A Phenotypic Screening Approach Using Human Treg Cells Identified Regulators of Forkhead Box p3 Expression.

Regulatory T (Treg) cells, expressing the transcription factor forkhead box p3 (FOXP3), are the key cells regulating peripheral autoreactive T lymphocytes by suppressing effector T cells. FOXP3+ Treg cells play essential roles controlling immune responses in autoimmune diseases and cancer. Several clinical approaches (e.g., polyclonal expansion of Treg cells with anti-CD3 and anti-CD28 coated beads in the presence of drugs) are under evaluation. However, expression of FOXP3, recognized as the master regulator of Treg cells, in induced Treg cells have been shown to be instable, and molecular targets involved in regulating FOXP3 expression and Treg cell function have not been well-defined. Thus, new targets directly regulating FOXP3 expression and the expression of its downstream genes (e.g., cytotoxic T-lymphocyte-associated protein 4 (CTLA4)) have the potential to stabilize the Treg cell phenotype and function. This report describes the development of an automated medium-throughput 384-well plate flow cytometry phenotypic assay meauring the protein expression of FOXP3 and CTLA4 in human Treg cells. Screening a library of 4213 structurally diverse compounds allowed us to identify a variety of compounds regulating FOXP3 and CTLA4 expression. Further evaluation of these and related small molecules, followed by confirmation using siRNA-mediated gene knockdown, revealed three targets: euchromatic histone-lysine N-methyltransferase (EHMT2) and glycogen synthase kinase 3 alpha/beta (GSK3α/ß) as potent positive regulators of FOXP3 expression, and bromodomain and extra-terminal domain (BET) inhibitors as negative regulators of FOXP3 and CTLA4 expression. These targets have potential implications for establishing novel therapies for autoimmune diseases and cancer.

Design and synthesis of ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of ß-amyloid peptides.

The evaluation of a series of aminoisoindoles as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aß40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 µM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of ß-amyloid peptides in mouse brain following oral dosing.