Synthetic Conjugates Research Articles

Microarray analysis of Akt PH domain binding employing synthetic biotinylated analogs of all seven phosphoinositide headgroup isomers

Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIPns), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIPn binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein–PIPn binding interactions is hindered by the complexity of the membrane environment and of the PIPn structures. Herein, we describe two rapid assays of use for characterizing protein–PIPn binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIPn headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P3 and PI(3,4)P2 as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIPn ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein–membrane binding interactions.

Anti-CD38 Pretargeted Radioimmunotherapy Demonstrates Therapeutic Efficacy In a Human Multiple Myeloma Mouse Xenograft Model

Abstract 1842Multiple myeloma (MM) remains incurable despite improved response rates and improved progression free survival in the era of therapy with novel agents, including bortezomib, thalidomide, and lenalidomide. Disease persistence is presumably due to residual malignant plasma cell clones that evade or develop resistance to available therapies. The efficacy of radioimmunotherapy (RIT) in the treatment of hematologic malignancies is well established and the radiosensitivity of malignant plasma cells has been demonstrated in both preclinical and clinical settings. The ectoenzyme receptor CD38 is a plasma cell antigen that exhibits relatively specific, stable and uniform expression (95–100%) at a high epitope density on myeloma cells, making it an attractive target for antibody based therapies, including RIT. Pretargeted RIT (PRIT), using a multi-step streptavidin (SA)-biotin targeting system enhances the therapeutic index of delivered radiation. We have generated an anti-CD38 antibody (Ab)-SA synthetic chemical conjugate (OKT10-SA). The OKT10-SA construct binds with high avidity to myeloma cells while retaining full biotin-binding capability for radiolabeled DOTA-biotin.Blood, tumor and nonspecific organ uptakes of OKT10-SA were directly measured in biodistribution experiments involving athymic nude mice bearing human MM xenograft tumors. Groups of 5 mice with s.c. L363 human MM (IgG) xenograft tumors received 1.4 nmol (300 μg) of either OKT10-SA (anti-CD38 SA) or an IgG1 isotype matched control Ab BHV1-SA (bovine herpes virus-1) 22 hrs prior to synthetic biotin-acetyl-galactosamine clearing agent (CA; 5.8 nmol [50 μg]) and 24 hrs prior to trace labeled 111In-DOTA-biotin (1 μg). The CA removed >95% of both unbound OKT10-SA and BHV1-SA from the mouse circulation within 30 minutes of administration. Animals were euthanized and comprehensive tissue biodistributions were assessed 2, 24, 48 and 96 hrs after 111In-DOTA-biotin injection. Tumors excised from mice pretargeted with OKT10-SA contained 13.1 ± 1.9 % of the injected dose of 111In-DOTA-biotin per gram (% ID/g) after 2 hrs and 8.8 ± 2.8 % ID/g after 24 hrs compared to 2.4 ± 0.6 % ID/g after 2 hrs and 0.9 ± 0.4 % ID/g after 24 hrs in tumors excised from control mice pretargeted with BHV1-SA. Tumor-to-normal organ ratios of absorbed radioactivity were 8:1; 10:1; 8:1; and 6:1 respectively for blood, lung, liver and kidney in mice pretargeted with OKT10-SA; compared to 0.6:1; 0.9:1; 0.8:1 and 0.4:1 respectively, in control mice pretargeted with BHV1-SA.Therapy studies were then performed in athymic nude mice (n=9-10/group) bearing s.c. L363 human MM xenograft tumors. Reagent concentrations and time-points for administration of OKT10-SA, BHV1-SA and CA were identical to those reported for the biodistribution studies. The high energy beta particle emitter 90Yttrium (t1/2 = 64 hrs) was used as the therapeutic radionuclide. 90Y-DOTA-biotin (2 μg) was labeled with 400 μCi, 800 μCi, or 1200 μCi per mouse in 3 OKT10-SA groups and 3 control groups (untreated control; 800 μCi or 1200 μCi 90Y-DOTA-biotin following BHV1-SA). All mice in the untreated control and BHV1-SA control groups experienced exponential MM tumor growth and 78% of the untreated control animals required euthanasia within 17 days. All mice pretargeted with OKT10-SA demonstrated tumor shrinkage by day 6 at all dose levels (see figure). After 17 days, 90% of the OKT10-SA treated animals in the 400 μCi and 1200 μCi groups and 100% of the animals in the 800 μCi remained alive. One animal treated with 1200 μCi was euthanized on day 10 due to weight loss, however the remaining 9 animals from that group were 106 ±9% of initial body weight on day 17. Objective remissions were observed within 6 days in 100% of the mice treated with OKT10-SA followed by 1200 μCi of 90Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in OKT10-SA treated mice compared to tumors that were 5240 ± 2495% of initial tumor volume in untreated control animals) by day 17.These studies represent the first application of both PRIT and CD38 targeted radioimmunotherapy in MM. Favorable OKT10-SA biodistribution findings correlate with early evidence of therapeutic efficacy. Tumor responses in this MM xenograft tumor model are encouraging, but long term toxicity and survival results are not yet mature. Future studies combining PRIT and novel agents are planned in xenograft and SCID-hu myeloma models. [Display omitted] Disclosures:Gopal:Millenium. Wood:BD Biosciences: Research Funding.

Polypeptide conjugates of d-penicillamine and idarubicin for anticancer therapy

We investigated anticancer therapy with a novel combination of d-penicillamine (d-pen) and Idarubicin (Ida) in a synthetic dual drug conjugate (DDC). d-pen and Ida were covalently linked to poly(α)-l-glutamic acid (PGA) via reducible disulfide and acid-sensitive hydrazone bonds, respectively. The DDCs showed cell uptake and sustained release of the bound drugs in conditions mimicking the intracellular release media (10mM glutathione and pH 5.2). The in-vitro cytotoxicity of DDCs was comparable to unconjugated Ida in several sensitive and resistant cancer cell lines and correlated with the rate of cell uptake. In a single equivalent-dose pharmacokinetic study, DDCs enhanced the drug exposure by 7-fold and prolonged the plasma circulation half-life (t1/2) by 5-fold over unconjugated Ida. The therapeutic index of DDCs was 2–3-fold higher than unconjugated drugs. DDCs caused 89% tumor growth inhibition compared to 60% by unconjugated Ida alone and led to significant enhancement in the median survival (17%) of athymic nu/nu mice bearing NCI-H460 tumor xenografts.

The design of semi-synthetic and synthetic glycoconjugate vaccines

Introduction:Glycoconjugate vaccines are among the safest and most efficacious vaccines developed during the last 30 years. They are a potent tool for prevention of life-threatening bacterial infectious diseases like meningitis and pneumonia. The concept of hapten–carrier conjugation is now being extended to other disease areas.Areas covered:This is an overview of the history and current status of glycoconjugate vaccines. The authors discuss the approaches for their preparation and quality control as well as those variables which might affect their product profile. The authors also look at the potential to develop fully synthetic conjugate vaccines based on the progress of organic chemistry. Additionally, new applications of conjugate vaccines technology in the field of non-infectious diseases are discussed. Through this review, the reader will have an insight regarding the issues and complexities involved in the preparation and characterization of conjugate vaccines, the variables that might affect their immunogenicity and the potential for future applications.Expert opinion:The immunogenicity of weak T-independent antigens can be increased in quantity and quality by conjugation to protein carriers, which provide T-cell help. Glycoconjugate vaccines are among the safest and most efficacious vaccines developed so far. Various conjugation procedures and carrier proteins can be used. Many variables impact on the immunogenicity of conjugate vaccines and a tight control through physicochemical tests is important to ensure manufacturing and clinical consistency. New and challenging targets for conjugate vaccines are represented by cancer and other non-infectious diseases.

Synthesis and α-amylase inhibitory activity of glucose–deoxynojirimycin conjugates

Inhibitors of α-amylase have attracted attention for their putative effects against diabetes mellitus. Although numerous studies have explored natural small molecule inhibitors, acarbose is currently the only compound with sufficient inhibitory potency and drug-like characteristics to be considered as a potential therapeutic agent. We have synthesized conjugates of the potent glucosidase inhibitor, 1-deoxynojirimycin, and glucose, with the aim of enhancing inhibitory activity against α-amylase. This synthetic conjugate showed increased inhibition of α-amylase compared to 1-deoxynojirimycin alone, suggesting that similar modifications of existing glucosidase inhibitors may yield more potent α-amylase inhibitors.

Development and Characterization of a Mouse Monoclonal Antibody Against Antimicrobial Peptide Tachyplesin I

Monoclonal antibodies against tachyplesin I (TP I) were developed to study its mechanisms of activity, a kind of cationic antimicrobial peptides (AMPs), in vivo or in vitro, and to purify TP I from expression products. The synthesized TP I was chemically conjugated with the carrier protein BSA and then injected into BALB/c mice. Positive hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using TP I and subcloned three times with limiting dilution. Five MAbs effective in detecting the native TP I (named 2D8, 3B8, 5H2, 6B12, and 8F5) were obtained. Isotyping of all obtained MAbs indicated that MAbs 2D8, 3B8, 5H2, and 8F5 belong to IgG1, and MAb 6B12 belongs to IgG2a. Specificity assay showed that MAb 8F5 had almost the same level of specificity to natural TP I, recombinant TP I, and synthesized TP I and TP II, but did not cross-react with control peptides. These results suggest that the synthetic AMP conjugates can elicit antibodies against native AMPs and can be used to detect antimicrobial peptides.

Antibody- and cell-mediated immune responses to a synthetic oligosaccharide conjugate vaccine after booster immunization

Memory formation to CRM-neoglycoconjugate, a synthetic branched tetrasaccharide of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS) that is conjugated to a CRM197 protein, was investigated using mice models. Mice were first immunized with the CRM-neoglycoconjugate and then boosted with either the same neoglycoconjugate or a native Pn14PS in order to investigate the effect of booster immunization. Boosting with the CRM-neoglycoconjugate resulted in increased levels of interleukin 5 (IL-5) in the serum on Day 1, followed by the appearance of high levels of specific anti-Pn14PS IgG antibodies on Day 7. Boosting with native Pn14PS resulted in neither IL-5 induction nor the generation of anti-Pn14PS IgG antibodies. In vitro (re)stimulation of spleen cells after booster injection with the neoglycoconjugate revealed the presence of IL-4 and IL-5. This was not seen in spleen cells obtained from mice boosted with the polysaccharide. When stimulated with heat-inactivated bacteria, however, the polysaccharide-boosted mice did have higher levels of IFN-γ and lower levels of IL-17 than both the CRM-neoglycoconjugate-boosted mice and the mock-immunized mice. In conclusion, neoglycoconjugate boosting is responsible for the activation of memory cells and the establishment of sustained immunity. Not only is a booster with native polysaccharide ineffective in inducing opsonic antibodies, but it also interferes with several immunoregulatory mechanisms.

Synthesis of Alkyne-Functionalized Helical Polycarbodiimides and their Ligation to Small Molecules using ‘Click’ and Sonogashira Reactions

New terminal alkyne side chain functionalized polycarbodiimides, have been synthesized and “alkyne” ligation tools—“click” (copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction) and Sonogashira couplings—were utilized to create functional polymers in one step postpolymerization modifications. Polycarbodiimides are interesting synthetic, helical, nanodimensional scaffolds that are capable of presenting a variety of functional groups in highly regular periodic fashion. Herein, we report the synthesis of the first alkyne substituted polycarbodiimides, Poly-1, Poly-2, and Poly-3 and the quantitative coupling of functional azides to the alkyne repeat units of these polymers, as evidenced by IR and 1H NMR and 13C NMR spectra. We are able to couple benzyl azide, carboxylic acid azide, and N-Boc amine azide by “click” and N-Boc-l-phenylalanine by Sonogashira reactions to the alkyne side chain of Poly 1 to provide Poly-1a, -1b, -1c and -1phe, respectively. Poly-1b and Poly-1c provide peptide-coupling sites and are precursors for synthetic helical polymer-peptide conjugates. This was demonstrated by coupling l-alanine methylester to Poly-1b with excellent conversion. Aliphatic-terminal alkyne functionalized polymer, Poly-2 with the flexible methylene linker to the backbone provides more degrees of steric freedom for the incorporated groups, fixes the groups closer to the chiral, helical backbone, and reduces the hydrophobic nature of the polymer. However, Poly-3 offers all of these advantages and with two clickable handles per repeat unit doubles the functional sites along the polymer backbone.

Mass Spectrometric Characterization of Circulating and Functional Antigens Derived from Piperacillin in Patients with Cystic Fibrosis

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

A novel dextran-oleate-cRGDfK conjugate for self-assembly of nanodrug

We report a novel synthetic biocompatible material: a conjugate with a fatty acid-substituted dextran decorated with cRGDfK peptide, which was used as a stable coating material instead of the conventional poly(ethylene glycol) for nanodrug preparation. This novel dextran-oleate-cRGDfK conjugate (DO-cRGDfk) could self-assemble into a micellar structure in aqueous solution, and was used as a surfactant to formulate nanodrug with poly(d,l-lactic- co-glycolic) acid as matrix to encapsulate paclitaxel with high drug-loading efficiency. The conjugate allowed the fabrication of nanodrug with a targeting moiety on its surface in a simple and robust step. The resultant nanoparticles could induce cellular apoptosis more effectively than that of the commercial paclitaxel formulation, Taxol. Thus, DO-cRGDfk could be used as an alternative to poly(ethylene glycol) as a biocompatible surface coating polymeric material for nanoparticle preparation. From the Clinical Editor The authors describe a novel synthetic biocompatible conjugate, which consists of a fatty acid-substituted dextran decorated with cRGDfK peptide. This conjugate was used as a stable coating material for nanodrug preparation, and can be used in place of conventional PEG.

Abstract 2321: Pyrrolobenzodiazepine (PBD) antitumour agents as chemical probes to determine the number of base pairs required for DNA minor groove structure

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective covalent-binding DNA-interactive agents that fit perfectly in the minor groove of double-stranded DNA due to their isohelical 3-dimensional shape. They possess an electrophilic N10-C11 imine (or the equivalent) that can form a covalent aminal bond between their C11-position and the C2-NH2 group of a guanine. One synthetic PBD dimer, SJG-136, has just entered Phase II clinical trials. Previous studies using HPLC/MS, CD spectroscopy, high-field NMR, X-ray crystallography and gel-based assays such as DNA footprinting and in vitro transcription stop are all consistent with the requirement for duplex DNA for covalent binding. Conversely, it is well-documented that PBDs will not interact with single stranded DNA which lacks minor groove structure. In this study, these observations served as a basis to use PBDs as chemical probes to determine the minimum number of bases required to create a minor groove environment within a DNA duplex. Thus we studied reaction of the naturally-occurring anthramycin (3 base pair span) and the synthetic C8-linked PBD bis-pyrrole conjugate GWL-78 (6 base pair span) with a range of designed duplex oligonucleotides of increasing length (3 to 11 base pairs), each containing an AGA binding triplet. Reaction was monitored by HPLC/MS which provided data on both the rate and extent of reaction, and the structure of DNA adducts formed. For both anthramycin and GWL-78, we found that a minimum of 7 DNA base pairs were required for reaction to occur, suggesting that this may represent the minimum necessary to provide a suitable minor groove environment. Furthermore, we found that the rate of reaction for both PBDs increased with the length of the oligonucleotide between 7 to 10 base pairs. It is noteworthy that GWL-78 is approximately twice the length of anthramycin but both require at least 7 base pairs in order to covalently bind. This suggests that both may be responding to the formation of a minor groove environment in an oligonucleotide that reaches 7 base pairs in length, rather than a direct relationship between the length of the PBD and the number of base pairs in the oligonucleotide. These observations were supported by molecular modeling which confirmed that 7 base pairs are required to provide minor groove structure. This is the first study to use a chemical probe of this type to monitor formation of a minor groove. This information should be useful in the design of future generations of PBDs and similar DNA-interacting anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2011-2321

The development of pyrrolobenzodiazepines as antitumour agents

Introduction: DNA interacting agents play a major role in cancer chemotherapy, either as single agents, in combination drug regimens, or as components of novel targeted therapies. The search for more selective and efficacious drugs that can deliver critical DNA damage with minimal side effects continues.Areas covered: The development of the pyrrolobenzodiazepines (PBDs) from their discovery as natural products in the 1960s, through synthetic PBD monomers, PBD hybrids and conjugates, and PBD dimers is described. The latter molecules are capable of forming sequence selective, non-distorting and potently cytotoxic DNA interstrand cross-links in the minor groove of DNA. In particular, the development of PBD dimer SJG-136 (SG2000), currently in Phase II clinical trials, is presented. Potential future cancer therapeutic applications of PBDs, including their use as components of targeting strategies, are also discussed.Expert opinion: The culmination of over four decades of study on structure–activity relationships of PBDs has led to a detailed understanding of how to introduce structural modification to enhance biological activity and potency. The challenge for the next phase in the development of the PBDs is to harness this activity and potency in a new generation of cancer therapeutics.

Optimization of the MTT Assay for B16 Murine Melanoma Cells and its Application in Assessing Growth Inhibition by Polyamines and Novel Polyamine Conjugates

As part of our continuing development of new cytotoxins with potential anticancer activity, we have synthesized polyamine conjugates containing both the linear tetra-amine spermine and an acridine unit. Studies of growth inhibition by these novel conjugates and spermine were carried out using B16 murine melanoma cells. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay has been optimized for this cell line with respect to incubation time for MTT metabolism, inoculum density and incubation period. A linear relationship has been established between the amount of formazan produced and the number of viable B16 murine melanoma cells. Furthermore, the optimal incubation time of cells with MTT is 3 h. Beyond this time no further significant amount of formazan was generated. The optimal seeding density was determined (4000 cells/well for 2- or 3- day experiments), and we estimate that the doubling time for B16 cells is approximately 24 h. In 48-h assays, spermine (1) inhibits cell growth with an EC50 of 450 μM, acylated analogue (5) has an EC50 of 5 μM, almost a hundred-fold increase in potency over spermine, and the aniline analogue (6) has an EC50 of 1 μM, a five-fold increase in potency over amide analogue (5). The EC50 for spermine (1) and aniline (6) changed little over 3 and 6 days compared with the EC50 over 2 days. We conclude that there is little significant metabolic influence (with time) on the observed toxicity data. We have also shown that the synthetic conjugates combining both an intercalator and groove binder show greater cytotoxicity than compounds that exhibit just one of these modes of binding. These analogues therefore offer a new lead in the design of cytotoxic polyamines with potential anticancer activity.

Arginine-specific protein modification using α-oxo-aldehyde functional polymers prepared by atom transfer radical polymerization

The residue-specific modification of peptides and proteins is a powerful strategy for preparing biomolecular–synthetic polymer conjugates with advanced properties. This manuscript aims at expanding the present toolbox of residue-selective protein modification reactions and targets arginine, a residue for which selective polymer coupling chemistry has only recently been established. To this end, a protected, α-oxo-aldehyde functionalized ATRP initiator that can be used for the preparation of a variety of α-oxo-aldehyde functionalized polymethacrylates has been developed. Polymerization kinetics for four different methacrylate monomers have been investigated in detail and optimized conditions for the chain-end deprotection to reveal the α-oxo-aldehyde end-group have been elaborated. As a final proof of concept, the residue-specific modification of a model protein, chicken egg white lysozyme (HEWL), at arginine residues has been demonstrated.

Strong supramolecular control over protein self-assembly using a polyamine decorated β-cyclodextrin as synthetic recognition element

The supramolecular host molecule heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-cyclodextrin provides strong control over protein self-assembly in synthetic supramolecular protein constructs. Mono-functionalization of this modified β-cyclodextrin with a cysteine residue allows for site-selective synthetic conjugation to a protein and formation of a highly stable synthetic protein complex with a lithocholic acid conjugated protein as the interaction partner.

Synthetic conjugates of genistein affecting proliferation and mitosis of cancer cells

This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsaturated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were obtained in a two-step synthesis: in a first step genistein was converted into an intermediate bearing an ω-hydroxyalkyl substituent, containing two, three or five carbon atoms, at position 7, while the second step involved Ferrier glycosylation reaction, employing glycals. Antiproliferative activity of several genistein derivatives was tested in cancer cell lines in vitro. The most potent derivative, Ram-3 inhibited the cell cycle, interacted with mitotic spindles and caused apoptotic cell death. Neither genistein nor the sugar alone were able to influence the mitotic spindle organization. Our results indicate, that conjugation of genistein with certain sugars may render the interaction of derivatives with new molecular targets.

Preparation and Characterization of Neutralizing Monoclonal Antibodies Against FMDV Serotype O with Synthetic Peptide Antigen

A short linear peptide was designed according to the antigenic site analysis of VP1 protein of foot-and-mouth virus (FMDV) serotype O and synthesized as the peptide immunogen. The peptide, which covers the region from amino acid 133 to 160 of VP1 of FMDV, was linked to the N-terminal cysteine and conjugated with the carrier protein of keyhole limpet hemocyanin (KLH). Normal 6- to 8-week-old BALB/c mice were immunized with the 20 μg dose conjugated peptide antigen four times. The splenocytes from the immunized mice were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned four times with limiting dilution. Five stable hybridoma cell lines, designated as 4F9, 1B11, 1E10, 1D4, and 4B8, were obtained. Isotyping of all obtained MAbs indicated that the MAbs of 4F9, 1E10, and 4B8 belonged to IgG2b; the 1B11 and 1D4 belonged to IgG1 and IgM, respectively. The micro-neutralization test indicated that the MAbs of 4F9, 4B8, and 1B11 were capable of neutralizing FMDV serotype O with neutralization indices ranging from 1.81 to 2.11. These results suggest that linear synthetic peptide conjugate can elicit antibodies against native FMDV virus and can be used as an alternative immunogen for production of MAbs with exact epitope.

The synthetic bile acid–phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide suppresses TNFα-induced liver injury

Excessive apoptosis and leukocyte-dependent inflammation mediated by pro-inflammatory cytokines, such as TNFα, are cardinal features of acute liver injury. This study evaluated the ability of the newly designed bile acid-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) to protect from hepatocellular injury in comparison to the known hepatoprotectant ursodeoxycholic acid (UDCA) and phosphatidylcholine (PC). Anti-apoptotic and anti-inflammatory properties of UDCA-LPE were evaluated after TNFα treatment of embryonic human hepatocyte cell line CL48 as well as of primary human hepatocytes. Acute liver injury was induced in C57BL/6 mice with d-galactosamine/lipopolysaccharide (GalN/LPS) in order to determine in vivo efficacy of the conjugate. UDCA-LPE inhibited TNFα-induced apoptosis and inflammation in hepatocytes in vitro and markedly ameliorated GalN/LPS-mediated fulminant hepatitis in mice, whereas UDCA or PC failed to show protection. The conjugate was able to decrease injury-induced elevation of phospholipase A(2) activity as well as its product lysophosphatidylcholine. Analysis of hepatic gene expression showed that UDCA-LPE treatment led to favourable inhibitory effects on expression profiles of key pro-inflammatory cytokines and chemokines, which are crucial for leukocyte recruitment and activation thereby inhibiting chemokine-mediated aggravation of parenchymal damage. Thus, UDCA-LPE as a synthetic bile acid-phospholipid conjugate may represent a potent anti-inflammatory agent that is more effective than UDCA and PC for treatment of liver diseases.

A synthetic ditryptophan conjugate that rescues bacteria from mercury toxicity through complexation

The synthesis of ditryptophan–pyridine conjugates and their binding to mercury ions is described. Conjugate 3 shows an excellent ability to sequester mercury from solution and rescue bacterial growth in a concentration-dependent survival assay. It is proposed that such compounds, composed primarily of bioessential/biodegradable components, could be potentially used as sequestrating agents for the removal of Hg(II) ions in detoxification strategies.

ChemInform Abstract: From the Laboratory to the Clinic: A Retrospective on Fully Synthetic Carbohydrate-Based Anticancer Vaccines

This review provides an account of our explorations into oligosaccharide and glycoconjugate construction for the creation and evaluation of vaccines based on carbohydrate-centered tumor antigens. Our starting point was the known tendency of transformed cells to express selective carbohydrate motifs in the form of glycoproteins or glycolipids. Anticancer vaccines derived from carbohydrate-based antigens could be effective targets for immune recognition and attack. Obtaining significant quantities of such structures from natural sources is, however, extremely difficult. With the total synthesis of tumor-associated carbohydrate antigens accomplished, we began to evaluate at the clinical level whether the human immune system can respond to such fully synthetic antigens in a focused and useful way. Toward this goal, we have merged the resources of chemistry and immunology in an attack on the problem. The synthesis and immunoconjugation of various tumor-associated carbohydrate antigens and the results of such constructs in mice vaccinations will be described. For fashioning an effective vaccine, conjugation to a suitable immunogenic carrier was necessary and conjugates of KLH (keyhole limpet cyanin) have consistently demonstrated the relevant immunogenicity. Preclinical and clinical studies with synthetic conjugate carbohydrate vaccines show induction of IgM- and IgG-antibody responses. Another approach to anticancer vaccines involves the use of clustered glycopeptides as targets for immune attack. Initial attention has been directed to mucin related O-linked glycopeptides. Synthetic trimeric clusters of glycoepitopes derived from the Tn-, TF- and Lewis(y)-antigens, appropriately bioconjugated, have been demonstrated to be immunogenic. The hope is that patients immunized in an adjuvant manner with synthetic carbohydrate vaccines would produce antibodies reactive with cancer cells and that the production of such antibodies would mitigate against tumor spread, thereby enabling a more favorable survival and quality of life prognosis.