Phospholipid-Dependent Functions of a Macrocyclic Analogue of the Ion-Channel-Forming Antibiotic Gramicidin A.

An ion-channel-forming natural peptide, gramicidin A (1), exhibits potent antimicrobial activity against Gram-positive bacteria, although medical applications are limited to topical use due to its mammalian cytotoxicity. We recently reported that the artificial macrocyclic analogue 2 provides a promising starting point for developing new ion-channel-based systemic antibacterial agents because of its low mammalian cytotoxicity compared to that of the parent 1. To dissect the molecular factors involved in the species selectivity of 2, we evaluated the ion transport activities, phospholipid affinities, and conformational properties of 1 and 2 using various compositions of phospholipids. A combination of lipid dot blot assays and circular dichroism (CD) analysis with H+/Na+ exchange assays revealed that the higher H+/Na+ exchange activity of 2 than that of 1 in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) is attributable to its higher affinity towards the phospholipids than that of 1. Notably, we also discovered that 2 showed weaker H+/Na+ exchange activity in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). CD analysis of 2 in liposomes indicated that the weak H+/Na+ exchange activity is induced by disturbance of the ion-conducting ß6.3-helical conformation in the POPE-containing lipid bilayer. These results suggest that the POPE-induced attenuation of the ion-conducting activity of 2 contributes to the alleviation of undesirable mammalian cytotoxicity of 2 compared to that of 1.

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

Photopharmacological Control of Cyclic Antimicrobial Peptides.

Gramicidin S is a naturally occurring antimicrobial cyclic peptide. Herein, we present a series of cyclic peptides based on gramicidin S that contain an azobenzene photoswitch to reversibly control secondary structure and, hence, antimicrobial activity. 1 H NMR spectroscopy and density functional theory calculations revealed a ß-sheet/ß-turn secondary structure for the cis configuration of each peptide, and an ill-defined conformation for all associated trans structures. The cis-enriched and trans-enriched photostationary states (PSSs) for peptides 1-3 were assayed against Staphylococcus aureus to reveal a clear relationship between well-defined secondary structure, amphiphilicity and optimal antimicrobial activity. Most notably, peptides 2 a and 2 b exhibited a fourfold difference in antimicrobial activity in the cis-enriched PSS over the trans-enriched equivalent. This photopharmacological approach allows antimicrobial activity to be regulated through photochemical control of the azobenzene photoswitch, thereby opening new avenues in the design and synthesis of future antibiotics.

Bioactivity of topologically confined gramicidin A dimers.

The d-/l-peptide gramicidin A (gA) is well known as a pivotal ion channel model and shows a broad spectrum of bioactivities such as antibiosis, antimalarial activity, as well as hemolysis. We applied inter-chain disulfide bonds to constrain the conformational freedom of gA into parallel and antiparallel dimeric topologies. Albeit the constructs were not found to be monoconformational, CD- and IR-spectroscopic studies suggested that this strategy indeed restricted the conformational space of the d-/l-peptide construct, and that ß-helical secondary structures prevail. Correlative testing of gA dimers in antimicrobial, antimalarial, and ion conduction assays suggested that the tail-to-tail antiparallel single stranded ß6.3 helix dominantly mediates the bioactivity of gA. Other conformers are unlikely to contribute to these activities. From these investigations, only weakly ion conducting gA dimers were identified that retained nM antimalarial activity.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles.

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

The pH-dependent induction of lipid membrane ionic permeability by N-terminally lysine-substituted analogs of gramicidin A.

Insertion of charged groups at the N-terminus of the gramicidin A (gA) amino acid sequence is considered to be fatal for peptide channel-forming activity because of hindrance to the head-to-head dimer formation. Here the induction of ionic conductivity in planar bilayer lipid membranes (BLM) was studied with gA analogs having lysine either in the first ([Lys1]gA) or the third ([Lys3]gA) position. If added to the bathing solution at neutral or acidic pH, these analogs, being protonated and thus positively charged, were unable to induce ionic current across BLM. By contrast, at pH 11 the induction of BLM conductivity was observed with both lysine-substituted analogs. Based on the dependence of the macroscopic current on the side of the peptide addition, sensitivity to calcium ions and susceptibility to sensitized photoinactivation, as well as on the single-channel properties of the analogs, we surmise that at alkaline pH [Lys1]gA formed channels with predominantly single-stranded structure of head-to-head helical dimers, whereas [Lys3]gA open channels had the double-stranded helical structure. CD spectra of the lysine-substituted analogs in liposomes were shown to be pH-dependent.

Fatty acyl-gramicidin S derivatives with both high antibiotic activity and low hemolytic activity.

In the present study, novel eight GS derivatives having the octanoyl-(Lys)(n)- moieties, cyclo{-Val-Orn-Leu-d-Phe-Pro(4ß-NH-X)-Val-Orn-Leu-d-Phe-Pro-} {X=-H (1), and -(Lys)(n)-CO(CH(2))(6)CH(3)n=0 (2), 1 (3), 2 (4), and 3 (5)} and cyclo{-Val-Orn-Leu-d-Phe-Pro(4α-NH-X)-Val-Orn-Leu-d-Phe-Pro-} {X=-H (6), and -(Lys)(n)-CO(CH(2))(6)CH(3)n=1 (7), and 2 (8)} were synthesized. Among them, 4, 5 and 8 result the high antibiotic activity against both Gram-positive and Gram-negative microorganisms tested. In addition, 4 and 5 showed very low hemolytic activity compared with that of GS. Thus, the introduction of the excess amino groups and the fatty acyl moiety to the γ-NH(2) group of Pro(5) residue in GS molecule lowered the unwanted hemolytic activity and enhanced the desired antibiotic activity.

'Inverted' analogs of the antibiotic gramicidin S with an improved biological profile.

A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.

Polycationic gramicidin S analogues with both high antibiotic activity and very low hemolytic activity.

The substitution of each constituent amino acid residue of gramicidin S (GS), cyclo(-Val(1,1')-Orn(2,2')-Leu(3,3')-D-Phe(4,4')-Pro(5,5')-)(2) with Lys residue indicated that each side chain structure of the constituent amino acid residues affect largely the antibiotic activity and hemolytic activity of GS. Further, the substitution of D-Phe(4,4') and Pro(5,5') residues with basic amino acid residues as a Lys residue results the high antibiotic activity and the very low hemolytic activity. Thus, we have found novel positions on the scaffold of GS at D-Phe(4,4') and Pro(5,5') residues whose modification will significantly increase the therapeutic index.

Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives.

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.

Novel cycloundecapeptides related to gramicidin S with both high antibiotic activity and low hemolytic activity.

To find candidates with high antimicrobial and low hemolytic activities, many gramicidin S (GS) analogs of various ring sizes have been designed and synthesized. However, syntheses of antimicrobially active analogues of GS having a disordered symmetry structure from C(2) have almost never been reported, because the stable, amphiphilic ß-sheet structure of GS with C(2) symmetry is considered essential for its strong antibacterial activity. In the present studies, novel thirteen cycloundecapeptides 1-13 related to GS were synthesized and examined. Among them, cyclo(-Va1(1)-Orn(2)-Leu(3)-D-Phe(4)-X(5)-Pro(6)-Val(7)-Orn(8)-Leu(9)-D-Phe(10)-Pro(11)-) (X=Lys (10), Orn (11), Arg (12) and Lys(Lys) (13)) resulted in high antibiotic activity against both Gram-positive and Gram-negative microorganisms tested. In addition, 11 showed low toxicity against sheep blood cells compared with that of GS. Further, circular dichroism (CD) spectra of 10-13 had a curve similar to each other, suggesting that the conformations of these analogues in methanol are similar to each other. However, CD spectra of 10-13 were different from that of GS in the 190-210 nm region. These results suggest that the presences of one added amino acid residue at position 5 of 10-13 might be partially effective through a structural change in the biological activity of 10-13. In addition, the structural modifications at position 5 lower the undesirable hemolytic activity and enhance the desirable antibiotic activity.

On resin amino acid side chain attachment strategy for the head to tail synthesis of new glutamine containing gramicidin-S analogs and their antimicrobial activity.

The alarming increase in infections caused by multiple drug resistant bacteria including methicillin-resistant Staphylococcus aureus has prompted a desperate search for new antimicrobials. Augmenting the discoveries of completely new scaffolds with antimicrobial activity are efforts aimed at modifying existing molecules to optimize activity or reduce toxicity. We report herein the parallel solid-phase synthesis of analogues of the cationic antimicrobial peptide gramicidin S (GS) using amino acid side chain attachment strategy. The ornithine (Orn) residues were replaced by glutamine (Gln) and the aromatic D-phenylalanine (Phe) were replaced by different aromatic D-amino acids. Additional Gln containing GS analogues with all the possible combinations of the hydrophobic amino acids valine and leucine were also synthesized. In this work we also report the antibacterial activity of these analogs against several clinically-important drug-resistant Gram-positive and Gram-negative pathogens.

Antimicrobially active cycloundecapeptides related to gramicidin S having a novel turn structure with cis D-Phe-Pro peptide bond.

Antimicrobially active cycloundecapeptides related to gramicidin S, cyclo(-Val1-Orn2-Leu3-X4-D-Phe5-Pro6-Val7-Orn8- Leu9-D-Phe10-Pro11-) (X= Leu (1), Ala (2), Orn (3), Lys (4) and Arg (5)), were synthesized. From the CD and NMR studies, 1-5 possess antiparallel -sheet conformation linked by a type II -turn around D-Phe10-Pro11 and a novel turn structure around X4-D-Phe5-Pro6 sequence with cis D-Phe-Pro peptide bond. The structural modifications at position 4 of 1-5 are beneficial to identification of novel antibiotic candidates without hemolytic activity.

Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Development of Therapeutic Gramicidin S Analogues Bearing Plastic ß,γ-Diamino Acids.

Gramicidin S (GS), one of the most widely investigated antimicrobial peptides (AMPs), is known for its robust antimicrobial activity. However, it is restricted to topical application due to undesired hemolytic activity. With the aim of obtaining nontoxic GS analogues, we describe herein a molecular approach in which the native GS ß-turn region is replaced by synthetic ß,γ-diamino acids (ß,γ-DiAAs). Four ß,γ-DiAA diastereomers were employed to mimic the ß-turn structure to afford GS analogues GS3-6, which exhibit diminished hemolytic activity. A comparative structural study demonstrates that the (ßR,γS)-DiAA is the most-stable ß-turn mimic. To further improve the therapeutic index (e. g., high antibacterial activity and low hemolytic activity) and to extend the molecular diversity, GS5 and GS6 were used as structural scaffolds to introduce additional hydrophobic or hydrophilic groups. We show that GS6K, GS6F and GS display comparable antibacterial activity, and GS6K and GS6F have significantly decreased toxicity. Moreover, antibacterial mechanism studies suggest that GS6K kills bacteria mainly through the disruption of the membrane.

Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified D-phenylalanine residues.

The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified D-phenylalanine residue, their antibacterial properties against several gram positive and negative strains, as well as their hemolytic activity is reported.

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes.

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic beta-sheet-beta-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar identical with Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic-exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K(b)). The K(b1) and K(b2) values for endothermic two-step binding processes corresponded to high and low binding affinities (K(b1) >or= 100 K(b2)). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S.

The cationic beta-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK(4) to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK(4) to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK(4) to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK(4) varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK(4) can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK(4) binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.

Chemically reactive derivatives of gramicidin A for developing ion channel-based nanoprobes.

Ion channel-forming peptides and proteins offer tremendous opportunities for fundamental and applied studies of function on individual molecules. An ongoing challenge in ion channel research is the lack of simple and accessible synthetic methods to engineer pores with tailored chemical and physical properties. This paper describes a practical synthetic route to rapidly generate C-terminally modified derivatives of gramicidin A (gA), an ion channel-forming peptide, through the use of two chemically reactive gA-based building blocks. These amine- and azide-containing building blocks can react readily with typical substrates for amidation and 1,3-dipolar cycloaddition ("click") reactions to present molecules with desired structure and functionality near the opening of a gA pore. These derivatives of gA are stable under typical aqueous conditions for ion channel recordings and retain characteristic single ion channel conductance properties in planar lipid bilayers. Additionally, the synthetic methods described here afford useful quantities of these gA derivatives in good purity and yield with minimal purification. We demonstrate that derivatives of gA can be used for studying, in situ, a change in conductance through a channel upon performing a "click" reaction on an azide moiety attached to the gA pore. We also demonstrate that these gA-based building blocks can be used to construct sensors for the recognition of specific protein-ligand binding interactions in solution. This widely accessible, enabling synthetic methodology represents a powerful new tool to study relationships between chemical structure and function on the single molecule level.