Fragment-Guided Discovery of Pyrazole Carboxylic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2 Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction.

The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization.

Gut-Restricted Selective Cyclooxygenase-2 (COX-2) Inhibitors for Chemoprevention of Colorectal Cancer.

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure.

Modular Protein Ligation: A New Paradigm as a Reagent Platform for Pre-Clinical Drug Discovery.

Significant resource is spent by drug discovery project teams to generate numerous, yet unique target constructs for the multiple platforms used to drive drug discovery programs including: functional assays, biophysical studies, structural biology, and biochemical high throughput screening campaigns. To improve this process, we developed Modular Protein Ligation (MPL), a combinatorial reagent platform utilizing Expressed Protein Ligation to site-specifically label proteins at the C-terminus with a variety of cysteine-lysine dipeptide conjugates. Historically, such proteins have been chemically labeled non-specifically through surface amino acids.

Development of a high-content imaging assay for screening compound aggregation.

Aggregated compounds can promiscuously and nonspecifically associate with proteins resulting in either false inhibition or activation of many different protein target classes. We developed a high-content imaging assay in a 384-well format using fluorescently labeled target proteins and an Operetta cell imager to screen for compound aggregates that interact with target proteins. The high-throughput assay can not only directly detect the interaction between compound aggregators and the target of interest, but also determine the critical aggregation concentration (CAC) of a given promiscuous small molecule.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy.

Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets.

SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism.

A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC.

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552.