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 nonnucleoside 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.The G protein 1 -sensitive, membrane-bound form of adenylyl cyclase consists of a large family; nine isoforms have been isolated and extensively studied (1-3). 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 (4); protein kinase C-sensitive type II is expressed in lungs (5); calmodulin-sensitive type I and type VIII are expressed exclusively in neuronal tissues (6); type III is expressed abundantly in olfactory tissues (7, 8) but also in other tissues including lungs (9), atria (10), and adipose tissue (11); type IV and VII are widely expressed (12, 13). 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 (14). 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 (C 1 and C 2 ) using a combination of hydropho...