Solid-Phase Synthesis of the Bicyclic Peptide OL-CTOP Containing Two Disulfide Bridges, and an Assessment of Its In Vivo µ-Opioid Receptor Antagonism after Nasal Administration.

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.

Exploring Glycan Binding Specificity of Odorranalectin by Alanine Scanning Library.

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.

In vitro characterization of odorranalectin for peptide-based drug delivery across the blood-brain barrier.

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.

Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159.

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.

Targeting cancer-specific glycans by cyclic peptide lectinomimics.

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.

Identification of activators of methionine sulfoxide reductases A and B.

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.

The designer leptin antagonist peptide Allo-aca compensates for short serum half-life with very tight binding to the receptor.

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.

Solid-phase synthesis of fusaricidin/LI-F class of cyclic lipopeptides: Guanidinylation of resin-bound peptidyl amines.

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-N″-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.

Direct access to side chain N,N'-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis.

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.

A practical synthesis of N α-Fmoc protected L-threo-ß-hydroxyaspartic acid derivatives for coupling via α- or ß-carboxylic group.

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available D: ,L: -tHyAsp racemic mixture by co-crystallization with L: -Lys, followed by ion exchange chromatography yielding enantiomerically pure L: -tHyAsp and D: -tHyAsp, and their selective orthogonal protection. In this way N ( α )-Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or ß-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via ß-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.

Novel Cyclic Lipopeptides Fusaricidin Analogs for Treating Wound Infections.

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.

Chemical synthesis of the organoarsenical antibiotic arsinothricin.

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.

Development of a pharmacologically improved peptide agonist of the leptin receptor.

Leptin, a hormone produced by adipose tissue, regulates energy balance in the hypothalamus and is involved in fertility, immune response and carcinogenesis. The existence of disorders related to leptin deficit and leptin overabundance calls for the development of drugs activating or inhibiting the leptin receptor (ObR). We synthesized four proposed receptor-binding leptin fragments (sites I, IIa and IIb, III), their reportedly antagonist analogs, and a peptide chimera composed of the two discontinuous site II arms. To assess the pharmacological utility of leptin fragments, we studied the peptides' ability to stimulate the growth of ObR-positive and ObR-negative cells. The combined site II construct and site III derivatives selectively reversed leptin-induced growth of ObR-positive cells at mid-nanomolar concentrations. However, these peptides appeared to be partial agonists/antagonists as they activated cell growth in the absence of exogenous leptin. A designer site III analog, featuring non-natural amino acids at terminal positions to decrease proteolysis and a blood-brain barrier (BBB) penetration-enhancing carbohydrate moiety, proved to be full agonist to ObR, i.e., stimulated proliferation of different ObR-positive but not ObR-negative cells in the presence or absence of leptin. This glycopeptide bound to isolated ObR on solid-phase assays and activated ERK-1/2 signaling in ObR-positive MCF-7 cells at 100-500 nM concentrations. The glycopeptide was stable in mouse serum, readily crossed endothelial/astrocyte cell layers in a cellular BBB model, and was distributed into the brain of Balb/c mice after intraperitoneal administration. These characteristics suggest a potential pharmaceutical utility of the designer site III glycopeptide in leptin-deficient diseases.

Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics.

The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3-APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of conventional antibiotics including chloramphenicol, beta-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent.

Peptidomimetics: Fmoc solid-phase pseudopeptide synthesis.

Peptidomimetic modifications or cyclization of linear peptides are frequently used as attractive methods to provide more conformationally constrained and thus more stable and bioactive peptides. Among numerous peptidomimetic approaches described recently in the literature, particularly attractive are pseudopeptides or peptide bond surrogates in which peptide bonds have been replaced with other chemical groups. In these peptidomimetics the amide bond surrogates possess three-dimensional structures similar to those of natural peptides, yet with significant differences in polarity, hydrogen bonding capability, and acid-base character. The introduction of such modifications to the peptide sequence is expected to completely prevent protease cleavage of amide bond and significantly improve peptides' metabolic stability. In this chapter we consider Fmoc solid-phase synthesis of peptide analogs containing the amide surrogate that tend to be isosteric with the natural amide. This includes synthesis of peptidosulfonamides, phosphonopeptides, oligoureas, depsides, depsipeptides, and peptoids.

Depsipeptide synthesis.

Naturally occurring cyclic depsipeptides, peptides that contain one or more ester bonds in addition to the amide bonds, have emerged as an important source of pharmacologically active compounds or promising lead structures for the development of novel synthetically derived drugs. This class of natural products has been found in many organisms, such as fungi, bacteria, and marine organisms. It is very well known that cyclic depsipeptides and their derivatives exhibit a diverse spectrum of biological activities, including insecticidal, antiviral, antimicrobial, antitumor, tumor-promotive, anti-inflammatory, and immunosuppressive actions. However, they have shown the greatest therapeutic potential as anticancer and particularly antimicrobial agents. Difficulties associated with isolation and purification of larger quantities of this class of natural products and, particularly, unlimited access to their synthetic analogs significantly hampered cyclic depsipeptides exploitation as lead compounds for development of new drugs. As an alternative, total solution or solid-phase peptide synthesis of these important natural products and combinatorial chemistry approaches can be employed to elucidate structure-activity relationships and to find new potent compounds of this class. In this chapter, methods for formation of depsipeptide ester bonds, hydroxyl group protection, and solid-phase reaction monitoring are described.

A novel strategy for the solid-phase synthesis of cyclic lipodepsipeptides.

A rapid and efficient Fmoc solid-phase synthesis of cyclic lipodepsipeptide analogue 1 to antibiotic fusaricidin A is described. Our synthetic approach includes resin attachment of the first amino acid via side chain, successful use of combination of four quasi-orthogonal removable protecting groups, stepwise solid-phase synthesis of linear peptide analogue, lipid tail attachment followed by depsipeptide bond formation and on-resin head-to-tail cyclization. Undesired O→N acyl shift, which may occur during Fmoc removal, was successfully avoided by the incorporation of the lipid tail into the linear peptide precursor prior to on-resin depsipeptide bond formation and the ring closure.

Identification of novel cyclic lipopeptides from a positional scanning combinatorial library with enhanced antibacterial and antibiofilm activities.

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.

Functional analysis of the lipoglycodepsipeptide antibiotic ramoplanin.

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.