Novel tricyclic glycal-based TRIB1 inducers that reprogram LDL metabolism in hepatic cells.

Increased expression of the Tribbles pseudokinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction. We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518, the initial lead compound from the tricyclic glycal class, exhibited stereochemically dependent activity and the potency far exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518 demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes. Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518 stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in vivo studies.

Modulators of hepatic lipoprotein metabolism identified in a search for small-molecule inducers of tribbles pseudokinase 1 expression.

Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Screening for inhibitors of an essential chromatin remodeler in mouse embryonic stem cells by monitoring transcriptional regulation.

The SWI/SNF-like adenosine triphosphate (ATP)-dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression-based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology.

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.