Doskey, Luke C.

BET bromodomain inhibitors are efficacious as anti-inflammatory compounds, but clinical translation is hampered by dose-limiting toxicities from non-selective inhibition. We herein report compounds 16 and 26, based on previously reported 4-methyl-1,2,3-triazole-based inhibitor, DW34. 16 has a BRD4 BD1 Ki of 10 nM, and up to 13–38-fold selectivity for BRD4 BD1 over other BD2 domains, and an improved hERG affinity of 4.24 µM over DW34. 26, A biscyclopropyl analog of 16 showed further enhancement of BD1 selectivity over BD2 (26–203-fold), but exhibited more potent hERG inhibition. Compound 16 had improved liver and plasma clearance over DW34, with good biodistribution to the liver, and had an intracellular target-engagement affinity Ki of 92 nM, with reduction of Ccl2, Cxcl1, AST, and ALT in in vitro and in vivo mouse models of liver inflammation. 16 represents a useful lead for treating inflammatory liver diseases, with improved specificity, drug-like properties, and reduced hERG affinity.

Background and Aims: Liver fibrosis and cirrhosis are major global health burdens with limited treatments. Activated HSCs drive fibrosis through increased proliferation, migration, collagen deposition and glycolysis. The aim of this study is to elucidate the signaling events that lead to enhanced glycolysis, subsequent HSC activation, and liver fibrosis. Approach and Results: Utilizing a phospho-kinase array, we identified with no lysine kinase 1 (WNK1) as a new kinase in primary human HSCs activated by the profibrotic PDGF-B. PDGF-mediated glycolysis in HSCs was attenuated by the WNK1 selective inhibitor, WNK-IN-11, as measured by live-cell metabolic assay, Glifon-300 fluorescence, Glucose-Glo assay, and glucose transporter 1 immunofluorescence. To understand how WNK1 promotes glycolysis, we employed chemical genetics where an engineered WNK1 carrying threonine 301 to glycine mutation (WNK1 T301G ) mutant with a modified ATP pocket accommodated a specific ATP-γ-S analogue to thio-phosphorylate its substrates. Thio-phosphorylated proteins were analyzed by mass spectrometry, which identified the glycolytic protein, triosephosphate isomerase 1 (TPI1), as a novel WNK1 direct substrate. TPI1 interacted with WNK1 in condensates, membrane-less phase separation structures. Condensate disruption and knockdown of TPI1 or WNK1 reduced HSC proliferation and migration in vitro. In vivo, pharmacological or genetic WNK1 inhibition significantly attenuated liver fibrosis in carbon tetrachloride and high-fat, choline-deficient, L -amino acid diet mouse models. Finally, WNK1 inhibition reduced fibrogenesis ex vivo in human precision-cut liver slices. Conclusions: These findings identify WNK1 as a previously unrecognized kinase that promotes HSC activation and liver fibrosis via TPI1-mediated glycolysis, highlighting the WNK1-TPI1 axis as a potential therapeutic target.

Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or antiinflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA-seq in a mouse model of liver injury and found that macrophage diversity was increased. Marco was among the most significantly upregulated genes, and a population of Marco hi macrophages increased with injury and spatially segregated to nonfibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow–derived macrophages decreased proinflammatory gene expression, increased antiinflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO + macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix–associated (ECM-associated) genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO – macrophages. Therefore, MARCO + macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a potential therapeutic pathway for liver fibrosis.

BET bromodomain inhibitors demonstrate significant promise as anti-inflammatory agents targeting a variety of inflammatory disorders. However, clinical data has demonstrated that the administration of non-selective BET bromodomain inhibitors has led to significant dose-limiting toxicity in clinical settings. More selective inhibitors of the second BET bromodomains, referred to as pan-D2 inhibitors, are better tolerated, however their efficacy varies significantly depending on disease indication. Here, we use three orally bioavailable inhibitors, 1–3, that are either selective for the first N-terminal bromodomain of BRD4 (BRD4-D1 selective), or pan-D1 biased, for assessing their cellular and in vivo efficacy and safety profile in two models of inflammatory liver disease. We compare their effects to known pan-D2 and pan-BET inhibitors. Our results show that pan-D1 biased inhibitor, 3, is as efficacious as the pan-BET inhibitor I-BET151 in reducing inflammation from LPS-induced and drug-induced toxicity, whereas pan-D2 inhibitors are less effective. BRD4-D1 selective inhibitors, 1 and 2, are also efficacious; however, new inhibitors with improved cellular target engagement will be necessary to more thoroughly assess their effects. Finally, BRD4-D1 selective inhibitors are better tolerated in a preclinical model of thrombocytopenia than pan-D1 biased inhibitors, while gastrointestinal (GI) toxicity may be a BRD4-driven effect. These results highlight the importance of assessing specific BET protein and bromodomain functions due to their varying roles in diverse disease models.

Human cholangiocytes, epithelial cells that line the bile ducts, were cultured as a model with which to study polarized small extracellular vesicle (sEV) communication. These cells release distinct sEV pools to mediate communication via their apical and basolateral domains. Furthermore, defective ESCRT function may contribute to disease states through altered sEV signaling.