Evaluation of a Three-Dimensional Primary Human Hepatocyte Spheroid Model: Adoption and Industrialization for the Enhanced Detection of Drug-Induced Liver Injury.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Discovery and SAR Study of Boronic Acid-Based Selective PDE3B Inhibitors from a Novel DNA-Encoded Library.

Human genetic evidence shows that PDE3B is associated with metabolic and dyslipidemia phenotypes. A number of PDE3 family selective inhibitors have been approved by the FDA for various indications; however, given the undesirable proarrhythmic effects in the heart, selectivity for PDE3B inhibition over closely related family members (such as PDE3A; 48% identity) is a critical consideration for development of PDE3B therapeutics. Selectivity for PDE3B over PDE3A may be achieved in a variety of ways, including properties intrinsic to the compound or tissue-selective targeting. The high (>95%) active site homology between PDE3A and B represents a massive obstacle for obtaining selectivity at the active site; however, utilization of libraries with high molecular diversity in high throughput screens may uncover selective chemical matter. Herein, we employed a DNA-encoded library screen to identify PDE3B-selective inhibitors and identified potent and selective boronic acid compounds bound at the active site.

Integrated Direct-to-Biology Platform for the Nanoscale Synthesis and Biological Evaluation of PROTACs.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co-opt the cell's natural proteasomal degradation mechanisms to degrade undesired proteins. A challenge associated with PROTACs is the time and resource-intensive optimization; thus, the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra-high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This 'direct-to-biology' (D2B) approach was validated and then exemplified in a medicinal chemistry campaign to identify novel BRD4 PROTACs. Using the D2B platform, the synthesis of 650 PROTACs was carried out in a 1536-well plate, and subsequent biological evaluation was performed by a single scientist in less than 1 month. Due to its ability to hugely accelerate the optimization of new degraders, we anticipate our platform will transform the synthesis and testing of PROTACs.

BET Bromodomain Inhibitors Suppress Inflammatory Activation of Gingival Fibroblasts and Epithelial Cells From Periodontitis Patients.

BET bromodomain proteins are important epigenetic regulators of gene expression that bind acetylated histone tails and regulate the formation of acetylation-dependent chromatin complexes. BET inhibitors suppress inflammatory responses in multiple cell types and animal models, and protect against bone loss in experimental periodontitis in mice. Here, we analyzed the role of BET proteins in inflammatory activation of gingival fibroblasts (GFs) and gingival epithelial cells (GECs). We show that the BET inhibitors I-BET151 and JQ1 significantly reduced expression and/or production of distinct, but overlapping, profiles of cytokine-inducible mediators of inflammation and bone resorption in GFs from healthy donors (IL6, IL8, IL1B, CCL2, CCL5, COX2, and MMP3) and the GEC line TIGK (IL6, IL8, IL1B, CXCL10, MMP9) without affecting cell viability. Activation of mitogen-activated protein kinase and nuclear factor-κB pathways was unaffected by I-BET151, as was the histone acetylation status, and new protein synthesis was not required for the anti-inflammatory effects of BET inhibition. I-BET151 and JQ1 also suppressed expression of inflammatory cytokines, chemokines, and osteoclastogenic mediators in GFs and TIGKs infected with the key periodontal pathogen Porphyromonas gingivalis. Notably, P. gingivalis internalization and intracellular survival in GFs and TIGKs remained unaffected by BET inhibitors. Finally, inhibition of BET proteins significantly reduced P. gingivalis-induced inflammatory mediator expression in GECs and GFs from patients with periodontitis. Our results demonstrate that BET inhibitors may block the excessive inflammatory mediator production by resident cells of the gingival tissue and identify the BET family of epigenetic reader proteins as a potential therapeutic target in the treatment of periodontal disease.