Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channels.

Chloride channels play important roles in homeostasis and regulate cell volume, transepithelial transport, and electrical excitability. Despite recent progress made in the genetic and molecular aspect of chloride channels, their pharmacology is still poorly understood. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated epithelial chloride channel for which mutations cause cystic fibrosis. Here we have synthesized benzo[c]quinolizinium and benzo[f]indolo[2,3-a]quinolizinium salts (MPB) and performed a SAR to identify the structural basis for activation of the CFTR chloride channel. Synthesized compounds were evaluated on wild-type CFTR and on CFTR having the glycine-to-aspartic acid missense mutation at codon 551 (G551D-CFTR), using a robot and cell-based assay. The presence of an hydroxyl group at position 6 of the benzo[c]quinolizinium skeleton associated with a chlorine atom at position 10 or 7 and an alkyl chain at position 5 determined the highest activity. The most potent product is 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride (8u, MPB-104). 8u is 100 times more potent than the parent compound 8a (MPB-07).

Isolation of drugs active against mammalian prions using a yeast-based screening assay.

We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs. The method allowed us to identify several compounds effective against budding yeast prions responsible for the [PSI+] and [URE3] phenotypes. These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine. Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay. The compounds isolated here were also active in promoting mammalian prion clearance. These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans.

Aloisines, a new family of CDK/GSK-3 inhibitors. SAR study, crystal structure in complex with CDK2, enzyme selectivity, and cellular effects.

Cyclin-dependent kinases (CDKs) regulate the cell cycle, apoptosis, neuronal functions, transcription, and exocytosis. The observation of CDK deregulations in various pathological situations suggests that CDK inhibitors may have a therapeutic value. In this article, we report on the identification of 6-phenyl[5H]pyrrolo[2,3-b]pyrazines (aloisines) as a novel potent CDK inhibitory scaffold. A selectivity study performed on 26 kinases shows that aloisine A is highly selective for CDK1/cyclin B, CDK2/cyclin A-E, CDK5/p25, and GSK-3 alpha/beta; the two latter enzymes have been implicated in Alzheimer's disease. Kinetic studies, as well as the resolution of a CDK2-aloisine cocrystal structure, demonstrate that aloisines act by competitive inhibition of ATP binding to the catalytic subunit of the kinase. As observed with all inhibitors reported so far, aloisine interacts with the ATP-binding pocket through two hydrogen bonds with backbone nitrogen and oxygen atoms of Leu 83. Aloisine inhibits cell proliferation by arresting cells in both G1 and G2.

Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07.

1. This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D-CFTR mutant. 2. We measured Cl(-) transport as the amplitude of short-circuit current in response to the membrane permeable cAMP analogue 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP) alone or in combination with a CFTR opener. The correction of G551D-CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. 3. In both types of epithelia, cAMP caused activation of Cl(-) transport that was inhibited by glibenclamide and not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid. After normalising for CFTR expression, the response of FRT-G551D epithelia was 1% that of wild type monolayers. 4. Addition of genistein (10-200 micro M), but not of 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1-100 micro M) or of the benzo[c]quinolizinium MPB-07 (10-200 micro M) to FRT-G551D epithelia pre-treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. 5. The genistein dose-response curve was bell-shaped due to inhibitory activity at the highest concentrations. The dose-dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. 6. On human nasal epithelia the correction of G551D-CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl(-) transport defect on G551D patients.

Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07).

The photodegradation of 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration, and temperature were used. MPB-07 concentration was determined at regular time intervals by reversed-phase HPLC. MPB-07 stability was also studied at pH 7.4 in the dark. Results showed that in all the conditions tested MPB-07 underwent rapid photodegradation, apparently following first-order kinetics. Rate constants were dependent on the initial MPB-07 concentration, temperature, and pH. At pH 7.4, and for concentrations from 1 to 125 microM, half-lives ranged from 0.681 +/- 0.047 to 4.54 +/- 0.28 h. The Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ x mol(-1). Analysis by chemical ionization-mass spectrometry showed that the chlorine atom of the MPB-07 molecule might be replaced by an OH group during the photodegradation process. In the dark, MPB-07 in solutions at pH 7.4 was found to be stable over a 6-week period. In conclusion, MPB-07 is a highly photolabile molecule that should be carefully protected from light when used.