The differential effect of roscovitine on cell cycle status in LTLTca compared

The profile of reference non selective gamma secretase inhibitors compared with the HTS hit and selective lead compounds in the SNC dual cell assay corroborated the biochemical enzymatic assay data, demonstrating APP selective inhibition of cellular gamma secretase by the screening hit and select lead compounds . Biochemical characterization JNJ-7706621 ic50 of the inhibitors revealed they behave as classical GSIs in that they elevate C99 with no effects on sAPPa or sAPPb, and are equipotent inhibitors of Ab40 and Ab42 . The compounds lowered secreted Ab in conditioned media at the concentrations tested for effects on APP metabolites . Binding site stu dies employing biotin labeled active site isostere as affi nity ligand for enriched gamma secretase enzyme confirmed competitive displacement of affinity ligand by its non biotinylated analog L 685,458, consistent with published results, as well as by very high con centrations of LY411575 .

Specifically, we observed a 50% displacement of a biotinylated active site isostere probe by L 685,458 at concentrations approxi mately three fold above its IC50 in the gammaAPP assay, whereas LY 411575 displaced 50% of biotinylated active site isostere Lenalidomide Revlimid probe at concentrations 500X to 1,000X above its IC50 value in the gammaAPP assay. Sulfonamides did not displace biotinylated active site isostere in competitive binding assays in the absence of substrate . However, in the pre sence of 1 Km MBP C125 substrate, ELN318463 and ELN475516 were able to displace the active site isostere at an ED50 of 23 uM and 0. 14 uM, respectively .

These values represent an approximately 2,000X and 67X multiple over the IC50 values of the two com pounds in the gammaAPP assay. In vivo testing of a benzene caprolactam sulfonamide, ELN318463, possessing favorable oral bioavailabilty, LY2228820 溶解度 revealed dose dependent acute reduc tion of brain Ab1 x species in PDAPP mice . In contrast, acute reduction of Abx 40 species in non transgene FVB mice was equivalent at both doses tested. Reduction of Abx 40 in PDAPP also was not dose responsive . The lack of a dose response in Abx 40 species in PDAPP FVB mice compared with Ab1 x species in PDAPP mice can not be explained by differences in terminal compound levels in the target organ at the two doses tested, nor by assay differences. Brain levels of ELN318463 at 30 mg kg were 0. 754 uM in FVB brains and 0.

69 uM in PDAPP brains, and at 100 mg kg dose the levels were 2. 7 uM in FVB brains and 1. 87 uM in PDAPP brains. Discordance between Ab1 40 and Abx 40 in BACE KO or wild type BACE inhibitor treated mice has been reported . However, our observations can not be explained by differences in assays employed for measuring Ab1 x vs. Abx 40, as observed by Nishitomi et al, because both assays used in this study are insensitive to the Ab P3 fragment by virtue of the capture antibody employed . Lead optimization toward discovery of more potent analogs revealed critical requirement for proton donor in caprolactam ring of benzene sulfonamides, corrobo rated by pyrazole substituted piperidines as scaffold replacements .