ABSTRACT
New strategies facilitate the design of cyclic peptides which can penetrate the brain. We have designed a bicyclic peptide, OL-CTOP, composed of the sequences of a selective µ-opioid receptor antagonist, CTOP (f-cyclo(CYwOTX)T) (X = penicillamine, Pen; O = ornithine) and odorranalectin, OL (YASPK-cyclo(CFRYPNGVLAC)T), optimized its solid-phase synthesis and demonstrated its ability for nose-to-brain delivery and in vivo activity. The differences in reactivity of Cys and Pen thiol groups protected with trityl and/or acetamidomethyl protecting groups toward I2 in different solvents were exploited for selective disulfide bond formation on the solid phase. Both the single step and the sequential strategy applied to macrocyclization reactions generated the desired OL-CTOP, with the sequential strategy yielding a large quantity and better purity of crude OL-CTOP. Importantly, intranasally (i.n.s.) administered OL-CTOP dose-dependently antagonized the analgesic effect of morphine administered to mice through the intracerebroventricular route and prevented morphine-induced respiratory depression. In summary, the results demonstrate the feasibility of our solid-phase synthetic strategy for the preparation of the OL-CTOP bicyclic peptide containing two disulfide bonds and reveal the potential of odorranalectin for further modifications and the targeted delivery to the brain.
Subject(s)
Solid-Phase Synthesis Techniques , Somatostatin , Mice , Animals , Administration, Intranasal , Somatostatin/pharmacology , Receptors, Opioid, mu , Peptides/pharmacology , Morphine/pharmacologyABSTRACT
Fluorescently labelled alanine scan analogues of odorranalectin (OL), a cyclic peptide that exhibits lectin like properties, were screened for binding BSA-conjugated monosaccharides using an enzyme-linked lectin assay (ELLA). Results revealed that Lys5, Phe7, Tyr9, Gly12, Leu14, and Thr17 were crucial for binding BSA-L-fucose, BSA-D-galactose and BSA-N-acetyl-D-galactosamine. Notably, Ala substitution of Ser3, Pro4, and Val13 resulted in higher binding affinities compared to the native OL. The obtained data also indicated that Arg8 plays an important role in differentiation of binding for BSA-L-fucose/D-galactose from BSA-N-acetyl-D-galactosamine. The thermodynamics of binding of the selected alanine analogues was evaluated by isothermal titration calorimetry. Low to moderate binding affinities were determined for the tetravalent MUC1 glycopeptide and asialofetuin, respectively, and high for the fucose rich polysaccharide, fucoidan. The thermodynamic profile of interactions with asialofetuin exhibits shift to an entropy-driven mechanism compared to the fucoidan, which displayed an enthalpyentropy compensation, typically associated with the carbohydratelectin recognition process.
ABSTRACT
Both acute and chronic cutaneous wounds are often difficult to treat due to the high-risk for bacterial contamination. Once hospitalized, open wounds are at a high-risk for developing hospital-associated infections caused by multi drug-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. Treating these infections is challenging, not only because of antibiotic resistance, but also due to the production of biofilms. New treatment strategies are needed that will help in both stimulating the wound healing process, as well as preventing and eliminating bacterial wound infections. Fusaricidins are naturally occurring cyclic lipopeptides with antimicrobial properties that have shown to be effective against a variety of fungi and Gram-positive bacteria, with low toxicity. Continuing with our efforts toward the identification of novel cyclic lipopeptides Fusaricidin analogs, herein we report the synthesis and evaluation of the antimicrobial activity for two novel cyclic lipopeptides (CLP), CLP 2605-4 and CLP 2612-8.1 against methicillin resistant S. aureus and P. aeruginosa, respectively, in in vivo porcine full thickness wound model. Both CLPs were able to reduce bacterial counts by approximately 3 log CFU/g by the last assessment day. Peptide 2612-8.1 slightly enhanced the wound healing, however, wounds treated with peptide 2605-4, have shown higher levels of inflammation and impaired wound healing process. This study highlights the importance of identifying new antimicrobials that can combat bacterial infection while not impeding tissue repair.
ABSTRACT
We report two routes of chemical synthesis of arsinothricin (AST), the novel organoarsenical antibiotic. One is by condensation of the 2-chloroethyl(methyl)arsinic acid with acetamidomalonate, and the second involves reduction of the N-acetyl protected derivative of hydroxyarsinothricin (AST-OH) and subsequent methylation of a trivalent arsenic intermediate with methyl iodide. The enzyme AST N-acetyltransferase (ArsN1) was utilized to purify l-AST from racemic AST. This chemical synthesis provides a source of this novel antibiotic for future drug development.
ABSTRACT
BACKGROUND: The use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders. However, the stability of siRNA and other small molecule therapeutics is challenged by their intrinsic instability and limited passage across the blood-brain barrier (BBB). Based on these premises, our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB. For this purpose, 5(6)-carboxyfluorescein-conjugated OL and scramble peptide were synthesized, and then their BBB cellular internalization/trafficking and stability were characterized versus temperature, pH and serum content in the media in hCMEC/D3 cells as a model of BBB endothelium. Specifically, integrity of the internalized peptide in cell lysates was analyzed by LC/MS while cellular distribution and intracellular trafficking of OL was examined by fluorescence microscopy with early-late endosome (pHRodo Red®) and lysosome (Lysotracker®) markers. RESULTS: Our data show that cellular uptake of OL increased linearly with the concentrations tested in this study at 37 °C and the uptake was two to threefolds higher when compared to scramble peptide. While there were no differences for scramble peptide, the uptake of OL decreased by 50% at 4 °C incubation (vs. 37 °C). No effects of pH were observed on endothelial uptake of OL. Immunofluorescence studies also indicated a significant cellular internalization of OL that remained intact (as evaluated by LC-MS/MS) and co-localized with endosomal, but not lysosome marker. Importantly, OL was found non-toxic to cells at all concentrations tested. CONCLUSIONS: In summary, our data suggest the existence of a receptor-mediated transcytosis pathway for cellular uptake of OL at the BBB endothelium. However, in vivo studies will be needed to assess the siRNA loading capacity of OL and its trans-BBB transport efficiency for targeted delivery in the brain.
Subject(s)
Blood-Brain Barrier/metabolism , Drug Delivery Systems/methods , Lectins/pharmacokinetics , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Stability , Endosomes/metabolism , Fluoresceins/chemistry , Humans , Lectins/blood , Lectins/chemistry , Lysosomes/metabolismABSTRACT
The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Thus, targeting glycosylation changes in cancer is likely to provide not only better insight into the roles of carbohydrates in biological systems, but also facilitate the development of new molecular probes for bioanalytical and biomedical applications. In the reported study, we have synthesized lectinomimics based on odorranalectin 1; the smallest lectin-like cyclic peptide isolated from the frog Odorrana grahami skin, and assessed the ability of these peptides to bind specific carbohydrates on molecular and cellular levels. In addition, we have shown that the disulfide bond found in 1 can be replaced with a lactam bridge. However, the orientation of the lactam bridge, peptides 2 and 3, influenced cyclic peptide's conformation and thus these peptides' ability to bind carbohydrates. Naturally occurring 1 and its analog 3 that adopt similar conformation in water bind preferentially L-fucose, and to a lesser degree D-galactose and N-acetyl-D-galactosamine, typically found within the mucin O-glycan core structures. In cell-based assays, peptides 1 and 3 showed a similar binding profile to Aleuria aurantia lectin and these two peptides inhibited the migration of metastatic breast cancer cell lines in a Transwell assay. Altogether, the reported data demonstrate the feasibility of designing lectinomimics based on cyclic peptides.
Subject(s)
Drug Delivery Systems , Lectins , Neoplasms/metabolism , Peptides, Cyclic/chemical synthesis , Peptidomimetics/chemical synthesis , Polysaccharides/metabolism , Binding, Competitive , Cell Line , Cell Movement/drug effects , Cell Survival/drug effects , Fucose/agonists , Fucose/metabolism , Hep G2 Cells , Humans , Inhibitory Concentration 50 , Lactams/chemistry , Lectins/chemistry , Lectins/metabolism , MCF-7 Cells , Molecular Docking Simulation , Neoplasms/drug therapy , Neoplasms/pathology , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Peptidomimetics/chemistry , Peptidomimetics/metabolism , Peptidomimetics/pharmacology , Polysaccharides/chemistry , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity RelationshipABSTRACT
Despite continuous efforts to control cariogenic dental biofilms, very few effective antimicrobial treatments exist. In this study, we characterized the activity of the novel synthetic cyclic lipopeptide 4 (CLP-4), derived from fusaricidin, against the cariogenic pathogen Streptococcus mutans UA159. We determined CLP-4's MIC, minimum bactericidal concentration (MBC), and spontaneous resistance frequency, and we performed time-kill assays. Additionally, we assessed CLP-4's potential to inhibit biofilm formation and eradicate preformed biofilms. Our results demonstrate that CLP-4 has strong antibacterial activity in vitro and is a potent bactericidal agent with low spontaneous resistance frequency. At a low concentration of 5 µg/ml, CLP-4 completely inhibited S. mutans UA159 biofilm formation, and at 50 µg/ml, it reduced the viability of established biofilms by >99.99%. We also assessed CLP-4's cytotoxicity and stability against proteolytic digestion. CLP-4 withstood trypsin or chymotrypsin digestion even after treatment for 24 h, and our toxicity studies showed that CLP-4 effective concentrations had negligible effects on hemolysis and the viability of human oral fibroblasts. In summary, our findings showed that CLP-4 is a potent antibacterial and antibiofilm agent with remarkable stability and low nonspecific cytotoxicity. Hence, CLP-4 is a promising novel antimicrobial peptide with potential for clinical application in the prevention and treatment of dental caries.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/growth & development , Dental Caries/prevention & control , Dental Plaque/prevention & control , Lipopeptides/pharmacology , Peptides, Cyclic/pharmacology , Streptococcus mutans/drug effects , Bacterial Proteins , Biofilms/drug effects , Cell Line , Dental Caries/drug therapy , Dental Caries/microbiology , Dental Plaque/drug therapy , Dental Plaque/microbiology , Depsipeptides/pharmacology , Humans , Microbial Sensitivity Tests , ProteolysisABSTRACT
The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB.
Subject(s)
Bacterial Proteins/chemistry , Depsipeptides/chemistry , Drug Discovery/methods , Methionine Sulfoxide Reductases/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Enzyme Activation , Enzyme Stability , Microfilament ProteinsABSTRACT
Treating bacterial infections can be difficult due to innate or acquired resistance mechanisms, and the formation of biofilms. Cyclic lipopeptides derived from fusaricidin/LI-F natural products represent particularly attractive candidates for the development of new antibacterial and antibiofilm agents, with the potential to meet the challenge of bacterial resistance to antibiotics. A positional-scanning combinatorial approach was used to identify the amino acid residues responsible for driving antibacterial activity, and increase the potency of these cyclic lipopeptides. Screening against the antibiotic resistant ESKAPE pathogens revealed the importance of hydrophobic as well as positively charged amino acid residues for activity of this class of peptides. The improvement in potency was especially evident against bacterial biofilms, since the lead cyclic lipopeptide showed promising in vitro and in vivo anti-biofilm activity at the concentration far below its respective MICs. Importantly, structural changes resulting in a more hydrophobic and positively charged analog did not lead to an increase in toxicity toward human cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Combinatorial Chemistry Techniques , Lipopeptides/pharmacology , Peptide Library , Peptides, Cyclic/pharmacology , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/chemistry , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Lipopeptides/chemical synthesis , Lipopeptides/chemistry , Microbial Sensitivity Tests , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.
ABSTRACT
The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C max of 8.9 µg/mL at 5 min corresponds to approximately 22% injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC50 value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with ka = 5 × 10(5) M(-1) s(-1) and kdiss = 1.5 × 10(-4) s(-1) values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.
Subject(s)
Leptin/antagonists & inhibitors , Peptides/chemistry , Receptors, Leptin/chemistry , Animals , Cattle , Cell Line, Tumor , Drug Design , Female , Half-Life , Humans , Kinetics , Leptin/chemistry , Leptin/metabolism , Mice , Peptides/blood , Peptides/metabolism , Peptides/pharmacokinetics , Receptors, Leptin/metabolismABSTRACT
A worldwide public health problem has resulted from the alarming spread of multi-drug resistant bacteria combined with the frequent occurrence of biofilm-type infections, creating a growing need for new therapies. In this study, we have demonstrated that novel cyclic lipopeptides, such as 1, cyclo-[D-Ala-(12-guanidinododecanoyl)Thr-D-Val-Val-DaThr-D-Asn], and 2, cyclo-[D-Ala-(12-guanidinododecanoyl)Dap-D-Val-Val-D-aThr-D-Asn], derived from the fusaricidin/ LI-F natural products efficiently inhibit the growth of Staphylococcus aureus biofilm in vitro at their minimum inhibitory concentrations (MICs). Complete S. aureus biofilm eradication was observed at 3 x MIC for 1 and 4 x MIC for 2. Promising in vivo activity was demonstrated by the ability of depsipeptide 1 to reduce the proliferation of methicillinresistant S. aureus US300 in a porcine wound model. Due to their unique structure and potent antibacterial and antibiofilm activities, cyclic lipopeptides that belong to the fusaricidin/LI-F family of antibiotics represent particularly attractive lead structures for the development of new antibacterial agents capable of treating complicated biofilm-associated infections.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Lipopeptides/chemistry , Lipopeptides/pharmacology , Staphylococcal Infections/drug therapy , Staphylococcus aureus/physiology , Amino Acid Sequence , Animals , Microbial Sensitivity Tests , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Staphylococcus aureus/drug effects , Swine , Wound Healing/drug effectsABSTRACT
Despite the enormous therapeutic potential, the clinical use of peptides has been limited by their poor bioavailability and low stability under physiological conditions. Hence, efforts have been undertaken to alter peptide structure in ways to improve their pharmacological properties. Inspired by the importance of basic amino acids in biological systems and the remarkable versatility displayed by lysine during the synthesis of complex peptide scaffolds, this chapter describes a simple procedure that enables rapid access to protected N,N'-diaminoalkylated basic amino acid building blocks suitable for standard solid-phase peptide synthesis. This procedure allows preparation of symmetrical, as well as unsymmetrical, dialkylated amino acid derivatives that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analog in which the lysine residue was substituted with its synthetic polyamino derivate. The substitution provided indolicidin analog with increase net positive charge, more ordered secondary structure in biological membranes mimicking conditions, and enhanced antibacterial activity without altering hemolytic activity. Taking into consideration the increasing interest for peptides with unusual structural features due to their improved biological properties, the described synthesis of polyfunctional amino acid building blocks is of particular practical value.
Subject(s)
Amino Acids, Basic/chemical synthesis , Peptides/chemical synthesis , Solid-Phase Synthesis Techniques , Alkylation , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/chemistry , Anti-Infective Agents/isolation & purification , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/isolation & purification , Oxidation-Reduction , Peptides/isolation & purificationABSTRACT
With the growing importance of peptides and peptidomimetics as potential therapeutic agents, a continuous synthetic interest has been shown for their modification to provide more stable and bioactive analogs. Among many approaches, peptide/peptidomimetic guanidinylation offers access to analogs possessing functionality with strong basic properties, capable of forming stable intermolecular H-bonds, charge pairing, and cation-π interactions. Therefore, guanidinium functional group is considered as an important pharmacophoric element. Although a number of methods for solid-phase guanidinylation reactions exist, only a few are fully compatible with standard Fmoc solid-phase peptide chemistry.In this chapter we summarize the solid-phase guanidinylation methods fully compatible with standard Fmoc-synthetic methodology. This includes use of direct guanidinylating reagents such as 1-H-pyrazole-1-carboxamidine and triflylguanidine, and guanidinylation with di-protected thiourea derivatives in combination with promoters such as Mukaiyama's reagent, N-iodosuccinimide, and N,N'-diisopropylcarbodiimide.
Subject(s)
Amino Acids/chemistry , Fluorenes/chemistry , Peptides/chemistry , Solid-Phase Synthesis Techniques , Amines/chemistry , Guanidines/chemistry , Lipopeptides/chemical synthesis , Peptides/chemical synthesis , Peptides, Cyclic/chemical synthesisABSTRACT
ß-Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug-resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of ß-sheet AMPs, we hypothesize that N-methylation of the intramolecular hydrogen bond(s)-forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model ß-sheet antimicrobial peptide, gramicidinâ S (GS), to study the N-methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N-methylated GS analogues by replacement of residues at the ß-strand and ß-turn regions with N-methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC50 values increased fivefold compared with that of GS, when the internal hydrogen-bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N-methylalanine in the ß-turn region. However, analogues containing N-methylleucine at ß-strand and N-methylalanine at ß-turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N-methylated GS analogues using (1)Hâ NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N-methylation of the internal hydrogen bond-forming amide affected the conformation, backbone shape, and side chain orientation of GS.
Subject(s)
Alanine/analogs & derivatives , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/pharmacology , Gramicidin/chemical synthesis , Gramicidin/pharmacology , Alanine/chemistry , Anti-Infective Agents/chemistry , Bacteria/drug effects , Gramicidin/analogs & derivatives , Hemolysis/drug effects , Magnetic Resonance Spectroscopy , Protein Structure, SecondaryABSTRACT
In order to provide effective treatment options for infections caused by multidrug-resistant bacteria, innovative antibiotics are necessary, preferably with novel modes of action and/or belonging to novel classes of drugs. Naturally occurring cyclic lipodepsipeptides, which contain one or more ester bonds along with the amide bonds, have emerged as promising candidates for the development of new antibiotics. Some of these natural products are either already marketed or in advanced stages of clinical development. However, despite the progress in the development of new antibacterial agents, it is inevitable that resistant strains of bacteria will emerge in response to the widespread use of a particular antibiotic and limit its lifetime. Therefore, development of new antibiotics remains our most efficient way to counteract bacterial resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Depsipeptides/pharmacology , Drug Resistance, Bacterial/drug effects , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacology , Bacterial Proteins/therapeutic use , Daptomycin/chemistry , Daptomycin/pharmacology , Daptomycin/therapeutic use , Depsipeptides/chemistry , Depsipeptides/therapeutic use , HumansABSTRACT
Cyclic lipopeptides derived from the fusaricidin/LI-F family of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents. In comparison with natural products, these derivatives may offer better stability under physiologically relevant conditions and lower nonspecific toxicity, while preserving their antibacterial activity. In this study we assessed the ability of cyclic lipodepsipeptide 1 and its analogues--amide 2, N-methylamide 3, and linear peptide 4--to interact with the cytoplasmic membranes of selected Gram-positive bacteria. We also investigated their bacteriostatic/bactericidal modes of action and inâ vivo potency by using a Galleria mellonella model of MRSA infection. Cyclic lipopeptides 1 and 2 depolarize the cytoplasmic membranes of Gram-positive bacteria in a concentration-dependent manner. The degree of membrane depolarization was influenced by the structural and physical properties of 1 and 2, with the more flexible and hydrophobic peptide 1 being most efficient. However, membrane depolarization does not correlate with bacterial cell lethality, suggesting that membrane-targeting activity is not the main mode of action for this class of antibacterial peptides. Conversely, substitution of the depsipeptide bond in 1 with an N-methylamide bond in 3, or its hydrolysis to peptide 4, lead to a complete loss of antibacterial activity and indicate that the conformation of cyclic lipopeptides plays a role in their antibacterial activities. Cyclic lipopeptides 1 and 2 are also capable of improving the survival of G.â mellonella larvae infected with MRSA at varying efficiencies, reflecting their inâ vitro activities. Gaining more insight into the structure-activity relationship and mode of action of these cyclic lipopeptides may enable the development of new antibiotics of this class with improved antibacterial activity.
Subject(s)
Amides/chemistry , Anti-Bacterial Agents/chemistry , Lipopeptides/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Cell Wall/drug effects , Circular Dichroism , Esters , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/metabolism , Larva/microbiology , Lepidoptera/growth & development , Lipopeptides/chemical synthesis , Lipopeptides/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Models, AnimalABSTRACT
Fusaricidins/LI-Fs and related cyclic lipopeptides represent an interesting new class of antibacterial peptides with the potential to meet the challenge of antibiotic resistance in bacteria. Our previous study (Bionda et al. ChemMedChem 2012, 7, 871-882) revealed the significance of the guanidinium group located at the termini of the lipidic tails of these cyclic lipopeptides for their antibacterial activities. Therefore, devising a synthetic strategy that will allow incorporation of guanidinium functionality into their structure is of particular practical importance. Since appropriately protected guanidino fatty acid building blocks are not commercially available, our strategy toward guanidinylated fusaricidin/LI-F analogs include solid-phase synthesis of a cyclic lipopeptide precursor possessing a lipidic tail with a terminal amino group followed by its conversion into corresponding guanidine. To find the optimal method for this conversion, we have examined commonly used guanidinylation reagents under the conditions compatible with standard solid-phase peptide synthesis. Described experimental results demonstrated superiority of N,N'-di-Boc-Nâ³-triflylguanidine in solid-phase preparation of fusaricidin/LI-F class of cyclic lipopeptides. The triflylguanidine reagent gave a single monoguanidinylated product in excellent yield independently of the type of solid-support.
Subject(s)
Lipopeptides , Solid-Phase Synthesis Techniques , Amines/chemistry , Anti-Bacterial Agents/chemistry , Peptides, CyclicABSTRACT
A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N'-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N (α)-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N (α)-(9H-fluorenyl-9-methoxycarbonyl)-N (ß),N (ß) '-bis[2-(tert-butoxycarbonylamino)ethyl]-L-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity.
Subject(s)
Amino Acids, Basic/chemistry , Anti-Bacterial Agents/chemical synthesis , Peptides/chemical synthesis , Alkylation , Amino Acids, Basic/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Erythrocytes/drug effects , Hemolytic Agents/chemical synthesis , Hemolytic Agents/chemistry , Hemolytic Agents/pharmacology , Humans , Molecular Structure , Peptides/chemistry , Peptides/pharmacology , Solid-Phase Synthesis TechniquesABSTRACT
Bacterial infections are becoming increasingly difficult to treat due to the development and spread of antibiotic resistance. Therefore, identifying novel antibacterial targets and new antibacterial agents capable of treating infections by drug-resistant bacteria is of vital importance. The structurally simple yet potent fusaricidin or LI-F class of natural products represents a particularly attractive source of candidates for the development of new antibacterial agents. We synthesized 18 fusaricidin/LI-F analogues and investigated the effects of structure modification on their conformation, serum stability, antibacterial activity, and toxicity toward human cells. Our findings show that substitution of an ester bond in depsipeptides with an amide bond may afford equally potent analogues with improved stability and greatly decreased cytotoxicity. The lower overall hydrophobicity/amphiphilicity of amide analogues in comparison with their parent depsipeptides, as indicated by HPLC retention times, may explain the dissociation of antibacterial activity and human cell cytotoxicity. These results indicate that amide analogues may have significant advantages over fusaricidin/LI-F natural products and their depsipeptide analogues as lead structures for the development of new antibacterial agents.