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K A Jacobson

Publications and source records attributed to K A Jacobson.

At least 253 records · Page 14Linked to original sources
PubMed1983

Conjugates of catecholamines. III. Synthesis and characterization of monodisperse oligopeptides conjugates related to isoproterenol.

A series of monodisperse oligopeptide conjugates related to the catecholamine, isoproterenol, has been synthesized. The peptide carrier molecules used were synthesized by stepwise and fragment condensation techniques and ranged in size from a single, blocked amino acid derivative to isomeric pentapeptides. The amino acid compositions and sequences of the carriers were chosen so as to provide specific information concerning the effects of molecular weight, hydrophilic/hydrophobic balance, charge, etc., on the biological activity of the final conjugates. The common point of attachment for the drug in all carriers was a p-aminophenylalanine residue. The peptide-catecholamine conjugates were prepared via the attachment of carboxyl-containing catecholamine congeners, to the peptide carriers by techniques described previously. The conjugates were purified rigorously by chromatographic techniques and characterized by high-field n.m.r. spectroscopy.

Catecholamines↗
PubMed1983

Conjugates of catecholamines. IV. In vitro and in vivo pharmacological activity of monodisperse oligopeptide conjugates.

Conjugates of low molecular weight amines with inert peptide carriers can be made to preserve some pharmacologic effects of the ligand. No previous studies of conjugates have systematically derivatized the ligand to optimize its activity or affinity to receptors; or have pharmacophores been selected for conjugation on the basis of their effects making them particularly interesting as ligands. Chemically pure and characterizable conjugates ranging from single blocked amino acid derivatives to isomeric pentapeptides were constructed and tested for beta adrenergic properties. Sixteen conjugates with varying water solubility, amino acid composition and sequence, charged groups and spacer length between ligand and carrier showed potencies and duration of action widely different from isoproterenol. In the in vitro S-49 mouse lymphoma assay a range of relative potencies (as compared to isoproterenol) from 10(-6) to 1 time as potent was obtained, whereas in the in vivo rat blood pressure assay these compounds were shown to be from one-fifth to 4 times as potent as isoproterenol. The most potent compound tested, Boc-Phe(NH)-Gly-NHCH3, was also tested in a guinea-pig atrial preparation as well as in an anesthetized cardiovascular dog preparation. The results indicated that the compound was approximately 3.5 times more potent in producing an inotropic response than isoproterenol. In some instances, ostensively trivial changes quite distant from the pharmacophores in amino acid sequence of the carrier made major changes in potency and efficacy of the compound.

Adrenergic beta-Antagonists↗
PubMed1983

Conjugates of catecholamines. II. In vitro and in vivo pharmacological activity of N-alkyl-functionalized carboxylic acid congeners and amides related to isoproterenol.

In this study, 10 congeners of isoproterenol were systematically synthesized and pharmacologically tested in both in vitro and in vivo systems. The aim was to produce compounds that were more potent and had effects different from those of the parent compound and that could ultimately be attached covalently to inert peptide carriers offering versatility in size, pK, and other physicochemical properties. The congeners synthesized were tested in the S49 mouse lymphoma assay for their ability to stimulate cyclic AMP accumulation and were shown to have potencies relative to isoproterenol ranging from 4 orders of magnitude less potent to 4 orders of magnitude more potent than isoproterenol in eliciting this response. Some of the congeners were also tested in a guinea pig isolated atrial preparation, a rat blood pressure assay, a guinea pig bronchodilation assay, and an anesthetized dog preparation. In these assays, the congeners were shown to have the same types of activities as in the S49 cell assay. The results of these studies indicate that structural modifications distant from the catecholamine moiety dramatically alter the pharmacological profile of the congeners versus the parent compound, resulting in a series of ligands with a wide range of activities.

Adrenergic beta-Antagonists↗
PubMed1981

Diffusion of injected macromolecules within the cytoplasm of living cells.

The diffusion of macromolecules introduced into the cytoplasm of human fibroblasts by erythrocyte-mediated microinjection was measured by the fluorescence recovery after photobleaching technique. The apparent diffusion coefficients for fluorescein-labeled IgG and fluorescein-labeled bovine serum albumin were approximately 10(-8) cm2/sec at 22 degrees C, consistent with the kinetics of spreading of the fluorescent probe following microinjection and approximately 1/70 the values in aqueous buffer. The diffusion of labeled bovine serum albumin was shown to be strongly dependent on temperature and, in fact, similar to that expected in a 61% aqueous sucrose solution. However, the marked reduction in diffusion at 5 degrees C could be fully reversed by incubation with 0.1 mM colchicine. These findings suggest that cytoplasmic diffusion rates are reduced relative to rates in aqueous media as a result of increased aqueous phase viscosity or the impedence provided by structural elements. Several simple models to account for the data are presented.

Cells, Cultured↗
PubMed1976

The effects of urine on the viability and activity of lymphocytes.

Circulating lymphocytes were incubated for 72 hours in either pure culture medium or a medium-urine mixture in the presence of phytohemagglutinin, concanavalin A or pokeweed mitogen, and assayed for blastogenesis and viability. The amount and osmolality of the urine were varied. Between 80 and 90 per cent of the cells remained viable during the entire study despite the culture mixture. If 50 percent of the culture was replaced by urine at an osmolality of 150, 300 or 500 mOsm, a severe but uniform depression of lymphocyte activity was seen. However, if only 25 percent or less of the medium was replaced by urine at 300 mOsm, the cells stimulated as well as they did in pure medium.

Adult↗
PubMedDate not supplied

Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors.

The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3',5'-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2'-Substitution with OH or OMe increases agonist efficacy over 2'-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation.

Binding Sites↗
PubMedDate not supplied

Apoptosis by 2-chloro-2'-deoxy-adenosine and 2-chloro-adenosine in human peripheral blood mononuclear cells.

Adenosine has profound effects on immune cells and has been implicated in the intrathymic apoptotic deletion of T-cells during development. In order to characterize adenosine effects on quiescent peripheral blood mononuclear cells (PBMC), we have evaluated the ability of the previously characterized adenosine receptor agonist 2-chloro-adenosine (2CA; Ceruti, Barbieri et al., 1997) and of the antineoplastic drug 2-chloro-2'-deoxy-adenosine (2CdA, cladribine) to trigger apoptosis of PBMC. Apoptosis was assessed by morphological changes, DNA fragmentation by agarose gel electrophoresis and appearance of hypodiploid DNA peak by flow cytometry. 2CA (10 microM) and 2CdA (1 microM) induced apoptosis in human PBMC, which are relatively insensitive to apoptosis. For both agents, the effect was concentration- and time-dependent, although 2CdA induced apoptosis more potently than 2CA. Evaluation of mitochondrial function in parallel samples using the mitochondrial membrane-potential-specific dye JC-1 showed that mitochondrial damage followed the same kinetics as apoptosis, hence an early damage of mitochondria is likely not responsible for adenosine-induced death of PBMC. The effect of 2CA was partially prevented by addition of dipyridamole (DP), a nucleoside transport inhibitor, hence some of the apoptotic effect of this nucleoside is, at least in part, due to intracellular action. Alternatively, DP did not affect 2CdA-induced apoptosis, suggesting that 2CdA may enter cells via a DP-insensitive transporter. 5-Iodotubercidin (5-Itu), a nucleoside kinase inhibitor, was also able to partially prevent the action of 2CA and was not able to affect 2CdA-induced apoptosis, suggesting a different role for phosphorylation in 2CA- vs 2CdA-induced apoptosis. To test the role of P1 receptors, agonists and antagonists selective at various P1 receptor subtypes were used. Data suggest that, for 2CA, apoptosis is partially sustained by activation of the A2A receptor subtype, whereas no role is exerted by P1 receptors in 2CdA-dependent apoptosis. Moreover, in these cells, apoptosis could also be triggered through intense activation of the A3 receptor via selective agonists such as 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), but this mechanism plays no role in either 2CA- or 2CdA-induced apoptosis. On the whole, our results suggest that 2CA and 2CdA follow different pathways in inducing apoptosis of immune cells. Moreover, our data also suggest that there are at least three different ways by which adenosine derivatives may induce apoptosis of human PBMC: (i) through an A2A-like extracellular membrane receptor; (ii) through entry of nucleosides into cells and direct activation of intracellular events involved in the apoptotic process; or (iii) through activation of the A3 receptor.

2-Chloroadenosine↗
PubMedDate not supplied

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors.

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.

Adenosine↗
PubMedDate not supplied

Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine.

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.

Animals↗
PubMedDate not supplied

"Cleavable trifunctional" approach to receptor affinity labeling: chemical regeneration of binding to A1-adenosine receptors.

A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionally-regenerated, receptor protein that also may contain a reporter group. The ligand recognition site of A1-adenosine receptors in bovine brain membranes was probed to demonstrated the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand [3H]CPX was preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with an IC50 of 0.29 M. Hydroxylamine displaced specific agonist ([3H]PIA) binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-](carboxymethyl)-oxy]phenyl]-1,3-dipropylxanthine) and shown to bind irreversibly to A1-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g., DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or an ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to A1-receptors was inhibited by these reactive xanthines in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [3H]CPX in A1-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.

Affinity Labels↗
PubMedDate not supplied

Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A3 adenosine receptors.

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure-activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 microM.

Animals↗
PubMedDate not supplied

Molecular modeling studies of human A3 adenosine antagonists: structural homology and receptor docking.

Molecular modeling studies were conducted on various chemically diverse classes of human A3 adenosine receptor antagonists (hA3ANTs), such as adenines, xanthines, triazoloquinazolines, flavonoids, thiazolopyridines, 6-phenyl-1, 4-dihydropyridines, and 6-phenylpyridines. Using a combination of ab initio quantum mechanical calculations, electrostatic potential map comparison, and the steric and electrostatic alignment (SEAL) method, a general pharmacophore map for hA3ANTs has been derived. Based on the proposed pharmacophore map, we hypothesize that the receptor binding properties of different A3 antagonist derivatives are due to recognition at a common region inside the receptor binding site and, consequently, a common electrostatic potential profile. A model of the human A3 receptor, docked with the triazoloquinazoline reference ligand CGS 15953 (9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine), was built and analyzed to help interpret these results. All other antagonist structures were docked inside the receptor according to the results obtained through the steric and electrostatic alignment (SEAL) approach using the structure of CGS 15953 as a template. The receptor model was derived from primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using rhodopsin as a template. An energetically refined 3D structure of the ligand-receptor complex was obtained using our recently introduced cross-docking procedure (J. Med. Chem. 1998, 41, 1456-1466), which simulates the ligand-induced reorganization of the native receptor structure.

Animals↗