao, N.; Ren, F.; Hourani, R.; Tsang Lee, M.; Shu, J.Y.; Mao, S.; Helms, B.A. Subnanometer porous thin films by the co-assembly of nanotube subunits and block copolymers. ACS Nano., 2011, 2, 1376-1384; j) Mizrahi, M.; Zakrassov, A.; Lerner-Yardeni, J.; Ashkenasy, N. Charge transport in vertically aligned, self-assembled peptide nanotube junctions. Nanoscale, 2012, 4, 518-524; k) Chapman, R.; Warr, G.G.; Perrier, S.; Jolliffe, K.A. Water-soluble and pH-responsive polymeric nanotubes from cyclic peptide templates. Chem.–Eur. J., 2013, 19, 1955-1961. Peptide sequences are written using the conventional one-letter code for amino acids, but residues having D stereochemistry are underlined. a) Seebach, D.; Matthews, J.L.; Meden, A.; Wessels, T.; Baerlocher, C.; McCusker, L.B. Cyclo--peptides: Structure and tubular stacking of cyclic tetramers of 3-aminobutanoic acid as determined from powder diffraction data. Helv. Chim. Acta., 1997, 80, 173182; b) Ishihara, Y.; Kimura, S. Peptide nanotube composed of cyclic tetra--peptide having polydiacetylene. Biopolymers, 2012, 98, 155-160; c) Gauthier, D.; Baillargeon, P.; Drouin, M.; Dory,

2660 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

[21]

[22]

[23]

[24]

[25]

[26]

[27] [28]

[29]

Y.L. Self-assembly of cyclic peptides into nanotubes and then into highly anisotropic crystalline materials. Angew. Chem. Int. Ed., 2001, 40, 4635-4638; d) Horne, W.S.; Stout, C.D.; Ghadiri, M.R. A Heterocyclic Peptide Nanotube. J. Am. Chem. Soc., 2003, 125, 9372-9376; e) Alfonso, I.; Bru, M.; Burguete, M.I.; GarcíaVerdugo, E.; Luis, S.V. Structural diversity in the self-assembly of pseudopeptidic macrocycles. Chem.–Eur. J., 2010, 16, 1246-1255; f) Semetey, V.; Didierjean, C.; Briand, J.P.; Aubry, A.; Guichard, G. Self-assembling organic nanotubes from enantiopure cycloN,N'-linked oligoureas: Design, synthesis, and crystal structure. Angew. Chem. Int. Ed., 2002, 41, 1895-1898; g) Fischer, L.; Decossas, M.; Briand, J.P.; Didierjean, C.; Guichard, G. Control of duplex formation and columnar self-assembly with heterogeneous amide/urea macrocycles. Angew. Chem. Int. Ed., 2008, 48, 16251628; h) López, J.L.; Pérez, E.M.; Viruela, P.M.; Viruela, R.; Ortí, E.; Martín, N. Controlled self-assembly of electron donor nanotubes. Org. Lett., 2009, 11, 4524-4527. a) Amorin, M.; Castedo, L.; Granja, J.R. New cyclic peptide assemblies with hydrophobic cavities: the structural and thermodynamic basis of a new class of peptide nanotubes. J. Am. Chem. Soc., 2003, 125, 2844-2845; b) Amorin, M.; Castedo, L.; Granja J.R. Self-assembled peptide tubelets with 7 Å pores. Chem.–Eur. J., 2005, 11, 6543-6551; c) Reiriz, C.; Brea, R.J.; Arranz, R.; Carrascosa, J.L.; Garibotti, A.; Manning, B.; Valpuesta, J.M.; Eritja, R.; Castedo, L.; Granja, J.R. ,-Peptide nanotube templating of one-dimensional parallel fullerene arrangements. J. Am. Chem. Soc., 2009, 131, 11335-11337; d) Montenegro, J.; Vázquez-Vázquez, C.; Kalinin, A.; Geckeler, K.E.; Granja, J.R. Coupling of carbon and peptide nanotubes. J. Am. Chem. Soc., 2014, 136(6), 2484-91. a) Brea, R.J.; Amorin, M.; Castedo, L.; Granja, J.R. Methylblocked dimeric ,-peptide nanotube segments: Formation of a peptide heterodimer through backbone-backbone interactions. Angew. Chem. Int. Ed., 2005, 44, 5710-5713; b) Brea, R.J.; PérezAlvite, M.J.; Panciera, M.; Mosquera, M.; Castedo, L.; Granja, J.R. Highly efficient and directional homo- and heterodimeric energy transfer materials based on fluorescently derivatized ,-cyclic octapeptides. Chem.–Asian. J., 2011, 6, 110-121. a) Brea, R.J.; Vazquez, M.E.; Mosquera, M.; Castedo, L.; Granja, J.R. Controlling multiple fluorescent signal output in cyclic peptide-based supramolecular systems. J. Am. Chem. Soc., 2007, 129, 1653-1657; b) Brea, R.J.; Castedo, L.; Granja, J.R.; Herranz, M.Á.; L. Sanchez, L.; Martin, N.; Seitz, W.; Guldi, D.M. Electron transfer in Me-blocked heterodimeric ,-peptide nanotubular donor-acceptor hybrids. Proc. Natl. Acad. Sci. USA, 2007, 104, 5291-5294; c) Aragay, G.; Pintre, I.; Guerra, A.; Ventura,V; Chiorboli, C.; García-Fandiño, R.; Flamigni, L.; Granja, J.R.; Ballester, P. Self-sorting of cyclic peptide homodimers into a heterodimeric assembly featuring an efficient photoinduced intramolecular electron-transfer process. Chem.–Eur. J., DOI: 10.1002/chem.201304200. a) Ghadiri, M.R.; Granja, J.R.; Buehler, L.K. Artificial transmembrane ion channels from self-assembling peptide nanotubes. Nature, 1994, 369, 301-304; b) Montenegro, J.; Ghadiri, M.R.; Granja, J.R. Ion channel models based on self-assembling cyclic peptide nanotubes. Acc. Chem. Res., 2013, 46, 2955-2965. a) Haris, P.I.; Chapman, D. The conformational analysis of peptides using FTIR. Biopoly. (Peptide Sci.) 1995, 37, 251-263; b) Krimm, S.; Bandekar, J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Pro. Chem., 1986, 38, 181-364; c) Bandekar, J. Amide modes and protein conformation. Biochim. Biophys. Acta, 1992, 1120, 123-143. Clark, T.D.; Buriak, J.M.; Kobayashi, K.; Isler, M.P.; McRee, D.E.; Ghadiri, M.R. Cylindrical -sheet peptide assemblies. J. Am. Chem. Soc., 1998, 120, 8949-8962. Hodkin, A.L.; Katz, B. The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol., 1949, 108, 3777. Sanchez-Quesada, J.; Isler, M.; Ghadiri, M.R. Modulating ion channel properties of transmembrane peptide nanotubes through heteromeric supramolecular assemblies. J. Am. Chem. Soc., 2002, 124, 10004-10005. a) Clark, T.D.; Buehler, L.K.; Ghadiri, M.R. Self-assembling cyclic 3-peptide nanotubes as artificial transmembrane ion channels. J. Am. Chem. Soc., 1998, 120, 651-656; b) Ranganathan, D. Designer hybrid cyclopeptides for membrane ion transport and tubular

Rodríguez-Vázquez et al.

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

structures. Acc. Chem. Res., 2001, 34, 919-930; c) Hennig, A.; Fischer, L.; Guichard, G.; Matile, S. Anion-macrodipole interactions: Self-assembling oligourea/amide macrocycles as anion Transporters that respond to membrane polarization. J. Am. Chem. Soc., 2009, 131, 16889-16895. a) Granja, J.R.; Ghadiri, M.R. Channel-mediated transport of glucose across lipid bilayers. J. Am. Chem. Soc., 1994, 116, 1078510786; b) Sanchez-Quesada, J.; Kim, H.S.; Ghadiri, M.R. A Synthetic pore-mediated transmembrane transport of glutamic acid. Angew. Chem. Int. Ed., 2001, 40, 2503-2506. a) García-Fandiño, R.; Granja, J.R.; D'Abramo, M.; Orozco, M. Theoretical characterization of the dynamical behavior and transport properties of ,-peptide nanotubes in solution. J. Am. Chem. Soc., 2009, 131, 15678-15686; b) García-Fandiño, R.; Granja, J.R. Effect of organochloride guest molecules on the stability of homo/hetero self-assembled ,-cyclic peptide structures: A computational study toward the control of nanotube length. J. Phys. Chem. C., 2013, 117, 10143-10162. García-Fandiño, R.; Amorín, M.; Castedo, L.; Granja, J.R. Transmembrane ion transport by self-assembling ,-peptide nanotubes. Chem. Sci., 2012, 3, 3280-3285. Hourani, R.; Zhang, C.; van der Weegen, R.; Ruiz, L.; Li, C.; Keten, S.; Helms, B.A.; Xu, T. Processable Cyclic Peptide Nanotubes with Tunable Interiors. J. Am. Chem. Soc., 2011, 133, 15296-15299. a) Rodríguez-Vázquez, N.; Salzinger, S.; Silva, L.F.; Amorín, M.; Granja, J.R. Synthesis of cyclic -amino acids for foldamers and peptide nanotubes. Eur. J. Org. Chem., 2013, 2013, 3477-3482; b) Reiriz, C.; Amorín, M.; García-Fandiño, R.; Castedo, L.; Granja, J.R. ,-Cyclic peptide ensembles with a hydroxylated cavity. Org. Biomol. Chem., 2009, 7, 4358-4361. a) Zaydman, M.A.; Silva, J.R.; Cui, J. Ion channel associated diseases: Overview of molecular mechanisms. Chem. Rev., 2012, 112, 6319-6333; b) Maffeo, C.; Bhattacharya, S.; Yoo, J.; Wells, D.; Aksimentiev, A. Modeling and simulation of ion channels. Chem. Rev., 2012, 112, 6250-6284. a) Engels, M.; Bashford, D.; Ghadiri, M.R. Structure and dynamics of self-assembling peptide nanotubes and the channel-mediated water organization and self-diffusion. A molecular dynamics study. J. Am. Chem. Soc., 1995, 117, 9151-9158; b) Tarek, M.; Maigret, B.; Chipot, C. Molecular dynamics investigation of an oriented cyclic peptide nanotube in DMPC bilayers. Biophys. J., 2003, 85, 2287-2298; c) Hwang, H.; Schatz, G.C.; Ratner, M.A. Steered molecular dynamics studies of the potential of mean force of a Na+ or K+ ion in a cyclic peptide nanotube. J. Phys. Chem. B., 2006, 110, 26448-26460; d) Hwang, H.; Schatz, G.C.; Ratner, M.A. Ion current calculations based on three dimensional PoissonNernstPlanck theory for a cyclic peptide nanotube. J. Phys. Chem. B., 2006, 110, 6999-7008; e) Chipot, C.; Tarek, M. Interaction of a peptide nanotube with a water–membrane interface. Phys. Biol., 2006, 3, S20-S25; f) Dehez, F.; Tarek, M.; Chipot, C. Energetics of Ion Transport in a Peptide Nanotube. J. Phys. Chem. B., 2007, 111, 10633-10635; g) Zhu, J.; Cheng, J.; Liao, Z.; Lai, Z.; Liu, B. Investigation of structures and properties of cyclic peptide nanotubes by experiment and molecular dynamics. J. Comput.Aided Mol. Des., 2008, 22, 773-781; h) Khalfa, A.; Treptow, W.; Maigret, B.; Tarek, M. Self assembly of peptides near or within membranes using coarse grained MD simulations. Chem. Phys., 2009, 358, 161-170; i) Liu, J.; Fan, J.; Tang, M.; Zhou, W. Molecular dynamics simulation for the structure of the water chain in a transmembrane peptide nanotube. J. Phys. Chem. A., 2010, 114, 2376-2383; j) Vijayaraj, R.; Damme, S. V.; Bultinck, P.; Subramaniam, V. Molecular dynamics and umbrella sampling study of stabilizing factors in cyclic peptide-based nanotubes. J. Phys. Chem. B., 2012, 116, 9922-9933; k) Cheng, J.; Zhu, J.; Liu, B. Molecular modeling investigation of adsorption of selfassembled peptide nanotube of cyclo-[(1R,3S)--Acc-D-Phe]3 in CHCl3 . Chem. Phys., 2007, 333, 105-110. García-Fandiño, R.; Castedo, L.; Granja, J.R.; Vázquez S. Interaction and dimerization energies in methyl-blocked ,peptide nanotube segments. J. Phys. Chem. B., 2010, 114, 49734983. García-Fandiño, R.; Outeiral, J.; Vázquez S.; Granja, J.R. Study of the transmembrane ion transport by biomimetic pores based on derivatized self-assembling ,-peptide nanotubes through Molecular Dynamics simulations. Manuscript in preparation.

Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes [39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

Kim, H.S.; Hartgerink, J. D.; Ghadiri, M.R. Oriented self-assembly of cyclic peptide nanotubes in lipid membranes. J.Am. Chem. Soc., 1998, 120, 4417-4424. Fernández-López, S.; Kim, H.-S.; Choi, E.C.; Delgado, M.; Granja, J.R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D.A.; Wilcoxen, K.M.; Ghadiri, M.R. Antibacterial agents based on the cyclic D,L--peptide architecture. Nature, 2001, 412, 452-455. It should be noted that although hemolytic activity is typically used as an indicator of toxicity, it has been found that some nonhemolytic antimicrobial cyclic D,L--peptides were acutely toxic in mice, see following reference [42]. Dartois, V.; Sánchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde, C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M.R.; Parr T.R. Systemic antibacterial activity of novel synthetic cyclic peptides. Antimicrob. Agents Chemother., 2005, 49, 33023310. Fletcher, J.T.; Finlay, J.A.; Callow, M.E.; Callow, J.A.; Ghadiri, M.R. A combinatorial approach to the discovery of biocidal sixresidue cyclic D,L--peptides against the bacteria methicillinresistant Staphylococcus aureus (MRSA) and E. coli and the Biofouling Algae Ulva linza and Navicula perminuta. Chem.–Eur. J., 2007, 13, 4008-4013. Horne, W.S.; Wiethoff, C.M.; Cui, C.; Wilcoxen, K.M.; Amorín, M.; Ghadiri, M.R.; Nemerow, G.R. Antiviral cyclic D,L-peptides: Targeting a general biochemical pathway in virus infections. Bioorg. Med. Chem., 2005, 13, 5145-5135. a) Koehn, F.E. New strategies and methods in the discovery of natural product anti-infective agents: the mannopeptimycins. J. Med. Chem., 2008, 51, 2613-2617; b) He, H. Mannopeptimycins, a novel class of glycopeptide antibiotics active against gram-positive bacteria. Appl. Microbiol. Biotechnol., 2005, 67, 444-452; c) He, H.; Williamson, R.T.; Shen, B.; Graziani, E.I.; Yang, H.Y.; Sakya, S.M.; Petersen, P.J.; Carter, G.T. Mannopeptimycins, novel antibacterial glycopeptides from Streptomyces hygroscopicus, LLAC98. J. Am. Chem. Soc., 2002, 124, 9729-9736. Bulet, P.; Dimarcq, J.L.; Hetru, C.; Lagueux, M.; Charlet, M.; Hegy, G.; van Dorsselaer, A.; Hoffmann, J.A. A novel inducible

Received: January 30, 2014

Accepted: October 20, 2014

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 23

[47]

[48] [49]

[50] [51]

[52]

[53]

[54]

[55]

2661

antibacterial peptide of Drosophila carries an O-glycosylated substitution. J. Biol. Chem., 1993, 268, 14893-14897. Motiei, L.; Rahimipour, S.; Thayer, D.A.; Wong, C.-H.; Ghadiri, M.R. Antibacterial cyclic D,L--glycopeptides. Chem. Commun., 2009, 3693–3695. Unpublished results. Amorín, M.; Castedo, L.; Granja, J.R. Folding Control in Cyclic Peptides through N-Methylation Pattern Selection: Formation of Antiparallel -Sheet Dimers, Double Reverse Turns and Supramolecular Helices by 3, Cyclic Peptides. Chem.–Eur. J., 2008, 14, 2100-2111. Guerra, A.; Brea, R.J.; Amorín, M.; Castedo, L.; Granja, J.R. Selfassembling properties of all -cyclic peptides containing sugar amino acid residues. Org. Biomol. Chem., 2012, 10, 8762-8766. von Roedern, E.G.; Lohof, E.; Hessler, G.; Hoffmann, M.; Kessler, H. Synthesis and conformational analysis of linear and cyclic peptides containing sugar amino acids. J. Am. Chem. Soc., 1996, 118, 10156-10157. Li, L.; Zhan, H.; Duan, P.; Liao, J.; Quan, J.; Hu, Y.; Chen, Z.; Zhu, J.; Liu, M.; Wu, Y.-D.; Deng, J. Self-Assembling Nanotubes Consisting of Rigid Cyclic -Peptides. Adv. Funct. Mater., 2012, 22, 3051-3056. Richman, M.; Wilk, S.; Chemerovski, M.; Wärmländer, S.K.T.S.; Wahlström, A.; Gräslund, A.; Rahimipour, S. In vitro and Mechanistic Studies of an Antiamyloidogenic Self- Assembled Cyclic D,L--Peptide Architecture. J. Am. Chem. Soc., 2013, 135, 3474-3484. Liu, H.; Chen, J.; Shen, Q.; Fu, W.; Wu, W. Molecular Insights on the Cyclic Peptide Nanotube-Mediated Transportation of Antitumor Drug 5-Fluorouracil. Mol. Pharmaceut., 2010, 7, 19851994. Wang, Y.; Yi, S.; Sun, L.; Huang, Y.; Lenaghan, S.C.; Zhang, M. Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells. J. Biomed. Nanotechnol., 2014, 10, 445-454.