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.

No abstract

No abstract

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.

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.