Effects of Lidocaine-Derived Organic Compounds on Eosinophil Activation and Survival.

Lidocaine, a local anesthetic, is known to possess anti-inflammatory properties. However, its clinical use is limited by inconveniences, such as its local synesthetic effects. This study evaluated lidocaine analogs designed and synthesized to overcome the disadvantages of lidocaine, having anti-inflammatory properties. Interleukin 5 (IL-5)-induced eosinophil activation and survival were evaluated using 36 lidocaine analogs with modified lidocaine structure on the aromatic or the acyl moiety or both. Eosinophil survival was evaluated using a CellTiter 96® aqueous cell proliferation assay kit. Superoxide production was determined using the superoxide dismutase-inhibitable reduction of cytochrome C method. Eosinophil cationic protein (ECP), IL-8, and transcription factor expression were determined using enzyme-linked immunosorbent assay. The platelet-activating factor (PAF)-induced migration assay was performed using a Transwell insert system. Compounds EI137 and EI341 inhibited IL-5-induced eosinophil survival and superoxide and ECP production in a concentration-dependent manner. These compounds also significantly reduced IL-8 production. Although compounds EI137 and EI341 significantly reduced phosphorylated ERK 1/2 expression, they did not influence other total and phosphorylated transcription factors. Moreover, 1000 µM of compound EI341 only inhibited PAF-induced migration of eosinophils. Lidocaine analogs EI137 and EI341 inhibited IL-5-mediated activation and survival of eosinophils. These compounds could be new therapeutic agents to treat eosinophilic inflammatory diseases.

Fatty Acid Conjugated Chalcones as Tubulin Polymerization Inhibitors: Design, Synthesis, QSAR, and Apoptotic and Antiproliferative Activity.

Cancer is often associated with an aberrant increase in tubulin and microtubule activity required for cell migration, invasion, and metastasis. A new series of fatty acid conjugated chalcones have been designed as tubulin polymerization inhibitors and anticancer candidates. These conjugates were designed to harness the beneficial physicochemical properties, ease of synthesis, and tubulin inhibitory activity of two classes of natural components. New lipidated chalcones were synthesized from 4-aminoacetophenone via N-acylation followed by condensation with different aromatic aldehydes. All new compounds showed strong inhibition of tubulin polymerization and antiproliferative activity against breast and lung cancer cell lines (MCF-7 and A549) at low or sub-micromolar concentrations. A significant apoptotic effect was shown using a flow cytometry assay that corresponded to cytotoxicity against cancer cell lines, as indicated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. Decanoic acid conjugates were more potent than longer lipid analogues, with the most active being more potent than the reference tubulin inhibitor, combretastatin-A4 and the anticancer drug, doxorubicin. None of the newly synthesized compounds caused any detectable cytotoxicity against the normal cell line (Wi-38) or hemolysis of red blood cells below 100 µM. It is unlikely that the new conjugates described would affect normal cells or interrupt with cell membranes due to their lipidic nature. A quantitative structure-activity relationship analysis was performed to determine the influence of 315 descriptors of the physicochemical properties of the new conjugates on their tubulin inhibitory activity. The obtained model revealed a strong correlation between the tubulin inhibitory activity of the investigated compounds and their dipole moment and degree of reactivity.

Chemical composition and anticholinesterase activity of Lepisanthes rubiginosa (Roxb.) Leenh. essential oil.

Essential oils obtained from medicinal plants show high therapeutic potential against several types of pathologies, including Alzheimer's disease. The purpose of this work was to study the chemical composition and anticholinesterase inhibitory activity of the essential oil obtained from Lepisanthes rubiginosa leaves collected from Malaysia. Twenty-four components were identified using gas chromatography-flame ionization detection (GC-FID) and gas chromatography/mass spectrometry (GC-MS), which represent 99.5% of the essential oil. The identified major components include α-cadinol (40.0%), safrole (12.6%), α-amorphene (9.5%), (E)-isosafrole (5.0%), δ-cadinene (4.2%), and t-muurolol (4.1%). Anticholinesterase activity was assessed using Ellman method, and the essential oil demonstrated a moderate inhibitory activity against acetylcholinesterase (I%: 75.2%) and butyrylcholinesterase (I%: 70.2%) at conconcetration of 1000 µg/mL. The current study is the first to report chemical composition and anticholinesterase activity of the essential oil obtained from L. rubiginosa, which may have implications on the characterization, pharmaceutical, and therapeutic applications of Lepisanthes genus essential oils.

Ketorolac-fluconazole: A New Combination Reverting Resistance in Candida albicans from Acute Myeloid Leukemia Patients on Induction Chemotherapy: In vitro Study.

BACKGROUND AND OBJECTIVES: Candida albicans is a significant source of morbidity and mortality for patients with acute myeloid leukemia (AML). Prolonged use of fluconazole as empirical antifungal prophylaxis in AML patients leads to overexpression of efflux pump genes that resulted in the emergence of azole-resistant species. Consequently, the introduction of a new strategy to improve the management of C. albicans infections is an urgent need. Nonsteroidal anti-inflammatory drug (NSAID) ketorolac is associated with a reduction in cancer relapses. The present study was performed to investigate the use of ketorolac-fluconazole combination to reverse fluconazole resistance in C. albicans isolated from AML patients on induction chemotherapy. PATIENTS AND METHODS: One hundred and seventy AML patients were evaluated. Fifty C. albicans were isolated and subjected to disc diffusion assay and broth microdilution for fluconazole alone and combined with different concentrations of ketorolac. Efflux pump gene (CDR1, CDR2, and MDR1) expressions were quantified by real-time PCR. RESULTS: The tested ketorolac acted synergistically with fluconazole against resistant C. albicans with the minimum inhibitory concentration (MIC) of fluconazole decreased from >160 µg/mL to 0.3-1.25 µg/mL in (93.8%) of resistant isolates with fractional inhibitory concentration index (FICI) value of 0.25. The majority of the resistant isolates overexpressed CDR1 (71.1%) and MDR1 (60%). CONCLUSION: Ketorolac-fluconazole in vitro combination would be a promising strategy for further clinical in vivo trials to overcome fluconazole resistance in AML patients on induction chemotherapy.

Synthesis of Amide Backbone-Modified Peptides.

Solubility is a key property of peptides and of central importance to the success of solid-phase peptide synthesis and subsequent peptide purification and handling. Substitution of the backbone amide bond can dramatically increase peptide solubility. Backbone amide bond protection works by preventing the formation of interchain association and can be used both to synthesize aggregation-prone peptide sequences on solid phase and to improve solubility of a peptide post synthesis. Improving peptide solubility by judicial use of backbone protection is of growing importance, particularly for chemical protein synthesis by chemical ligation.

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP.

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

A backbone amide protecting group for overcoming difficult sequences and suppressing aspartimide formation.

A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material. Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine.

Automated synthesis of backbone protected peptides.

The synthesis of peptides rich in aggregation prone sequences can be improved with backbone protection. We report the automated introduction of backbone protection to a peptide. This new method was applied in a fully-automated synthesis, giving improved handling, quality and yield of several challenging target sequences.

Immune and anticancer responses elicited by fully synthetic aberrantly glycosylated MUC1 tripartite vaccines modified by a TLR2 or TLR9 agonist.

The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.

Structure-activity relationship for the development of a self-adjuvanting mucosally active lipopeptide vaccine against Streptococcus pyogenes.

Infection with group A streptococcus (GAS) can result in a number of diseases, some of which are potentially life-threatening. The oral-nasal mucosa is a primary site of GAS infection, and a mucosally active vaccine candidate could form the basis of an antidisease and transmission-blocking GAS vaccine. In the present study, a peptide from the GAS M protein (J14) representing a B cell epitope was incorporated alongside a universal T cell helper epitope and a Toll-like receptor 2 targeting lipid moiety to form lipopeptide constructs. Through structure activity studies, we identified a vaccine candidate that induces J14-specific mucosal and systemic antibody responses when administered intranasally without additional adjuvants. The systemic antibodies elicited were capable of inhibiting the growth of GAS. In addition, J14-specific mucosal antibodies corresponded with reduced throat colonization after respiratory GAS challenge. These preclinical experiments show that this lipopeptide could form the basis of an optimal needle-free mucosal GAS vaccine.

Design of fully synthetic, self-adjuvanting vaccine incorporating the tumor-associated carbohydrate Tn antigen and lipoamino acid-based Toll-like receptor 2 ligand.

Overexpression of certain tumor-associated carbohydrate antigens (TACA) caused by malignant transformation offers promising targets to develop novel antitumor vaccines, provided the ability to break their inherent low immunogenicity and overcome the tolerance of the immune system. We designed, synthesized, and immunologically evaluated a number of fully synthetic new chimeric constructs incorporating a cluster of the most common TACA (known as Tn antigen) covalently attached to T-cell peptide epitopes derived from polio virus and ovalbumin and included a synthetic built-in adjuvant consisting of two 16-carbon lipoamino acids. Vaccine candidates were able to induce significantly strong antibody responses in mice without the need for any additional adjuvant, carrier protein, or special pharmaceutical preparation (e.g., liposomes). Vaccine constructs were assembled either in a linear or in a branched architecture, which demonstrated the intervening effects of the incorporation and arrangement of T-cell epitopes on antibody recognition.

Immunological evaluation of lipopeptide group A streptococcus (GAS) vaccine: structure-activity relationship.

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Simple synthetic toll-like receptor 2 ligands.

Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune responses against pathogens followed by slow but specific adaptive immune responses. Development of synthetic TLR2 agonists/antagonists would be useful in the prevention of different infectious and immunologic disorders. The current study reports synthesis and TLR2 activity of two simple TLR2 ligands, which feature minimal structural requirement for TLR2 activity (two long lipid chains) and stimulate agonistic activity at nanomolar concentration.

Design of three-component vaccines against group A streptococcal infections: importance of spatial arrangement of vaccine components.

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4(+) T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.

Structure-activity relationship of lipopeptide Group A streptococcus (GAS) vaccine candidates on toll-like receptor 2.

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-kappaB (NF-kappaB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure-function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-kappaB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons)>LAA 2 (14 carbons)>LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. N(epsilon)-terminus of the central lysine residue) does not significantly affect NF-kappaB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFkappaB activation.

Synthesis and immunological evaluation of self-adjuvanting glycolipopeptide vaccine candidates.

Synthesis of four glycolipids with different number of lauroyl groups on glucose or cellobiose as scaffolds is described. Their immunological evaluations either admixed with or covalently linked to J8, a peptide antigen derived from the C-terminus of the antiphagocytic M-protein of group A streptococcus, are also investigated. Administration of mixtures of J8 and glycolipids to B10BR (H-2(k)) mice induced low-levels of J8-specific IgG antibodies. While glycolipopeptides, in which J8 was covalently linked to the synthetic glycolipids, demonstrated high-levels of antibody titers comparable with the co-administration of these glycolipopeptides with complete Freund's adjuvant, suggesting clearly the strong potency of the synthesized glycolipids as self-adjuvanting moieties.

Synthesis and in vivo studies of carbohydrate-based vaccines against group A streptococcus.

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.

Development of a liposaccharide-based delivery system and its application to the design of group A streptococcal vaccines.

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.

Structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates.

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.