SummaryMetazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that cross-links collagens and elastin in the extracellular matrix and is a critical mediator of tumor growth and metastatic spread. LOX is a target for cancer therapy, and thus the search for therapeutic agents against LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High-throughput screening provided the initial hits. Structure–activity relationship (SAR) studies led to the discovery of AMT inhibitors with sub-micromolar half-maximal inhibitory concentrations (IC50) in a LOX enzyme activity assay. Further SAR optimization yielded the orally bioavailable LOX inhibitor CCT365623 with good anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy.

Here, we describe an approach to identify novel selective estrogen receptor downregulator (SERD) compounds with improved properties such as oral bioavailability and the potential of increased efficacy compared to currently marketed drug treatments. Previously, methodologies such as Western blotting and transient cell reporter assays have been used to identify and characterize SERD compounds, but such approaches can be limited due to low throughput and sensitivity, respectively. We have used an endogenous cell-imaging strategy that has both the throughput and sensitivity to support a large-scale hit-to-lead program to identify novel compounds. A screening cascade with a suite of assays has been developed to characterize compounds that modulate estrogen receptor α (ERα)-mediated signaling or downregulate ERα levels in cells. Initially, from a focused high-throughput screening, novel ERα binders were identified that could be modified chemically into ERα downregulators. Following this, cellular assays helped determine the mechanism of action of compounds to distinguish between on-target and off-target compounds and differentiate SERDs, selective estrogen receptor modulator (SERM) compounds, and agonist ERα ligands. Data are shown to exemplify the characterization of ERα-mediated signaling inhibitors using a selection of literature compounds and illustrate how this cascade has been used to drive the chemical design of novel SERD compounds.

The lysyl oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing the formation of crosslinks in fibrillar elastin and collagens leading to extracellular matrix (ECM) stabilization. These enzymes have also been implicated in tumor progression and metastatic disease and have thus become an attractive therapeutic target for many types of invasive cancers. Following our recently published work on the discovery of aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors, we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz) scaffold. Incorporation of a thiazole core leads to improved potency toward LOXL2 inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved pharmacokinetic properties and excellent antitumor efficacy, with significantly reduced tumor growth in a spontaneous breast cancer genetically engineered mouse model.