Biomedical subjects
Publications and source records attributed to M W Beukers.
The synthesis and receptor binding of novel adenosine receptor antagonists is described. We found that non-xanthine 4-phenyl-2-(phenylcarboxamido)-1,3-thiazole derivatives may have high affinity and substantial selectivity for the adenosine A(1) receptor.
A new preparative synthetic route for the irreversible adenosine A1 antagonist 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, 1) is described. The availability of ample amounts of the irreversible antagonist FSCPX allowed us to use FSCPX as a research tool for adenosine A1 receptors in in vivo experiments. After verification of the irreversible antagonistic function of FSCPX in in vitro experiments, FSCPX was used successfully as a 'receptor knock-down' tool in in vivo experiments on conscious rats.
Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N(6)-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 +/- 0.10 microM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 microM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A(2B) receptors.
Explore the source record for details and available documents.
A thermodynamic analysis of the binding of a full agonist (N6-cyclopentyladenosine), a partial agonist (8-butylamino-N6-cyclopentyladenosine) and an antagonist (8-cyclopentyltheophylline) to human wild-type and mutant (mutation of a threonine (Thr) to an alanine (Ala) residue at position 277) adenosine A1 receptors expressed on Chinese hamster ovary (CHO) cells, and to rat brain adenosine A1 receptors was undertaken. The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium to rat brain receptors were determined by means of affinity measurements carried out at four different temperatures (0, 10, 20 and 25 degrees) and van't Hoff plots. Two temperatures (0 and 25 degrees) were considered for human receptors. Affinity constants were obtained from inhibition assays on membrane preparations of rat brain and CHO cells by use of the antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as selective adenosine A1 receptor radioligand. As for rat brain receptors, full agonist binding was totally entropy driven, whereas antagonist binding was essentially enthalpy driven. Partial agonist binding appeared both enthalpy and entropy driven. As for human receptors, full agonist affinity was highly dependent on the presence of Thr277. Moreover, affinity to both wild-type and mutant receptors was enhanced by temperature increase, suggesting a totally entropy-driven binding. Antagonist binding did not depend on the presence of Thr277. Antagonist affinity decreased with an increase in temperature, suggesting a mainly enthalpy-driven binding. Partial agonist binding was significantly dependent on the presence of Thr277 at 25 degrees, whereas such a dependence was not evident at 0 degrees. It is concluded that Thr277 contributes only to the binding of adenosine derivatives and that its role changes drastically with the receptor conformation and with the type of agonist (full or partial) interacting with the adenosine A1 receptors.
The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm
1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.
To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H2 receptor down-regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP-dependent and independent pathways of H2 receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down-regulation.
Ecto-nucleotidases are plasma membrane-bound enzymes that sequentially dephosphorylate extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides obscures the characterization and classification of P2 (nucleotide) receptors. We therefore studied suramin and several of its analogs, divalent cations and ATP gamma S for their ability to inhibit ecto-ATPase in human blood cells. Suramin itself and Ni2+ were the more potent, non-competitive inhibitors with micromolar affinity. ATP gamma S also displayed micromolar affinity and inhibited ecto-ATPase competitively. The data obtained with the divalent cations demonstrate that coordination of the phosphate chain but not the N7 of the adenine ring is required for the breakdown of ATP by ecto-ATPase. Divalent cations that coordinate both the phosphate chain and N7 inhibit ecto-ATPase in a non-competitive manner.
1. FPL 67156 (6-N,N-diethyl-beta, gamma-dibromomethylene-D-ATP), is a newly synthesized analogue of ATP. 2. In a rabbit isolated tracheal epithelium preparation, measuring P2U-purinoceptor-dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5-fold in the presence of 100 microM FPL 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3. FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma 32P]-ATP dephosphorylation. The compound inhibited [gamma 32P]-ATP degradation with a pIC50 of 4.6. 4. FPL 67156 was then tested for its effects on ATP and alpha, beta-methylene-ATP responses at P2X-purinoceptors in the rabbit isolated ear artery. In the concentration range 30 microM-1 mM, the compound potentiated the contractile effects of ATP but not those of alpha, beta-methylene-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/[A] curves. 5. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analyzed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pKi for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X- and P2T-purinoceptors (pA2 ~ 3.3 and 3.5 respectively), and weak agonist effects at P2u-purinoceptors(p[A 50]~ 3.5).7. The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.8.This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.
Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) (ADP beta 35S) to P2 gamma purinoceptors on turkey erythrocyte membranes was used to determine the affinity of suramin and various suramin congeners belonging to different structure classes (large urea, small urea, dibenzamides and benzamides) for these receptors. Suramin was shown to be a competitive antagonist with a Ki value of 7.3 +/- 2.2 microM. The simple benzamide compound XAMR0721 (8-(3,5-dinitrophenylene carbonylimino)-1,3,5-naphthalene trisulfonate, trisodium salt) displays a high affinity for the P2 gamma purinoceptor (Ki value of 19 +/- 6 microM). Similar to suramin, compound XAMR0721 is a competitive antagonist at P2 gamma purinoceptors. In contrast to suramin, which is a potent inhibitor of the ecto-nucleotidase activity in human blood cells (44 +/- 2% residual activity at 100 microM), compound XAMR0721 is hardly active in this assay (93 +/- 1% residual activity at 100 microM). So XAMR0721, the first competitive antagonist for P2 purinoceptors that is able to discriminate between P2 purinoceptor affinity and ecto-nucleotidase activity, is an interesting pharmacological tool for the characterization of P2 purinoceptor mediated effects.
In this study, we determined whether R75231, (+/-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis( 4-fluoro- phenyl)pentyl]-1-piperazineacetamide, and its two enantiomers, all nucleoside transport inhibitors, could play a role as anti-aggregatory agents. First, we determined the binding characteristics of [3H]nitrobenzylthioinosine, also a nucleoside transport inhibitor, on intact human erythrocytes. The Kd value was 0.27 +/- 0.04 nM and the Bmax was 23.5 +/- 5.1 pmol/10(9) erythrocytes. Second, we studied the ability of R75231 and its enantiomers R88021 ((-)-R75231, or draflazine) and R88016 ((+)-R75231), to displace [3H]nitrobenzylthioinosine. R75231 had an IC50 value of 2.2 +/- 0.3 nM. R88021 was twice as potent as R75231 and R88016 was approximately 20-fold less potent than R75231. Finally, the ability of these nucleoside transport inhibitors to enhance anti-aggregatory effects of adenosine was examined in whole human blood. Adenosine alone, 10 microM, had no effect on ADP-induced platelet aggregation. However, in the presence of 1 microM R75231, 10 microM of adenosine inhibited the aggregatory response completely. Dose-response curves indicated that the IC50 values of draflazine and R88016 were approximately 0.5 microM and 10 microM, respectively. R75231 and its enantiomers are valuable research tools to assess the role of the nucleoside transporter. Moreover, R75231 and draflazine (R88021) may prove to be useful as anti-aggregatory agents.
1. Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X-purinoceptor in the rabbit ear artery. Those studies employed alpha,beta-methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist. 2. Suramin, in the concentration range 30-1000 microM, potentiated the contractile effects of ATP, producing a 3-fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100-1000 microM, produced a significant but small (approximately 3-fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using alpha,beta-methylene ATP in which 30-fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301. 3. Suramin and FPL 66301 were tested as ecto-ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3. 4. The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKB estimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay. 5. From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of 'self-cancellation' of the potentiating (enzyme inhibitory) and rightward-shifting (receptor antagonistic) properties.6. The analysis also indicated that the presence of ecto-ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4,whereas the 'true' value, that is the value which would be obtained in the absence of ecto-ATPase activity, was 6.0, some 400-fold higher.7 Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes.Secondly, the agonist potency order currently used to designate P2X- purinoceptors may require modification.
Ecto-ATPase (EC 3.6.1.15) is a plasma membrane-bound enzyme which degrades extracellular triphosphate nucleotides. Although its physiological function is still unclear, the enzyme obscures the study of P2 purinoceptors (i.e. receptors for ATP and other di- and triphosphate nucleotides), since it is capable of metabolizing the pharmacological ligands, such as ATP, for these receptors. We characterized the ecto-ATPase activity on human blood cells with a [gamma 32P]ATP assay and HPLC measurements. We also determined whether ecto-ATPase activity could affect the anti-aggregatory role of ATP in whole human blood. The Km for ATP of the ecto-ATPase on human blood cells was 8.5 +/- 2.3 microM and the maximum degradation rate, at 37 degrees, was 2.7 +/- 1.1 nmol ATP/(min x mL whole blood). In whole blood the major part of ATP was broken down by the blood cells, predominantly by the leukocytes. ATP and UTP were broken down equally well, mainly yielding the corresponding di- and monophosphates. In search of inhibitors for the ecto-ATPase, we studied several analogs of ATP. 8-Bromo-ATP as well as 2'- and 3'-deoxy-ATP were substrates for the enzyme. In contrast, modification of the phosphate side chain yielded inhibitors. Subsequently, a possible role of the ecto-ATPase in platelet aggregation was verified. To assess the role of the plasma membrane-bound enzyme, platelet aggregation was determined in whole blood instead of platelet-rich plasma. In the presence of ATP alone, an antagonist of ADP-induced platelet aggregation, some aggregation was still observed. As breakdown of ATP by the ecto-ATPase leads to gradual formation of ADP, as mentioned above, we compared the effects of a stepwise versus bolus addition of ADP. Subsequent dosing of ADP (1.5, 2.5, 5 and 10 microM) resulted in platelet aggregation but to a much smaller extent, at most approximately 60%, compared to the amount of platelet aggregation obtained with a bolus addition of ADP (10 microM). In conclusion, human blood cells possess a high affinity ecto-ATPase which degrades ATP as well as ATP analogs with modified base and ribose moieties. ATP analogs with a modified phosphate chain are inhibitors of the ecto-ATPase. A direct role of the ecto-ATPase activity on platelet aggregation is probably small, as degradation of ATP to ADP proceeds slowly and cumulative addition of ADP to platelets in whole blood results in a modest amount of aggregation.(ABSTRACT TRUNCATED AT 400 WORDS)
Amiloride, a potassium sparing diuretic, inhibits adenosine A1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A1 receptor in a manner different from classical A1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their Ki values in displacement studies, decrease the affinity of the A1 antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor-ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the Ki value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A1 receptor were investigated at the level of cyclic adenosine 3',5'-monophosphate (cAMP) formation. The amiloride analogue 5-(N-butyl-N-methyl) amiloride (MBA) reversed A1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 microM. This value is in fair agreement with a Ki value of 3.5 microM in binding assays under similar conditions. In conclusion, amiloride inhibits A1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A1 receptor ligands may at least partially overlap.(ABSTRACT TRUNCATED AT 250 WORDS)
The binding affinities of seven analogues of recombinant human insulin-like growth factor II (hIGF-II) were characterized for the IGF type-I and type-II receptors and insulin receptors, as well as for IGF-binding protein (IGFBP)-1, IGFBP-2, IGFPB-3 and human serum IGFBPs. A switch of two of the three cysteine bridges in hIGF-II, 9-47 and 46-51 to 9-46 and 47-51, severely impaired the binding of this analogue to all receptors and to the IGFBPs. The affinities for the IGF type-I receptor and the IGFBPs were decreased over 100-fold, while the binding to the insulin receptor and the IGF type-II receptor was less affected, with a 6-10-fold decrease in affinity. Slight modifications of the N-terminus had only minor effects upon the binding of hIGF-II to the IGFBPs or to the receptors. Deletion of both the N-terminal amino acid and the two C-terminal amino acids resulted in moderate decreases in affinity, with a 60% decrease in affinity for IGFBP-1 and the IGF type-I receptor. Acetylation of the N-terminus of Ala1 and the epsilon-nitrogen of Lys65 decreased the affinity, by 60-90%, of hIGF-II for all of the IGFBPs and receptors. The experiments involving acetylation of IGF-II or switching of its cysteine bridges indicated that these modifications (no substitution, deletion or addition of any of the 67 amino acids of hIGF-II) may lead to a severe impairment of the binding affinity of IGF-II for both the IGFBPs and the receptors. Acetylation of the epsilon-nitrogen of Lys65, which causes a charge change, or alteration of the three-dimensional structure, as shown by the cysteine bridge switch, lead to a severe impairment of the binding affinity for the binding proteins and for the receptors. In general, care should be taken with the synthesis of analogues and the interpretation of resulting binding data, since affinity alterations ascribed to amino acid changes may instead be caused by alterations of the charge or the three-dimensional structure of the protein.
Explore the source record for details and available documents.
Insulin-like growth factor-II (IGF-II) is able to interact with three different receptors: the type-I and type-II IGF receptors, and the insulin receptor, although with a lower affinity. This cross reactivity obscures the mechanisms via which the biological activities of IGF-II are mediated. We have synthesized an IGF-II analog, [Leu27]IGF-II, that is highly selective for the type-II IGF receptor. Receptor binding experiments demonstrate a high affinity for the type-II IGF receptor, analogous to synthetic (syn) and recombinant (rec) IGF-II, but no affinity for the type-I IGF and the insulin receptor at concentrations up to 50 and 200 ng/ml, respectively. The lack of affinity for these two receptors is confirmed by biological studies which demonstrated that this analog, in contrast with synIGF-II, did not stimulate [3H]thymidine incorporation in Balb/c 3T3 cells. [Leu27]IGF-II opens new ways to identify which actions of IGF-II are mediated via the type-II receptor and which are due to cross reactivity with the type-I IGF or the insulin receptor.