Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography

Fluorescent esters of the prostablandins D₂, E₂, F₂α, and 6-keto-F₁α and of thromboxane B₂ have been prepared using the reagent 4-bromomethyl-7-methoxycoumarin. All of these derivatives can be separated in a single run either by thin-layer or high-performance liquid chromatography (TLC or HPLC). As little as 20 ng of PGE₂ can be detected after derivatization and HPLC analysis. Identification of thromboxane B₂ produced by human platelets and of 6-keto-PG F₁α produced by bovine aortic microsomes has been achieved with this method.     

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. ³H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.     

Synthesis and biological activity of amino acid conjugates of abscisic acid

We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA.     

Study of prostaglandins and thromboxanes B2 using mass-spectrometry of secondary ions

Prostaglandins E, F, I2 and thromboxane B2 have been studied by secondary ion mass spectrometry. It is shown that the method is suitable for direct identification of these compounds either as free acids or as their sodium salts. The spectra of the former reveal their structural features, while with the latter information on the molecular weight can be obtained. The limit of detection (about 1 microgram) allows the analysis of prostaglandin solutions of 1 microgram/microliter concentrations used in pharmacological tests.     

Synthesis of dihydroquinopimaric acid conjugates with amino acids

Synthesis of dihydroquinopimaric acid amides and their 2β-succinyl and 2β-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.     

Design,synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC₅₀ values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.     

Synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates

Beta-carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further enhance the cytotoxic potency and bioavailability of beta-carboline, a series of novel beta-carboline amino acid ester conjugates were designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines. In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The beta-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental beta-carbolines. In particular, the Lys/Arg conjugates were the most potent analogs with an IC(50) value of 4 and 1 microM against human cervical carcinoma cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken together, this study provided new insights into structure-activity relationships in the beta-carboline amino acid ester conjugates and identified the beta-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.     

Use of ion-exchange resins for removing prostaglandins from human urine prior to radioimmunoassay

Investigation of prostaglandin and prostaglandin metabolite levels in human urine by radioimmunoassay requires the removal of prostaglandins from urine in a predictable, reproducible manner. Screening of eighteen ion-exchange resins for their ability to remove tritiated prostaglandins from human urine was undertaken. Two resins, Amberlite IRP-64M and Amberlite CG-50 were found acceptable.     

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Synthesis of amino acid conjugates of 1,1′-dimethylferrocene: New chiral conjugates

Syntheses of diastereomeric mixtures of 1,1′-dimethylferrocene-2-Ala-OMe (3) and 1,1′-dimethylferrocene-3-Ala-OMe (4) are reported by peptide coupling of L-Ala-OMe to the enantiomeric mixtures of the corresponding acids, 1,1′-dimethylferrocene-2-carboxylic acid (1) and 1,1′-dimethylferrocene-3-carboxylic acid (2).     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Measurement of prostaglandins in embryonic tissue using radioimmunoassay

Although prostaglandins appear to play an important role in numerous physiological processes in the adult, neonate, and fetus, very little is known about the role of these compounds in the embryo. This study demonstrates that rat embryo homogenates synthesized 6-oxo-PGF_1α; PGE and PGF in markedly different amounts from endogenous substrate. Synthesis was inhibited by indomethacin (10 μM) in varying degrees (70–89%) depending on the prostaglandin. The metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGF_2α-M), was produced in limited amounts in the absence of exogenous NAD. In the presence of exogenous NAD and PGF however, embryonic homogenates produced PGF_2α-M. The potential role of prostaglandins during embryogenesis is discussed.     

Variables associated with radioimmunoassay of prostaglandins in plasma

This study examined a number of variables which influence radioimmunoassay measurements of prostaglandins in plasma. Comparative assays in specimens obtained from normal subjects and processed by different methods demonstrated that prostaglandin measurements were uniformly lower when blood was anticoagulated with EDTA, aspirin or indomethacin was added to inhibit further synthesis of prostaglandins , samples were centrifuged at 4°C at speeds which removed platelets from the sample, and assays were performed when plasma was thawed for the first time. Examination of assay results also revealed that the presence of unextracted plasma enhanced the binding of labeled ligands to antisera (%B_o) but thereafter, the displacement of ligand by increasing quantities of the reference prostaglandin paralleled the results in buffer throughout the range of the assay. In subsequent studies therefore the %B_o for plasma assays was adjusted on the basis of ligand binding in the presence of comparable volumes of pooled plasma from aspirin-treated subjects. Results of 7 prostaglandin assays in plasma and serum of normal subjects are reported.     

Anti-HIV activity of a series of cosalane amino acid conjugates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.