TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Cristalli, G.
A1  - Grifantini, M.
A1  - Vittori, S.
A1  - Lohse, M. J.
T1  - Photoaffinity labeling of A\(_1\) adenosine receptors
JF  - The journal of biological chemistry
N2  - The ligand-binding subunit of the A\(_1\)-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R- \(N^6\)-phenylisopropyladenosine, R-2-azido-\(N^6\)-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific Iigand for A\(_1\)-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R·AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A\(_1\)-subtype. It competes for [\(^3\)H].\(N^6\)- phenylisopropyladenosine binding to Arreceptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [\(^3\)H)\(N^6\)-phenylisopropyladenosine binding afterextensive washing; the K; value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity Iabel of high specific radioactivity (\(^{125}\)I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for \(^{125}\)I-AHPIA binding to rat brain membranes with an order of potency characteristic for A\(_1\)-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of M\(_r\) = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A\(_1\)-subtype. The results indicate that \(^{125}\)I-AHPIA identifies the ligand-binding subunit of the A\(_1\)-adenosine receptor, which is a peptide with M\(_r\) = 35,000.
KW  - Toxikologie
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60198
VL  - 27
IS  - 260
ER  - 
TY  - JOUR
A1  - Cristalli, G.
A1  - Franchetti, P.
A1  - Grifantini, M.
A1  - Vittori, S.
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
T1  - Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine
N2  - In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions.
KW  - Toxikologie
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60262
ER  - 
TY  - JOUR
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
A1  - Schwabe, U.
A1  - Cristalli, G.
A1  - Vittori, S.
A1  - Grifantini, M.
T1  - 2-Chloro-N\(^6\)-cyclopentyladenosine: a highly selective agonist at A\(_1\) adenosine receptors
N2  - 2-Chloro-N\(^6\)-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for At adenosine receptors. Binding of [\(^3\)H]PIA to A1 receptors of rat brain membranes was inhibited by CCP A with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N\(^6\)-cyclopentyladenosine (CPA). Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A\(_1\)-selectivity of CCPA. CCP A inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A\(_1\) receptor, with an IC\(_{50}\)-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC\(_{50}\)-value of 3500 nM. The more than 100-fold A\(_1\)-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A\(_1\) adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard At receptor agonist R-N\(^6\) -phenylisopropyladenosine (R-PIA). CCP A represents the agonist with the highest selectivity for A\(_1\) receptors reported so far.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Adenylate cyclase
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60279
ER  - 
TY  - JOUR
A1  - Klotz, Karl-Norbert
A1  - Lohse, M. J.
A1  - Schwabe, U.
A1  - Cristalli, G.
A1  - Vittori, S.
A1  - Grifantini, M.
T1  - 2-Chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) - a high affinity agonist radioligand for A\(_1\) adenosine receptors
N2  - The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCP A was prepared with a specifi.c radioactivity of 1.58 TBqjmmol ( 43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity K0 -value of 0.2 nmol/1. In the presence of GTP a K0 -value of 13 nmol/1 was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confrrmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgC12, as has been shown for the agonist [ 3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K0 -value of 0.4 nmol/1 and a Bmax-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmoljl, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [ 3H]CCPA proved to be a useful agonist radioligand for characterization of A 1 adenosine receptors also in tissues with very low receptor density.
KW  - Toxikologie
KW  - Adenosine receptors
KW  - Radioligauds
KW  - agonists
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60328
ER  - 
TY  - JOUR
A1  - Cristalli, G.
A1  - Eleuteri, A.
A1  - Vittori, S.
A1  - Volpini, R.
A1  - Lohse, M. J.
A1  - Klotz, Karl-Norbert
T1  - 2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamides as selective agonists at A\(_2\) adenosine receptors
N2  - In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.
KW  - Toxikologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60412
ER  -