Radiolabeled Form Research Articles

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes

Characteristics of GABA_B receptor binding sites on rat whole brain synaptic membranes

Volume-regulatory amino acid release from the protozoan parasite Crithidia luciliae.

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue alpha-aminoisobutyrate. alpha-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of alpha-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated alpha-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for alpha-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated alpha-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mM. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells.

Technepine: a high-affinity 99m-technetium probe to label the dopamine transporter in brain by SPECT imaging.

Increasing evidence suggests that the dopamine transporter, localized on dopamine neurons, is a marker for a number of physiological and pathological states (KaufmanandMadras, 1991,1993; Madras et al., 1990a, Schoemaker et al., 1985; Singer et al., 1991). With the development of sensitive probes, brainimaging and mea- surement of the transporter have become feasible in re- cent years (Brownell et al., in press; Innis et al., 1991; Frost et al., 1993; Madras et al., 1991; Morns et al., sub- mitted; Seibyl et al., 1995; van Dyke et a1.,1995; Wonget al., 1993,1995). Drugs of many chemical classes, includ- ing cocaine, bind to the dopamine transporter (Seeman, 1993). Nevertheless, effective imaging agents have been developed almost exclusively from the phenyltropane analogue of cocaine WIN 35,428 or CFT, a potent dopa- mine transport inhibitor (Clarke et al., 1973; Heikkila et al., 1979). The impetus for developing [llC]WIN 35,428 as a PET ligand (Hantraye et al., 1992; Madras, 1994; Madras et al., 1991, 1994; Wong et al., 1993; Meltzer et al., 1993) and y-emitting analogues for SPECT imaging (e.g., RTI-55, the 4-iodophenyl analogue of WIN 35,428, Canfield et al., 1990; Boja et al., 1991; Innis et al., 1991) arose directly from our observations of the binding of WIN 35,428 to the dopamine transporter. Unlike previ- ous dopamine transport inhibitors (noncocaine conge- ners) proposed for brain imaging(Kuhar et a1.,1990), the radiolabeled form of WIN 35,428 binds to the dopamine transporter in brain striatum with very low levels of non- specific binding (Madras et al., 1989a,b) and distributes principally to dopamine-rich regions of brain, as we re- ported in 1989 (Canfield et al., 1989) and subsequently (Canfield et al., 1990; Kaufman et al., 1991; Kaufman and Madras, 1992). SPECT imaging techniques are more practical than PET for routine clinical studies because of the lesser

Vector-mediated delivery of 125I-labeled beta-amyloid peptide A beta 1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the A beta 1-40/vector complex.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue beta-amyloid peptide A beta 1-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Transport of 125I-labeled A beta 1-40 (125I-A beta 1-40) through the BBB was found to be negligible by experiments with both an intravenous injection technique and an internal carotid artery perfusion method in anesthetized rats. In addition, 125I-A beta 1-40 was rapidly metabolized after either intravenous injection or internal carotid artery perfusion. BBB transport was increased and peripheral metabolism was decreased by conjugation of monobiotinylated 125I-A beta 1-40 to a vector-mediated drug delivery system, which consisted of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the 125I,bio-A beta 1-40/SA-OX26 conjugate was 0.15 +/- 0.01, a level that is 2-fold greater than the brain uptake of morphine. The binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was demonstrated by both film and emulsion autoradiography performed on frozen sections of AD brain. Binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was completely inhibited by high concentrations of unlabeled A beta 1-40. In conclusion, these studies show that BBB transport and access to amyloid within brain may be achieved by conjugation of A beta 1-40 to a vector-mediated BBB drug delivery system.

Coupled Movements of Glucose 6-Phosphate and Triose Phosphate through the Envelopes of Plastids from Developing Embryos of Pea ( Pisum sativum L.)

Summary The aim of this work was to identify the system responsible for the import of glucose 6-phosphate by plastids from developing embryos of pea ( Pisum sativum L.). Uptake was assayed after rapid separation of plastids from incubation media by centrifugation through silicone oil. Uptake of glucose 6-phosphate was fast and specific and was inhibited by dihydroxyacetone phosphate, phosphate, and phosphoenolpyruvate. Dihydroxyacetone phosphate also entered plastids. The movements of glucose 6-phosphate and dihydroxyacetone phosphate were linked; import of one was accompanied by release of 14 C-label from plastids preincubated with the other in radiolabelled form. Addition of phosphate or 3-phosphoglycerate to plastids preincubated with [U- 14 C] glucose 6-phosphate also caused the release of 14 C-label. The simplest explanation of these results is that plastids from developing embryos of pea have a counter-translocator accepting phosphate, triose phosphate, 3-phosphoglycerate, and glucose 6-phosphate as substrates. We also discuss the possible existence of other translocators, particularly a phosphate translocator similar to that of chloroplasts.

In vivo labeling of nicotinic acetylcholine receptors in brain with [ 3H]epibatidine

In vivo imaging of nicotinic acetylcholine receptors in brain has been hampered by lack of an adequate radioligand. In the present study, [ 3H]epibatidine was administered to mice intravenously, and its time-course in brain regions and sensitivity to blockade by nicotinic drugs were studied. The distribution of radioligand accumulation in brain, and the pharmacological characteristics of binding indicate that radiolabeled forms of epibatidine would be exceptionally promising ligands for the study of nicotinic acetylcholine receptors in vivo.

3H]Paroxetine and [3H]Citalopram as Markers of the Human Brain 5-HT Uptake Site

The binding of [3H]paroxetine and [3H]citalopram to the brain serotonin (5-HT) uptake site was characterized and compared in human hypothalamus, frontal cortex and caudate nucleus. Our results showed that the binding exclusively related to the 5-HT uptake site (specific or 5-HT displaceable binding) was identical for both [3H]ligands. The selective 5-HT uptake inhibitor citalopram displayed the highest potency (Ki) in inhibiting the [3H]paroxetine and [3H]citalopram binding, as compared with the values obtained for desipramine and norzimelidine. This fact is in accordance with the reported blockage of these compounds of the 5-HT uptake. Furthermore, similar Bmax values were obtained for both radioligands in the brain regions under study, indicating their binding to the same presynaptic membrane protein. These findings give evidence of the suitability of both [3H]paroxetine and [3H]citalopram as markers of the 5-HT transporter. However, the higher affinity of [3H] paroxetine (10 to 30-fold with respect to [3H]citalopram) confirms that the radiolabelled form of this compound is the best tool for the study of the 5-HT uptake site available today.

Predominant labeling of beta- over alpha-tubulin from porcine brain by a photoactivatable taxoid derivative.

An [(azidophenyl)ureido]taxoid (TaxAPU) was synthesized in a radiolabeled form by coupling an aminotaxoid to tritiated N-methyl-N-(chloroformyl)-p-azidoaniline. TaxAPU was used to photolabel polymerized porcine brain tubulin. This newly synthesized probe possesses taxoid properties as demonstrated by its effect, in the absence of light, on the kinetics of tubulin assembly and microtubule disassembly and on the critical concentration of tubulin. TaxAPU apparently competes with Taxol for the same binding site with an equilibrium dissociation constant of 6 microM. The photoactivation of 266 nm of the radiolabeled probe in the presence of microtubules led to a covalent incorporation of radioactivity. Analysis of the radiolabeled polypeptides by electrophoresis under denaturing conditions revealed a specific incorporation of tritium in both the alpha-and beta-subunits of tubulin. A dependence on probe concentration was observed for the irreversible radioactivity incorporated into both subunits and maintained essentially a ratio of 2.5:1 between beta/alpha. Therefore, TaxAPU constitutes a true photoaffinity probe for the taxoid binding site on microtubules. Our results complement those reported by Rao et al. (1992) of photo-cross-linking experiments with unmodified Taxol.

The post-translational processing and intracellular sorting of PC2 in the islets of Langerhans.

Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.

D 1-receptor antagonists: Comparison of [ 3H]SCH39166 to [ 3H]SCH23390

A radiolabeled form of the benzonaphthazephine, SCH39166 was used to characterize the binding of this D 1 antagonist in cortex, and an autoradiographic comparison of the localization of [ 3H]SCH39166 to [ 3H]SCH23390 (D 1 antagonist and forerunner of SCH39166) binding was performed. The K d for [ 3H]SCH39166, calculated from dissociation and association rate constants (1.09 n m), was comparable to the K d value derived from Scatchard analyses of saturation data (1.74 n m). [ 3H]SCH39166 binds to brain tissue in a saturable manner with high affinity and low non-specific binding. Inhibition of [ 3H]SCH39166 binding by dopaminergic and serotonergic agents supports the hypothesis that this is indeed a D 1-specific compound with little overlap onto serotonin (5-HT) receptors. The affinity of [ 3H]SCH39166 for 5-HT 2 and 5-HT 1c receptors is at least an order of magnitude lower than the affinity of [ 3H]SCH23390 for these same receptor sites. Quantitative autoradiographic analysis of [ 3H]SCH39166 and [ 3H]SCH23390 binding indicates high D 1-receptor density in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra and entopeduncular nucleus. Low levels of binding (not significantly above back ground) were detected with [ 3H]SCH39166 in lamina IV of the cortex and in choroid plexus; areas which had significant [ 3H]SCH23390 binding and are known to have a high density of 5-HT (5-HT 2 and 5-HT 1c respectively) receptors.

Estrogen platinum-diamine complexes: Preparation of a non-steroidal estrogen platinum—diamine complex labeled with platinum-191 and a study of its binding to the estrogen receptor in vitro and its tissue distribution in vivo

We have prepared in radiolabeled form (platinum-191) a non-steroidal estrogen platinum—diamine complex (Pt—diamine complex) that is reported to have selective cytostatic activity in estrogen receptor positive mouse mammary tumors. We then studied the interaction of this metal radiolabeled complex with the estrogen receptor in vitro and its distribution in immature rats in vivo. The radiolabeled complex was prepared by incubation of the non-steroidal estrogen diamine with [ 191 Pt]( II)Cl 4 −2(t 1 2 = 2.96 days, sp. act. 7.54 Ci/mmol) in dimethylformamide (DMF)/H 2O, followed by purification by HPLC. The final radiolabeled product coeluted with an authentic standard of the unlabeled Pt—diamine complex, with a retention time distinct from those of the precursor diamine and chloroplatinate. In competitive radiometric receptor binding assays with rat uterine estrogen receptor, samples of the unlabeled diamine and Pt—diamine complex have apparent binding affinities of 53±3% and 32 ± 11%, respectively, relative to estradiol (RBA = 100% as standard). However, attempts to observe the binding of the 191Pt—diamine complex with the estrogen receptor were complicated by a very high level of non-receptor binding, an irreversible binding to proteins in the receptor preparation, and a degradation of the platinum complex that, in part, releases the diamine. As a result, it is difficult to be certain whether the binding affinity measured for the Pt—diamine complex in the competitive binding assays is due to the complex itself, or whether it arises from diamine released upon degradation of the complex. In tissue distribution studies in immature female rats, much of the 191Pt—diamine complex was deposited in the liver; there was no evidence of selective uptake of this compound by estrogen target tissues. Thus, it is not clear, from these studies, that the observed bioactivity of this complex arises from the interaction of the Pt complex or the diamine ligand with the estrogen receptor.

Autoradiographic localization of [ 3H]quinpirole binding to dopamine D 2 and D 3 receptors in rat brain

A radiolabelled form of the dopamine agonist, quinpirole (LY17155), has been evaluated as a ligand for dopamine receptors in the rat brain. Quinpirole has been reported to be a selective D 2 dopamine agonist; however, a recent report has indicated that it may have high affinity for a novel dopamine binding site which has been termed D 3. In rat brain sections, [ 3H]quinpirole binding exhibited a distribution similar to that described for dopamine D 2 receptors using either agonist or antagonist labelling. High densities of binding could be found in caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. When the labelling was done in the presence of 10 μM guanylyl-5′-imidodiphosphate to convert the dopamine D 2 receptor to a ‘low affinity agonist conformation’, binding was inhibited in most brain regions with the notable exception of the islands of Calleja which retained most of the [ 3H]quinpirole binding. The guanine nucleotide insensitivity of this binding and distribution of this site indicates that [ 3H]quinpirole is binding to dopamine D 3 receptors in this region of the brain. Therefore, these results indicate that [ 3H]quinpirole labels a high affinity agonist conformation of dopamine D 2 receptors as well as dopamine D 3 receptors in rat brain. In addition, this study provides the first detection the dopamine D 3 receptor protein in the brain.

Basic biochemical investigations as rationale for the design of original antimalarial drugs. An example of phospholipid metabolism.

The future of antimalarial chemotherapy is particularly alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaying resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate the phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug able to interfere with this Plasmodium membranes. Any drug able to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity threshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphoblastoid cell line, demonstrating a total absence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20- to 100-fold lower than their acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.

Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [ 3H] deltorphin II

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe 3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a K d value of 1.9 nM, and the B max was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.

Bioavailability in Vivo of Benzo[a]Pyrene Adsorbed to Diesel Particulate

To evaluate health risks associated with exposure to particulates in the environment, it is necessary to quantify the bioavailability of carcinogens associated with the particulates. Direct analysis of bioavailability in vivo is most readily accomplished by adsorbing a radiolabeled form of the carcinogen to the particulate. A sample of native diesel particulate collected from an Oldsmobile diesel engine that contained 1.03 micrograms benzo[a]pyrene (BaP)/g particulate was supplemented with exogenous [3H]-BaP to produce a particulate containing 2.62 micrograms BaP/g. To insure that elution of BaP from native and [3H]-BaP-supplemented particulate was similar, in vitro analyses were performed. When using phospholipid vesicles composed of dimyristoylphosphatidylcholine (DMPC), 1.52% of total BaP was eluted from native particulate into the vesicles in 18 hrs; from [3H]-BaP supplemented particulate, 1.68% was eluted. Using toluene as eluent, 2.55% was eluted from native particulate, and 8.25% from supplemented particulate, in 6 hrs. Supplemented particulate was then instilled intratracheally into male Sprague-Dawley rats and distribution of radioactivity was analyzed at selected times over 3 days. About 50% of radioactivity remained in lungs at 3 days following instillation, with 30% being excreted into feces and the remainder distributed throughout the organs of the rats. To estimate the amount of radioactivity that entered feces through swallowing of a portion of the instilled dose, [3H]-BaP-supplemented particulate was instilled intratracheally into rats that had a cannula surgically implanted in the bile duct. Rate of elimination of radioactivity into bile was monitored; 10.6% of radioactivity was recovered in 6 hr, an amount slightly lower than the 12.8% excreted in 6 hrs into feces of animals with intact bile ducts. Our studies provide a quantitative description of the distribution of BaP and its metabolites following intratracheal instillation of diesel particulate. Because rates of elution of BaP in vitro are similar for native diesel particulate and particulate with supplemental [3H]-BaP, our results provide a reasonable estimate of the bioavailability in vivo of BaP associated with diesel particulate.

Synthesis and characterization of NBD-PS: A fluorescent analog of cerebroside sulfate for diagnosis of arylsulfatase a deficiency disorders

N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBD-PS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.

Kinetic analysis of the binding of nomegestrol acetate to the progesterone receptors in rat uterus by competition studies

The characteristics of binding (Kinetic and equilibrium binding analysis) of nomegestrol acetate (NOM, 17 alpha-acetoxy-6 alpha-methyl-19-nor-pregna-4.6-diene-3.20-dione) to the progesterone receptor (PgR) in rat uterine cytosolic fraction were determined in comparison to progesterone (P), to fully appreciate the amplitude and specificity of the induced biological response. Since an appropriate radio-labelled form of this steroid molecule was not available, competition studies were performed against the synthetic progestin: [3H]-Organon 2058 [( 3H]-ORG). This allowed a direct comparison between the unlabelled forms of NOM and P, the kinetic constants of which were respectively: Inhibition constant (Ki): 22.8 and 34.3 nM; Association rate constant (k1): 0.39 X 10(3) and 0.21 X 10(3) M-1.s-1; Dissociation rate constant (k-1): 1.81 X 10(-5) and 2.16 X 10(-5) s-1. These results are much more informative than the mere determination of relative binding affinities which only reflect the specificity of the PgR. It was concluded that NOM behaves like the natural hormone in the cytosol of rat uterus.

Detection of neuropeptide release in the central nervous system with antibody microprobes

Antibody microprobes are glass micropipettes bearing a uniform layer of immobilized antibodies on their outer surfaces. When a microprobe is placed in the central nervous system, localized release of a ligand for the particular antibody produces binding at a localized area of the microprobe. This is detected on microprobe autoradiographs as deficits in the binding of a radiolabelled form of the ligand in which probes are incubated after withdrawal from the brain or spinal cord. An image analysis system scans autoradiographs quantitatively. Experiments are best done under near sterile conditions as peptides and proteases occur in inflammatory exudates.

Potential antipsychotic agents. 7. Synthesis and antidopaminergic properties of the atypical highly potent (S)-5-bromo-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide and related compounds. A comparative study

(S)-5-Bromo-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide (6) and some related compounds, i.e. the R isomer 7, the 3-hydroxy analogue 8, the desbromo derivative 9, the monomethoxy compound 10, and the 2,4-dimethoxy analogue 11, have been synthesized from the corresponding benzoic acids. The benzamides, lacking o-hydroxy groups, were evaluated for their affinity for the [3H]spiperone binding site and for their inhibition of apomorphine-induced behavioral responses in relation to the effect of the corresponding salicylamides. Besides the 2-hydroxy-3-methoxybenzamide 12 and the related 1,4-benzodioxane (13) and 2,3-dihydrobenzofuran (14), carboxamides were investigated in order to evaluate the stereoelectronic requirements on the 2-methoxy group for the receptor interaction. The study supports the view that the o-methoxy group may adopt coplanar, as well as perpendicular orientations, and maintain the intramolecular hydrogen bonding required in the bioactive conformation. The benzamide 6 was found to be equipotent with the analogous highly active salicylamide 3 (FLB 463) both in vitro and in vivo. In addition, 6 displayed a preferential inhibition of the hyperactivity component of the behavioral syndrome, which is regarded to indicate a low tendency to induce extrapyramidal side effects in man at antipsychotically effective doses. The benzamide class of compounds (6-10) were found to be somewhat more sensitive to the structural modifications than the salicylamide class, i.e. the o-hydroxy-substituted benzamides (2-5). The potent and selective benzamide 6 (FLB 457) is highly suitable for investigations of dopamine D-2 mediated responses and, in radiolabeled form, for receptor binding studies in vitro and in vivo.

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [ tyrosyl-3,5- 3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH 2, which is based on the p17–p24 cleavage site found in the viral polyprotein substrate Pr55 gag . Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [ tyrosyl-3,5- 3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.