Tritium-labeled Analog Research Articles

Characterization of a New Positive Allosteric Modulator of AMPA Receptors - PAM-43: Specific Binding of the Ligand and its Ability to Potentiate AMPAR Currents.

Currently, the most dynamic areas in the glutamate receptor system neurobiology are the identification and development of positive allosteric modulators (PAMs) of glutamate ionotropic receptors. PAM-based drugs are of great interest as promising candidates for the treatment of neurological diseases, such as epilepsy, Alzheimer's disease, schizophrenia, etc. Understanding the molecular mechanisms underlying the biological action of natural and synthetic PAMs is a key point for modifying the original chemical compounds as well as for new drug design. We are trying to elaborate a system of molecular functional screening of ionotropic glutamate receptor probable PAMs. The system will be based on the radioligand - receptor method of analysis and will allow rapid quantification of new AMPAR probable PAMs molecular activity. We plan to use a tritiumlabeled analogue of recently elaborated ionotropic GluR probable PAM ([3H]PAM-43) as the main radioligand. Here, we characterized the specific binding of the ligand and its ability to potentiate ionotropic GluR currents. The existence of at least two different sites of [3H]PAM-43 specific binding has been shown. One of the above sites is glutamate-dependent and is characterized by higher affinity. "Patchclamp" technique showed the ability of PAM-43 to potentiate ionotropic GluR currents in rat cerebellar Purkinje neurons in a concentration-dependent manner. The possibility of using PAM-43 as a model compound to study different allosteric effects of potential regulatory drugs (AMPAR allosteric regulators) was shown. [3H]PAM-43 based screening system will allow rapid selection of new AMPAR probable PAM structures and quantification of their molecular activity.

Synthesis of Tritium-Labeled Non-Natural Analogs of Purine and Pyrimidine Nucleosides

Convenient preparative ways to synthesize tritium-labeled non-natural derivatives containing the cytosine and guanine moieties have been developed. The main distinction of these compounds from those already known consists in that they contain a chiral center. The tritium-labeled 2'-desoxy-3'-oxacytidine can was synthesized by the reaction of catalytic dehalogenation in solution of the corresponding 5-bromine-substituted compound; for 6-aminocyclopropyl-2'-desoxy-3'-oxaguanosine, the reaction of catalytic heterogeneous catalytic exchange in solution was used; for rest of compounds, the reaction of solid-phase catalytic hydrogenation (SCH) was employed.

A biotechnological procedure for preparing short peptides and their radioactively labeled analogs for estimating specific binding of Semax and its metabolites in various sections of rat brain

The peptides His-Phe-Pro-Gly-Pro and Phe-Pro-Gly-Pro, as well as their tritium-labeled analogs, were prepared by biotechnological methods from peptides of the melanocortin series using amino-acylarylamidase (E.C. 3.4.11.2) (yield >60%) and glutamate-specific aminopeptidase Bacillus intermedius Glutamyl Endopeptidase (E.C. 3.4.21.19) (yield 100%). With the latter enzyme, it is possible to prepare the peptides and their modified analogs from precursors containing a Glu residue in the amino acid sequence. The biotechnological method allows synthesis of peptides in preparative amounts in a high yield, without formation of byproducts. With radioactively labeled compounds as substrate, the biosynthesis yields peptides identical in the radiochemical characteristics. Experiments with labeled peptides revealed the existence of centers of specific binding in different sections of rat brain for different Semax metabolites.

Characterization of the Tritium-Labeled Analog of L-threo-β-Benzyloxyaspartate Binding to Glutamate Transporters

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo-beta-benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([3H]ETB-TBOA). [3H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na+-dependence of [3H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na+, suggesting that Na+ binding in the ligand binding step is different from Na+ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [3H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

The Design and Synthesis of YC-1 Analogues as Probes for Soluble Guanylate Cyclase.

Soluble guanylate cyclase (sGC) is highly activated in the presence of both YC-1 (1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole) and CO. In this report, the design, synthesis, and activity (i.e., sGC activation) of photolabile analogues of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) are presented. Initial results with 6-azido-3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole led to the synthesis of a tritium-labeled analogue. When photoactivated, this analogue labeled the alpha-subunit of sGC.

The design and synthesis of YC-1 analogues as probes for soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is highly activated in the presence of both YC-1 (1-benzyl-3-(5′-hydroxymethyl-2′-furyl)-indazole) and CO. In this report, the design, synthesis, and activity (i.e., sGC activation) of photolabile analogues of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) are presented. Initial results with 6-azido-3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole led to the synthesis of a tritium-labeled analogue. When photoactivated, this analogue labeled the α-subunit of sGC.

Synthesis of deuterium- and tritium-labeled 25,26,26,26,27,27,27-heptafluorocholesterol

25,26,26,26,27,27,27-Heptafluorocholesterol (F7-cholesterol) is a novel analog of cholesterol with potential value in biological investigations. We have combined an isotope-labeling strategy described for 15-ketosterols with modifications in the original synthesis of F7-cholesterol to prepare analogs containing deuterium or tritium at the C-23 position. 1H, 2H, and 13C nuclear magnetic resonance spectra indicated high chemical purity of the deuterium-labeled analog and confirmed the presence of deuterium only at C-23. The tritium-labeled analog was synthesized in high radiochemical purity with a specific activity of 472 mCi/mmol. Copyright © 1999 John Wiley & Sons, Ltd.

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

N-Arylazido-.beta.-alanyl-Nad+, a new nad+ photoaffinity analog. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.

Development of a radioreceptor assay for the D2-selective dopamine agonist N-0437.

N-0437 is a recently developed dopamine (D2) agonist, theoretically attractive in the therapy of Parkinson's disease and glaucoma. Since its high potency allows small doses of the compound in clinical use and as extensive metabolism occurs in animals, a highly sensitive assay method was required for drug-monitoring purposes. To this end we developed a radioreceptor assay (RRA), a sensitive tool for the assessment of the sample's (dopaminergic) bioactivity. The RRA is based on competition between N-0437 and its tritium-labeled analogue for binding to dopamine receptors. The assay has been optimized for the preparation of the receptor suspension and the incubation conditions. Direct application of the assay for biological samples was impossible because of matrix interferences. Therefore, a solid-phase extraction method was developed in which the combination of a polar Si column and dichloromethane as eluent resulted in an effective elimination of the interferences. Recoveries were better than 90 and 95% for plasma and urine, respectively, even at concentrations at the determination limit of the method (300 pg/ml). Relative standard deviations were less than 15%. Because RRAs are stereoselective, the method discriminates between active and inactive species.

Evidence for the ionization steps in monoterpene cyclization reactions using 2-fluorogeranyl and 2-fluorolinalyl pyrophosphates as substrates

Conversion of geranyl pyrophosphate to cyclic monoterpenes is considered to involve the preliminary isomerization of this acyclic precursor to enzyme-bound linalyl pyrophosphate and the cyclization of this tertiary intermediate. 2-Fluorogeranyl pyrophosphate and 2-fluorolinalyl pyrophosphate are effective competitive inhibitors of the cyclization of geranyl pyrophosphate by several different monoterpene cyclases, and the electron withdrawing α-fluorine substituent was shown to suppress the rate of cyclic product formation from both tritium-labeled analogs by at least two orders of magnitude. These results indicate that both steps of the coupled isomerization-cyclization sequence are initiated by ionization of an allylic pyrophosphate, and they confirm the electrophilic nature of this enzymatic reaction type and its similarity to the prenyltransferase reaction.

An optimized method for measuring cyclosporin A with 125I-labeled cyclosporin.

We evaluated the use of the new iodinated ligand for the in vitro measurement of cyclosporin A by radioimmunoassay (RIA). Substitution of the iodinated cyclosporin (125I-CyA) for the corresponding tritium-labeled analog (3H-CyA) considerably simplifies and accelerates the currently available RIA, and improves its precision. Analysis of the respective dose-response curves showed that the 50% B0 value was lower for the 125I-CyA assay than for the 3H-CyA assay (37 vs 77 micrograms/L). Use of whole-blood specimens minimized interferences from temperature and hematocrit. We conclude that the use of 125I-CyA in a commercially available RIA for whole-blood specimens is accessible to most laboratories and provides rapid, reproducible data for management of transplant patients.

Photoaffinity labeling of the V1 vasopressin receptor in plasma membranes from rat liver.

Photoaffinity labeling experiments were performed with membranes from rat liver containing V1 vasopressin receptors. Photoreactive analogues of [1-beta-mercaptopropionic acid]vasopressin [( Mpa1], vasopressin, or deamino-vasopressin) retaining a high binding affinity (apparent dissociation constants: 5 X 10(-9) M-3 X 10(-8) M) and agonistic properties were used. The tritium-labeled analogue [Mpa1,Lys(N epsilon-4-azidobenzoyl)8]vasopressin preferentially and specifically labels a 30-kDa polypeptide and with lower efficiency a 38-kDa polypeptide. The analogue [Mpa1,Dab4(N gamma-(N-4-azido-2-nitrophenyl-beta-Ala4]arginine-vasopressin specifically labels the 38-kDa polypeptide. The labeling of these two membrane proteins is completely suppressed by an excess of arginine-vasopressin; bradykinin or angiotensin II do not inhibit the incorporation of the reactive vasopressin analogues into these proteins. The results suggest that the rat hepatic V1 receptor exists in the plasma membrane in an oligomeric form composed of two subunits with a molecular mass of 30 and 38 kDa.

Pyrazolo[1,5-a]pyrimidines: Receptor binding and anxiolytic behavioral studies

Pyrazolo[1,5-a]pyrimidines (PZP) have been reported to be specific anxiolytic agents which do not potentiate ethanol or barbiturates. To further investigate these compounds, three of the most promising analogs were synthesized and a tritium-labeled analog of one of them prepared by a new synthetic procedure. These analogs did not compete with [ 3H]flunitrazepam or [ 3H]β-carboline ethyl ester binding nor did they potentiate the [ 3H]flunitrazepam binding. Receptor binding studies with the [ 3H]PZP revealed a low affinity receptor site, distinct from that of the benzodiazepines, but with only a small fraction (20%) of specific binding. Behavioral tests using three different animal models for anxiety: muricide, approach/avoidance conflict and two-chamber exploration tests gave conflicting results, positive in the first and negative in the latter two. Furthermore, these compounds were found not to be antagonists of diazepam's anticonvulsant activity. Taken together, these results, while provocative, do not support evidence that these analogs are promising specific anxiolytic agents.

Separation of unlabeled metabolites of arachidonic acid from their deuterium- and tritium-labeled analogs by argentation high-pressure liquid chromatography

Deuterium- and tritium-labeled analogs of high isotopic purity are required for the analysis of metabolites of arachidonic acid (20:4) by mass spectrometry and radioimmunoassay. We prepared a number of 2H-labeled standards by incubating [5,6,8,9,11,12,14,15- 2H]20:4 with soybean lipoxygenase, polymorphonuclear leukocytes, or homogenates of rat spleen or bovine lung. The unlabeled and 2H-labeled forms of the following products were completely resolved by argentation high-pressure liquid chromatography: 20:4; 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5h-20:4); 11h-20:4; 12h-20:4; 15h-20:4; 12-hydroxy-5,8,10-heptadecatrienoic acid; leukotriene B 4; prostaglandin D 2 (PGD 2); and PGF 2α. The corresponding forms of PGE 2 and thromboxane B 2 were also separated, but not quite as well as the above compounds. Analysis by mass spectrometry indicated that the amounts of unlabeled material in approx 1:1 mixtures of unlabeled and 2H-labeled products could be reduced to 0.1% or less after two steps of argentation high-performance liquid chromatography. Metabolites of 20:4 labeled with tritium were also separated from their unlabeled counterparts and had retention times longer than those of the corresponding 2H-labeled analogs.

Quantitative analysis of two dinor urinary metabolites of prostaglandin I 2

The synthesis of deuterium- and tritium-labeled analogs of 2,3-dinor-6-keto-prostaglandin F 1α and of 6,15-diketo-13,14-dihydro-2,3-dinor-prostaglandin F 1α is described. These analogs were used as internal standards in the assay of the corresponding unlabeled metabolites in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry. In male subjects the 24-h urinary excretion of the two metabolites was found to be 719 ± 264 and 314 ± 115 ng, respectively. The method offers a noninvasive approach to the study of prostaglandin I 2 synthesis in man.

Method for quantitative determination of the four known juvenile hormones in insect tissue using gas chromatography—mass spectroscopy

A rapid, specific, and sensitive method is described for the extraction, purification, and quantitative determination of the three known juvenile hormones in insect tissue, using combined gas chromatography—mass spectroscopy (GC—MS) in the electron impact mode with selected ion-monitoring (SIM) for detection. A fourth, new, naturally occuring juvenile hormone was detected (and subsequently identified by other means) in this way. After initial extraction of biological material, a fast and efficient purification sequence utilizing non-aqueous reversed-phase and alumina mini-column chromatography achieves quickly a 10 3–10 4 purification factor. Derivatization for analysis by acid-catalyzed methan-d 3-olysis of the 10,11-epoxide moiety, followed by liquid chromatography separation of the resultant 11-methoxy-d 3-10-hydroxy (methoxyhydrin) derivatives, completes the sample work-up. The methoxyhydrins are then directly analyzed by GC—MS (SIM). A tritium-labeled analogue is added during the extraction as an internal standard, which allows determination of recovery for each sample and simultaneously assures quantitative accuracy. Recoveries are 75–80%, and detection limits of 10–40 pg per gram of tissue are normally achievable.