12 research outputs found
A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context
Role of P-Glycoprotein and Breast Cancer Resistance Protein-1 in the Brain Penetration and Brain Pharmacodynamic Activity of the Novel Phosphatidylinositol 3-Kinase Inhibitor GDC-0941
Preclinical Assessment of the Absorption and Disposition of the Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor GDC-0980 and Prediction of Its Pharmacokinetics and Efficacy in Human
Preclinical Disposition of GDC-0973 and Prospective and Retrospective Analysis of Human Dose and Efficacy Predictions
Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma
Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR
Inhibition of phosphoinositide 3-kinase
(PI3K) signaling is an appealing approach to treat brain tumors, especially
glioblastoma multiforme (GBM). We previously disclosed our successful
approach to prospectively design potent and blood–brain barrier
(BBB) penetrating PI3K inhibitors. The previously disclosed molecules
were ultimately deemed not suitable for clinical development due to
projected poor metabolic stability in humans. We, therefore, extended
our studies to identify a BBB penetrating inhibitor of PI3K that was
also projected to be metabolically stable in human. These efforts
required identification of a distinct scaffold for PI3K inhibitors
relative to our previous efforts and ultimately resulted in the identification
of GDC-0084 (<b>16</b>). The discovery and preclinical characterization
of this molecule are described within
The Design and Identification of Brain Penetrant Inhibitors of Phosphoinositide 3-Kinase α
Role of P-Glycoprotein on the Brain Penetration and Brain Pharmacodynamic Activity of the MEK Inhibitor Cobimetinib
Identification, Characterization, and Implications of Species-Dependent Plasma Protein Binding for the Oral Hedgehog Pathway Inhibitor Vismodegib (GDC-0449)
Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design
The <i>N</i>-methyl-d-aspartate receptor (NMDAR)
is a Na<sup>+</sup> and Ca<sup>2+</sup> permeable ionotropic glutamate
receptor that is activated by the coagonists glycine and glutamate.
NMDARs are critical to synaptic signaling and plasticity, and their
dysfunction has been implicated in a number of neurological disorders,
including schizophrenia, depression, and Alzheimer’s disease.
Herein we describe the discovery of potent GluN2A-selective NMDAR
positive allosteric modulators (PAMs) starting from a high-throughput
screening hit. Using structure-based design, we sought to increase
potency at the GluN2A subtype, while improving selectivity against
related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptors (AMPARs). The structure–activity relationship of
channel deactivation kinetics was studied using a combination of electrophysiology
and protein crystallography. Effective incorporation of these strategies
resulted in the discovery of GNE-0723 (<b>46</b>), a highly
potent and brain penetrant GluN2A-selective NMDAR PAM suitable for <i>in vivo</i> characterization