Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

The sterol 27-hydroxylase (EC 1

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Dé saubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028 -14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2,5-dideoxyadenosine (2,5-ddAdo) > ␤-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3-polyphosphates inhibitory potency followed the order Ado < 2-dAdo < 2,5-ddAdo and 3-mono-< 3-di-< 3-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2,5-dd-3-ATP with IC 50 values of 40 -300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact purine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4 -6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.P-site-mediated inhibition has been characterized pharmacologically (1, 2, 4, 12-16). Inhibition requires an intact adenine moiety, and potency of inhibition is increased substantially for deoxyribose and especially 3Ј-phosphorylribose adenine nucleosides. Inhibitory potency follows the order: 3Ј-mono-Ͻ 3Ј-di Ͻ 3Ј-triphosphate and adenosine (Ado) Ͻ 2Ј-deoxy (d) 1 -Ado Ͻ 2Ј,5Ј-ddAdo, with 2Ј,5Ј-dd-3Ј-ATP being the most potent ligand and exhibiting an IC 50 ϳ40 nM (15). In addition, tolerance for large substitutions at the 3Ј-position and for other ribose modifications has been demonstrated (1, 2, 4).We reported previously that levels of 2Ј-d-3Ј-AMP and 3Ј-AMP varied considerably in different tissues and were dependent on the metabolic state of the animal (17). Moreover, sensitivity o...

No abstract

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.