Synthesis of substituted 5[H]phenanthridin-6-ones as potent poly(ADP-ribose)polymerase-1 (PARP1) inhibitors.

1-, 2-, 3-, 4-, 8-, or 10-Substituted 5(H)phenanthridin-6-ones were synthesized and found to be potent PARP1 inhibitors. Among the 28 compounds prepared, some showed not only low IC(50) values (compound 1b, 10 nM) but also desirable water solubility characteristics. These properties, which are superior to the common PARP1 inhibitors such as benzamides and isoquinolin-1-ones, are essential for potential therapeutic usage. The variety of compounds allows SAR analysis of favored substituents and substituted positions on 5(H)phenanthridin-6-one ring.

GPI 6150 prevents H(2)O(2) cytotoxicity by inhibiting poly(ADP-ribose) polymerase.

GPI 6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one) is a novel inhibitor of poly(ADP-ribose) polymerase (PARP). It has demonstrated efficacy in rodent models of focal cerebral ischemia, traumatic brain injury, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine damage to dopaminergic neurons, regional myocardial ischemia, streptozotocin-induced diabetes, septic shock, and arthritis. Here we report the structure of GPI 6150, its enzymatic characteristics, and biochemical property in cytoprotection. As a competitive PARP inhibitor (K(i) = 60 nM), GPI 6150 protected the P388D1 cells against hydrogen peroxide cytotoxicity, by preventing PARP activation and the depletion of NAD(+), the substrate for PARP. To address the concerns of potential side effects of PARP inhibition, we tested GPI 6150 and found it had no effect on the repair and expression of a plasmid DNA damaged by N-methyl-N'-nitro-N-nitrosoguanidine. Neither did it affect dehydrogenases with NAD co-enzyme. GPI 6150 was much less potent to inhibit mono-ADP-ribosyltransferase. There was no selectivity for GPI 6150 between PARP isozymes. These attributes render GPI 6150 a useful tool to probe the functions of PARP.

A yeast three-hybrid method to clone ternary protein complex components.

The yeast two-hybrid system is a powerful technique to detect binary protein interactions. In order to facilitate ternary complex analysis, we have developed the yeast three-hybrid system. Using epidermal growth factor (EGF) receptor, Grb2 and Sos proteins as an example, we demonstrated the use of the three-hybrid system to detect formation of a three-component complex, i.e., the Grb2-mediated interactions between EGF receptor and Sos. We then used the three-hybrid system to screen a library with Gal4-DNA binding domain -EGF receptor and Gal4 activation domain-Sos fusion proteins and isolated a plasmid encoding Grb2. Thus, the three-hybrid system is useful for characterizing and cloning components of ternary protein complexes.

Ligand autoradiographic receptor screening: receptor cDNA expression in replicas of transfected COS cells.

In this paper we validate a methodology, ligand autoradiographic receptor screening (LARS), for detecting expression of full length receptor cDNAs in COS cells. The method involves transfection of COS cells with receptor cDNAs by spheroplast fusion, production of filter replicas of the cell fragments, ligand binding to the receptors expressed in the membranes, and autoradiographic detection of bound ligand. A beta-adrenergic receptor cDNA cloned into a eukaryotic expression vector reliably induces high levels of beta-adrenergic receptor expression in 2-12% of COS cell colonies transfected with this plasmid after experimental conditions are optimized. Receptor expression is monitored by autoradiographic detection of 125iodocyanopindolol binding to COS cell fragments immobilized on polyester filter replicas. Binding displays appropriate pharmacological properties. The number of high-density binding spots per filter depends on the fraction of the spheroplasts in the fusion mixture that contain the beta-adrenergic receptor cDNA. A 100-plate LARS experiment can assess receptor expression in more than 10(4) transfected colonies. Thus detection of receptor-encoding sequences present in libraries in proportions as low as 1 in 10(4) should be possible. This technique may therefore be useful in detecting expression of other receptor cDNAs for which selective radioligands are available.