Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase.
7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Laurenza
A
A
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
Robbins
J D
JD
Seamon
K B
KB
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism / Q000648:ultrastructure; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002219:Carbamates / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D004224:Diterpenes; D004347:Drug Interactions; D004789:Enzyme Activation; D006801:Humans; D007457:Iodine Radioisotopes; D008566:Membranes / Q000378:metabolism; D014316:Tritium
D002219:Carbamates; D004224:Diterpenes; D007457:Iodine Radioisotopes; D014316:Tritium; C073123:3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-(2-aminoethyl)aminocarbonylforskolin; D005576:Colforsin; D000262:Adenylyl Cyclases