Agonist Neuroscience

RDEA is represented by large sticks, whereas the MEK residues are represented by thin lines and color coded in the same manner as A. Proteinligand ternary complex formation and cocrystallization.Frozen MEKDRDEA cocrystals have unit cell dimensions of a. Data collection and structure determination.The initial model was refined against the data initially by treating the MEK peptide as a rigid body.Additional rounds of torsion angle annealing and manual adjustment to the model revealed strong difference density for one molecule of ATP, one magnesium ion, and RDEA.Ligand atoms were added to the model accordingly and included in further rounds of refinement.for the model, which includes MEK residues, one molecule of ATP, one magnesium ion, water molecules, and the inhibitor RDEA.MEK activity was assayed by measuring the phosphorylation of ERK and ERK using a phosphoERK ELISA kit. After d, AL of H tetrazolium, inner salt reagent were added per well.After hat jC, absorbance at nm was determined on the M plate reader. Tumor volumes were monitored by caliper measurewidth andl length in mm of the tumor.For efficacy analysis, treatment was initiated when tumors were to mm.Homogenates were incubated on ice for hand centrifuged at gfor min.Lysates were transferred to microcentrifuge tubes and further clarified by centrifugation at, gfor min.Blood samples were collected for plasma bioanalysis of RDEA and PD concentrations by centrifugation at, rpm for min.Plasma supernatants were aspirated and placed in labeled tubes and stored at jC until analysis.Brain and lung samples were also collected, weighed, and flash frozen in liquid nitrogen for analysis of total ERK and pERK levels.Plasma samples were analyzed for RDEA using a liquid chromatographytandem mass spectrometry, protein precipitation with acetonitrile, and final analysis by highperformance LCMSMS.An API triple quadrupole mass spectrometer was used to monitor the precursorproduct ion transitions of mz and mz for RDEA and RDEA in positive electrospray ion mode.The calibration curves covered the concentration range from to, ngmL.Results RDEA is an orally bioavailable small molecule with a molecular weight of gmol.We conducted a variety of preclinical in vitro and in vivo studies to determine antiproliferative activity in cell culture systems using both cancer and normal cells, pharmacokinetic relationships between antitumor efficacy and brain retention, and downstream consequences of MEK inhibition on proliferation andor apoptosis.To determine the mechanism of inhibition of RDEA, we cocrystallized RDEA with a truncated version of MEK that was amenable to crystallization. This interaction site is similar to the one identified for the MEK inhibitor PD. Four other kinases showed inhibition of; however, these used MEK in a cascade assay XY1 format and the inhibition in these assays is due to MEK inhibition rather than direct inhibition of the other kinases by RDEA.The kinasespecific assay conditions use a cascade format that has RAFMEKERK all together in the assay, which phosphorylate an ERK substrate.Growth inhibition potency and effect of BRAF status.RDEA inhibited anchoragedependent growth of human cancer cell lines harboring the gainoffunction VE BRAF mutant with GI values ranging from to nmolL. In contrast, RDEA had significantly less growthinhibitory potency against cell lines with wildtype BRAF enhancement of inherent kinase activity.

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