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• W1503606784 abstract "Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2′-deoxyadenosine 3′-monophosphate, a classical and 3′-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination. Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2′-deoxyadenosine 3′-monophosphate, a classical and 3′-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination. heterotrimeric guanine nucleotide-binding protein the α subunit of G protein that stimulates adenylyl cyclase guanosine 5′-(γ-thio) triphosphate 9-(tetrahydro-2-furyl) adenine 9-(cyclopentyl) adenine α,β-methylene adenosine 5′-triphosphate 2′-deoxyadenosine 3′-monophosphate adenosine 3′-monophosphate The G protein1-sensitive, membrane-bound form of adenylyl cyclase consists of a large family; nine isoforms have been isolated and extensively studied (1Ishikawa Y. Homcy C.J. Circ. Res. 1997; 80: 297-304Crossref PubMed Scopus (133) Google Scholar, 2Tang W.J. Hurley J.H. Mol. Pharmacol. 1998; 54: 231-240Crossref PubMed Scopus (161) Google Scholar, 3Hurley J.H. J. Biol. Chem. 1999; 274: 7599-7602Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). These isoforms are characterized by distinct biochemical properties and tissue distribution. For example, calcium-inhibitable type V is expressed in the heart as a major isoform (4Ishikawa Y. Sorota S. Kiuchi K. Shannon R.P. Komamura K. Katsushika S. Vatner D.E. Vatner S.F. Homcy C.J. J. Clin. Invest. 1994; 93: 2224-2229Crossref PubMed Scopus (120) Google Scholar); protein kinase C-sensitive type II is expressed in lungs (5Feinstein P.G. Schrader K.A. Bakalyar H.A. Tang W.J. Krupinski J. Gilman A.G. Reed R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10173-10177Crossref PubMed Scopus (224) Google Scholar); calmodulin-sensitive type I and type VIII are expressed exclusively in neuronal tissues (6Cali J.J. Zwaagstra J.C. Mons N. Cooper D.M. Krupinski J. J. Biol. Chem. 1994; 269: 12190-12195Abstract Full Text PDF PubMed Google Scholar); type III is expressed abundantly in olfactory tissues (7Bakalyar H.A. Reed R.R. Science. 1990; 250: 1403-1406Crossref PubMed Scopus (525) Google Scholar, 8Defer N. Marinx O. Poyard M. Lienard M.O. Jegou B. Hanoune J. FEBS Lett. 1998; 424: 216-220Crossref PubMed Scopus (61) Google Scholar) but also in other tissues including lungs (9Ishikawa Y. Grant B.S. Okumura S. Schwencke C. Yamamoto M. Mol. Cell. Endocrinol. 2000; 162: 107-112Crossref PubMed Scopus (12) Google Scholar), atria (10Xia Z. Choi E.J. Wang F. Storm D.R. Neurosci. Lett. 1992; 144: 169-173Crossref PubMed Scopus (75) Google Scholar), and adipose tissue (11Chaudhry A. Granneman J.G. Am. J. Physiol. 1997; 273: R762-R767PubMed Google Scholar); type IV and VII are widely expressed (12Gao B.N. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10178-10182Crossref PubMed Scopus (277) Google Scholar, 13Watson P.A. Krupinski J. Kempinski A.M. Frankenfield C.D. J. Biol. Chem. 1994; 269: 28893-28898Abstract Full Text PDF PubMed Google Scholar). Therefore, it is now accepted that the content and mixture of adenylyl cyclase isoforms provide a biochemical signature of tissue cyclic AMP generation. Forskolin, like digitalis, is a natural plant extract that has been used in traditional medicine (14Seamon K.B. Padgett W. Daly J.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3363-3367Crossref PubMed Scopus (1470) Google Scholar). Forskolin directly activates adenylyl cyclase to increase the concentration of intracellular cyclic AMP. This mechanism for activation is now explained as follows. Forskolin binds to the catalytic core at the opposite end of the same ventral cleft that contains the active site and activates the enzyme by gluing together the two cytoplasmic domains in the core (C1and C2) using a combination of hydrophobic and hydrogen bond interactions (15Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (325) Google Scholar). Although the efficacy of forskolin was confirmed in human studies (16Bristow M.R. Ginsburg R. Strosberg A. Montgomery W. Minobe W. J. Clin. Invest. 1984; 74: 212-223Crossref PubMed Scopus (109) Google Scholar, 17Feldman M.D. Copelas L. Gwathmey J.K. Phillips P. Warren S.E. Schoen F.J. Grossman W. Morgan J.P. Circulation. 1987; 75: 331-339Crossref PubMed Scopus (422) Google Scholar), its poor tissue selectivity has hampered its clinical use. Recently, however, a water-soluble forskolin derivative 6-[3-(dimethylamino)propionyl]forskolin (NKH477) was introduced to treat human heart failure (18Shafiq J. Suzuki S. Itoh T. Kuriyama H. Circ. Res. 1992; 71: 70-81Crossref PubMed Scopus (60) Google Scholar, 19Sanbe A. Takeo S. J. Pharmacol. Exp. Ther. 1995; 274: 120-126PubMed Google Scholar). NKH477 is a forskolin derivative in which a 3-(dimethylamino)propionyl group was attached to forskolin at the C6 position. Furthermore, NKH477 was found to have enhanced type V selectivity (20Toya Y. Schwencke C. Ishikawa Y. J. Mol. Cell. Cardiol. 1998; 30: 97-108Abstract Full Text PDF PubMed Scopus (53) Google Scholar). As predicted by a recent crystallographic study, there is a relatively large open space between the C6/C7 positions of forskolin and its binding site within adenylyl cyclase (15Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (325) Google Scholar, 21Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (676) Google Scholar), implying that a forskolin derivative modified in these positions might have altered isoform selectivity without disrupting their activity; this is consistent with the findings on NKH477 (20Toya Y. Schwencke C. Ishikawa Y. J. Mol. Cell. Cardiol. 1998; 30: 97-108Abstract Full Text PDF PubMed Scopus (53) Google Scholar). In contrast, P-site inhibitors are adenosine analogs that inhibit adenylyl cyclase (22Londos C. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5482-5486Crossref PubMed Scopus (489) Google Scholar). P-site inhibitors bind to the same binding site as the substrate ATP within the adenylyl cyclase molecule (23Tesmer J.J. Dessauer C.W. Sunahara R.K. Murray L.D. Johnson R.A. Gilman A.G. Sprang S.R. Biochemistry. 2000; 39: 14464-14471Crossref PubMed Scopus (95) Google Scholar); as yet the mode of inhibition is either un- or non-competitive with respect to ATP as shown by kinetic analysis (24Johnson R.A. Shoshani I. J. Biol. Chem. 1990; 265: 11595-11600Abstract Full Text PDF PubMed Google Scholar). P-site inhibitors occupy the site where cyclic AMP is accommodated, forming a dead-end complex with pyrophosphate (25Florio V.A. Ross E.M. Mol. Pharmacol. 1983; 24: 195-202PubMed Google Scholar, 26Dessauer C.W. Gilman A.G. J. Biol. Chem. 1997; 272: 27787-27795Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Most importantly, a recent study demonstrated selective inhibition of adenylyl cyclase isoforms by certain P-site inhibitors (27Johnson R.A. Desaubry L. Bianchi G. Shoshani I. Lyons E.J. Taussig R. Watson P.A. Cali J.J. Krupinski J. Pieroni J.P. Iyengar R. J. Biol. Chem. 1997; 272: 8962-8966Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). These findings suggested that P-site ligands can serve as isoform- and, therefore, tissue-selective regulators of cyclic AMP signaling. A major concern, however, is that P-site ligands require the presence of an intact adenine ring moiety to retain inhibition. Such molecules might therefore be expected to lack specificity and affect other pathways within the cells, including DNA synthesis. Indeed, a recent study demonstrated that acyclic derivatives of adenine possess both antiviral and adenylyl cyclase inhibitory effects (28Shoshani I. Laux W.H. Perigaud C. Gosselin G. Johnson R.A. J. Biol. Chem. 1999; 274: 34742-34744Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The above findings have prompted us to search for forskolin derivatives with enhanced isoform selectivity and non-nucleoside inhibitors that may mimic P-site ligands. Based upon the findings from a prior crystallographic study (15Zhang G. Liu Y. Ruoho A.E. Hurley J.H. Nature. 1997; 386: 247-253Crossref PubMed Scopus (325) Google Scholar) and computer-assisted drug design, we have identified forskolin derivatives that have enhanced selectivity for type II, type III, and type V, respectively. Furthermore, we have found a novel non-nucleoside inhibitor of the type V isoform, which was obtained after virtual screening of more than 850,000 compounds on the basis of the pharmacophore analysis of adenylyl cyclase and P-site ligands. We have also found enhanced selectivity in regulating tissue adenylyl cyclase catalytic activity with the use of these compounds. Forskolin, 2′-d-3′-AMP, 3′-AMP, Ap(CH2)pp, and GTPγS were purchased from Sigma. More than 200 forskolin derivatives, 9-(tetrahydro-2-furyl)adenine (THFA or SQ 22,536), 9-(cyclopentyl)adenine (CPA), and 2-amino-CPA were synthesized by Nippon Kayaku Co., Ltd. (Tokyo, Japan). 2-Amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80) was purchased from Chem Ster Ltd. (Moscow, Russia).cis-N-(2-Phenylcyclopentyl)-azacyclotridec-1-en-2-amine (MDL 12330A) was purchased from Research Biochemicals International (Natick, MA).N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was purchased from Biomol Research Lab., Inc. (Plymouth Meeting, PA). Overexpression of each adenylyl cyclase isoform and Gsα were performed as previously described (20Toya Y. Schwencke C. Ishikawa Y. J. Mol. Cell. Cardiol. 1998; 30: 97-108Abstract Full Text PDF PubMed Scopus (53) Google Scholar). High Five cells were washed twice with ice-cold phosphate-buffered saline and homogenized in a buffer containing 50 mm Tris/HCl (pH 8.0), 1 mm EGTA, 1 mm EDTA, 1 mmdithiothreitol, 200 mm sucrose, and a protease inhibitor mixture containing 20 μg/ml 1-chloro-3-tosylamido-7-amino-l-2-heptanone, 10 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, 50 units/ml egg white trypsin inhibitor, and 2 μg/ml aprotinin (Buffer A). Cells were disrupted with a sonicator and centrifuged at 500 × g for 10 min at 4 °C. The supernatants were further centrifuged at 100,000 × g for 40 min at 4 °C. The resultant pellets were resuspended in the same buffer without EGTA (Buffer B). For Gsα preparation, we used the supernatant fraction after ultracentrifugation. The crude membrane and the Gsα-rich supernatant were stored at −80 °C until use. Protein concentration was measured with the Bio-Rad protein assay system. The overexpression of recombinant adenylyl cyclase isoform in insect cells increased catalytic activity by ∼56-fold for type II, 48-fold for type III, and 360-fold for type V over that of control cell membranes as determined in the presence of Gsα·GTPγS-forskolin (50 μm). Thus under these conditions, each isoform was examined as the dominant positive adenylyl cyclase isoform in these cells. Male Wistar rats were purchased from Charles River Japan, Inc. (Yokohama, Japan). Tissues were minced and homogenized with a Polytron for 3 × 10 s in Buffer A followed by centrifugation at 500 × g for 10 min at 4 °C. The supernatants were retained and further centrifuged at 100,000 × g for 40 min at 4 °C. The crude membrane preparations were made by resuspending the pellet in Buffer B and stored at −80 °C until use. Animals used in this study were maintained in accordance with the guidelines of the animal experiment committee of the Yokohama City University School of Medicine. Adenylyl cyclase catalytic activity was measured as previously described with some modification (29Kawabe J. Ebina T. Ismail S. Kitchen D.B. Homcy C.J. Ishikawa Y. J. Biol. Chem. 1994; 269: 24906-24911Abstract Full Text PDF PubMed Google Scholar). Briefly, the reaction mixtures contained 20 mm HEPES (pH 8.0), 0.5 mm EDTA, 0.1 mm ATP containing [α-32P]ATP (1 × 106 cpm), 0.1 mm cyclic AMP, 1 mmcreatine phosphate, 8 units/ml creatine phosphokinase, 5 mmMgCl2 or 15 mm MnCl2, and 4 μg (for insect cells) or 8 μg (for tissues) of membrane protein in a final volume of 100 μl. Gsα-enriched supernatant obtained from insect cells overexpressing Gsα was used in an amount that stimulated adenylyl cyclase maximally in the presence of 1 μm GTPγS. Further details are provided in the figure legends. Assays were performed at 30 °C for 20 min and terminated by the addition of 10 μl of ice-cold 2.2 n HCl. The product32P-cyclic AMP was separated with single acidic alumina columns (30Alvarez R. Daniels D.V. Anal. Biochem. 1992; 203: 76-82Crossref PubMed Scopus (74) Google Scholar). In brief, the samples were applied to the acidic alumina columns (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and washed with 0.005 n HCl to remove any unbound contaminants. The bound cyclic AMP was eluted with 0.1 m ammonium acetate (pH 7.0). The radioactivity of the eluted samples was measured by scintillation counting. Determinations of sample recovery using 3H-cyclic AMP were omitted because high cyclic AMP recovery (86–93%) was typically achieved with this system in a validation study, and the data were similar to that obtained using the conventional two-column method (31Salomon Y. Adv. Cyclic Nucleotide Res. 1979; 10: 35-55PubMed Google Scholar). Results were obtained from quadruplicate determinations unless specified and are shown as the means ± S.D. Amino acid sequences of rat adenylyl cyclase type II and type III and canine adenylyl cyclase type V were obtained from sequence data bases (PIR and SWISSPROT). The three-dimensional structures of adenylyl cyclases were modeled using the homology modeling method. The crystal structure of the catalytic core of adenylyl cyclase, which consists of the two homologous cytoplasmic domains (C1a and C2a), was used as a template. In this structure the C1a and the C2a domains were those from canine type V and rat type II, respectively. The coordinates of the crystal structure were retrieved from the Protein Data Bank (entry: 1AZS). The sequences of the three isoforms of adenylyl cyclase were aligned for 1AZS with Chem-X (Chemical Design Ltd., Oxon, England) using default parameters. Because there is no atomic coordinates for the structures between PRO:B954 and GLU:B963 of the C2a domain of 1AZS, the region was removed from the alignment. As a result, there is neither gap nor insertion in the alignment between the target sequences and the template sequence. Therefore, the amino acids of the template were simply mutated into those of the target at different amino acid sites using Chem-X without altering the backbone conformation. Hydrogen atoms were added to the model in Insight II 98.0 (Molecular Simulations Inc., San Diego, CA). As the first step of refinement, all close contacts caused by the mutation of side chains were fixed by searching the most suitable conformer of the side chains from the established rotamer libraries of Biopolymer module within Insight II. The model was relaxed by energy minimization using Discover with the force field of the consistent valence force field according to the following protocol: (i) minimization of all hydrogen atoms with all heavy atoms fixed, (ii) minimization of the side chains of mutated residues with main chain fixed, (iii) minimization of all the side chains with main chain fixed, (iv) minimization of the whole system using a harmonic force constraint of 10 kcal/mol Å2 on all the backbone atoms, (v) minimization of the whole system without any constraint. In these minimization steps, a maximum derivative of 1.0 kcal/mol Å2 was used as a convergence criteria with a dielectric constant of 4r. Forskolin derivatives were manually docked into the binding site guided by an overlay of the fused rings onto forskolin in the pocket of adenylyl cyclase from the crystal structure. After the minimization of the complex of a forskolin derivative with adenylyl cyclase, water molecules were added within a sphere of 25 Å from the center of the forskolin derivative. The solvated system was minimized, and then the MD simulation at 298 K was performed with Discover to search the low energy docking mode. Based upon a previous study in which the interaction of 2′-d-3′-AMP to adenylyl cyclase was examined (21Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (676) Google Scholar), we used a pharmacophore (=C2H-N1=C6(NH2)-) that we presumed necessary for the inhibition of adenylyl cyclase in our virtual screening. More than 850,000 chemical compounds available from an existing data base were screened using ISIS (MDL Information Systems Inc., San Leandro, CA), and potential candidates were subjected to adenylyl cyclase assays. The pharmacophore structure (=C2H-N1=C6(NH2)-) of the identified compounds was then superimposed on that of 2′-d-3′-AMP in the crystal structure of the complex consisting of adenylyl cyclase and 2′-d-3′-AMP followed by minimization of the complex consisting of the type V enzyme and the compound using Discover. As to the isoform selectivity of adenylyl cyclase, there has been little data available about the structure/function relationship of forskolin. Therefore, to develop an approach for modifying forskolin that might increase isoform selectivity, we synthesized more than 200 derivatives of forskolin that were modified at the positions of C1, C6, C7, C9, C11, and C13 and examined their effects on the catalytic activity of the adenylyl cyclase type II, type III, and type V, which belong to different subgroups within the adenylyl cyclase family. The relative stimulatory activity of each derivative versus forskolin (% forskolin activity) is shown in the following results. It is important to note that most of the newly synthesized forskolin derivatives either had no isoform selectivity or lost their ability to stimulate adenylyl cyclase. In general, the modification at the C1, C9, C11, and C13 positions of forskolin resulted in loss of adenylyl cyclase stimulatory activity, whereas a small enhancement in isoform selectivity was noted in a few cases. An example was 1,9-dideoxyforskolin, which is known to be an inactive forskolin derivative (32Seamon K.B. Daly J.W. Metzger H. De Souza N.J. Reden J. J. Med. Chem. 1983; 26: 436-439Crossref PubMed Scopus (134) Google Scholar, 33Robbins J.D. Boring D.L. Tang W.J. Shank R. Seamon K.B. J. Med. Chem. 1996; 39: 2745-2752Crossref PubMed Scopus (18) Google Scholar). In contrast, 11-deoxo-11-hydroxyforskolin was a weak stimulator and had a small enhancement in isoform selectivity; the relative potency of stimulating each isoform versus forskolin (% forskolin activity) was 44% for type II, 19% for type III, and 55% for type V. Forskolin derivatives that were substituted with an alkyl group at the C13 position such as 13-devinyl-13-aminomethylforskolin, 13-devinyl-13-hydroxymethylforskolin, and 14,15-dihydro-15-chloroforskolin were mostly inactive on any isoform. An exception was that with an unsubstituted ethyl group at the position C13 (14,15-dihydroforskolin), which has been used in radioligand binding assays (34Ho R.J. Shi Q.H. J. Biol. Chem. 1984; 259: 7630-7636Abstract Full Text PDF PubMed Google Scholar). It exhibited decreased stimulatory activity with a small enhancement in isoform selectivity; the relative potency of stimulation versus forskolin was 39% for type II, 25% for type III, and 41% for type V. As shown in a previous crystallographic study (21Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (676) Google Scholar), a relatively large open packing space exists between adenylyl cyclase and the C6 position of forskolin, whereas a tight hydrogen bond exists between adenylyl cyclase and other positions of forskolin such as the hydrogen group of C1 and the carbonyl group of C11. There is also a tight hydrogen bond between the carbonyl group of C7 and adenylyl cyclase; however, there exists an open packing space between the methyl group of C7 and adenylyl cyclase. Thus, we thought that modification of these residues (C6 and C7, see Table I), unlike the earlier modifications, may promote isoform selectivity without loss of potency. Indeed, that was the case. Results from representative derivatives (FD1-FD6) are summarized in Table I and Fig.1.Table IThe chemical structure of representative forskolin derivativesThe abbreviation of forskolin derivatives used in the text and the modification of their residues at R6, R7, and R13 are summarized. FD1, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl]forskolin; FD2, 6-(4-acrylbutyryl)forskolin; FD3, 7-deacetyl-7-hydroxamylforskolin; FD4, 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolin; FD5, 6-[3-(dimethylamino)propionyl]forskolin (NKH477); FD6, 6-[3-(dimethylamino)propionyl]-14 15-dihydroforskolin. Lower the structure of forskolin and the position of each residue modified (R6, R7, and R13) are indicated. Open table in a new tab The abbreviation of forskolin derivatives used in the text and the modification of their residues at R6, R7, and R13 are summarized. FD1, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl]forskolin; FD2, 6-(4-acrylbutyryl)forskolin; FD3, 7-deacetyl-7-hydroxamylforskolin; FD4, 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolin; FD5, 6-[3-(dimethylamino)propionyl]forskolin (NKH477); FD6, 6-[3-(dimethylamino)propionyl]-14 15-dihydroforskolin. Lower the structure of forskolin and the position of each residue modified (R6, R7, and R13) are indicated. 6-[N-(2-Isothiocyanatoethyl)aminocarbonyl]forskolin (FD1) was originally reported to irreversibly inhibit forskolin binding to the type I isoform of adenylyl cyclase; its effect on the other isoforms remained unexamined (35Sutkowski E.M. Robbins J.D. Tang W.J. Seamon K.B. Mol. Pharmacol. 1996; 50: 299-305PubMed Google Scholar). We found that this derivative exhibited enhanced stimulation of type II, whereas it very weakly stimulated type III and type V; the relative potency of stimulation of this derivative versus forskolin was 219% for type II, 46% for type III, and 21% for type V (Fig. 1). We thus investigated the mechanism that led to increased selectivity for type II. The isothiocyanate group at the C6 position of this derivative can interact with functional groups with high nucleophilicity such as the ε-amino group of lysine or the thiol group of cysteine. To examine whether this isothiocyanate group contributes to increased selectivity for type II, we synthesized forskolin derivatives in which the isothiocyanate group at C6 was inactivated; an example was 6-(2-thioureidoethylaminocarbonyl) forskolin. This derivative was still active but lost type II selectivity (57% on type II, 60% on type III, and 92% on type V). We also synthesized a forskolin derivative that had an α,β-unsaturated carbonyl group at the same position (C6), which is functionally similar to the isothiocyanate group (FD2, 6-(4-acrylbutyryl)forskolin) (see Table I). This derivative retained similar type II selectivity (Fig.1). Furthermore, a docking study of FD1 with different adenylyl cyclase isoforms predicted that Lys896, unique to type II, may interact with the isothiocyanate group at the C6 position of this derivative. These findings suggest that to enhance type II selectivity forskolin needs to be replaced with a functional group at C6 that can productively interact with Lys896 of type II. A point mutation study of type II adenylyl cyclase at this residue (Lys896), however, will be necessary to address this issue directly. We also examined forskolin derivatives that were modified at the C7 position with various functional groups. We found that derivatives to which a polar group was attached at the C7 position, i.e. 7-deacetyl-7-hydroxamylforskolin (FD3) or 5,6-dehydroxy-7-deacetyl-7-nicotinoylforskolin (FD4), enhanced their selectivity for type III (Table I and Fig. 1). The stimulatory activity of other isoforms (types II and V) remained similar; the relative potency of stimulation of FD4 versus forskolin was 116% for type II, 307% for type III, and 77% for type V. Similarly, dehydroxyl modification at the positions of C5 and C6 also enhanced selectivity for type III (5,6-dehydroxyforskolin, 86% for type II, 166% for type III, and 78% for type V). We thus speculate that the polar substitution at the C7 position as well as the attachment of carbon-carbon double bonds to the ring core of forskolin (C5 and C6) contributes to type III selectivity. We previously reported that 6-[3-(dimethylamino)propionyl]forskolin (NKH477, FD5) had enhanced stimulation of type V, whereas the potency of stimulating other isoforms (types II and III) remained similar (Table I and Fig. 1) (20Toya Y. Schwencke C. Ishikawa Y. J. Mol. Cell. Cardiol. 1998; 30: 97-108Abstract Full Text PDF PubMed Scopus (53) Google Scholar). It should be noted that NKH477 (FD5) is now used to stimulate cardiac adenylyl cyclase in patients with congestive heart failure (19Sanbe A. Takeo S. J. Pharmacol. Exp. Ther. 1995; 274: 120-126PubMed Google Scholar, 36Hosono M. Takahira T. Fujita A. Fujihara R. Ishizuka O. Tatee T. Nakamura K. J. Cardiovasc. Pharmacol. 1992; 19: 625-634Crossref PubMed Scopus (81) Google Scholar). Several other forskolin derivatives in which a positively charged group such as 3-(dimethylamino)propionyl group was attached to the position of C6 or C7 showed a similar enhancement in type V selectivity. Thus, modification of the C6 or the C7 positions with a positively charged residue resulted in enhanced type V selectivity without losing potency for other adenylyl cyclase isoforms. As previously stated, 14,15-dihydroforskolin has a weak stimulatory effect on adenylyl cyclase but showed a small enhancement in type V selectivity. We thus combined the two modifications; a 3-(dimethylamino)propionyl group was placed at the C6 position of 14,15-dihydroforskolin. The resulting forskolin derivative (FD6, 6-[3-(dimethylamino)propionyl]-14,15-dihydroforskolin) had a further enhancement in selectivity for type V; the relative potency of stimulation of this derivative versus forskolin was 51% for type II, 22% for type III, and 139% for type V (Table I and Fig. 1). Thus, combining the two modifications, i.e. a minor modification at the C13 position and the 3-dimethylaminopropionyl modification at the C6 position, had additive effects in enhancing isoform selectivity. In summary, our findings strongly suggest that the modification of a specific residue(s) of forskolin increases selectivity for different adenylyl cyclase isoforms, and the combination of multiple modifications further enhances isoform selectivity. The relative potency of each forskolin derivative (FD1, -4, and -6) in comparison to that of forskolin is shown in Fig. 2. Forskolin stimulated each adenylyl cyclase isoform in a concentration-dependent manner. The selectivity of FD1 for type II," @default.
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