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Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

GPBAR1 (also known as TGR5) is a G-protein-coupled receptor (GPCR) that triggers intracellular signals upon ligation by various bile acids. The receptor has been studied mainly for its function in energy expenditure and glucose homeostasis, and there is little information on the role of GPBAR1 in the context of inflammation. After a high-throughput screening campaign, we identified isonicotinamides exemplified by compound 3 as nonsteroidal GPBAR1 agonists. We optimized this series to potent derivatives that are active on both human and murine GPBAR1. These agonists inhibited the secretion of the proinflammatory cytokines TNF-α and IL-12 but not the antiinflammatory IL-10 in primary human monocytes. These effects translate in vivo, as compound 15 inhibits LPS induced TNF-α and IL-12 release in mice. The response was GPBAR1 dependent, as demonstrated using knockout mice. Furthermore, agonism of GPBAR1 stabilized the phenotype of the alternative, noninflammatory, M2-like type cells during differentiation of monocytes into macrophages. Overall, our results illustrate an important regulatory role for GPBAR1 agonists as controllers of inflammation.

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) ( Nature 2011 , 475 , 524 ; 519 ). EBI2 is highly expressed in immune cells ( J. Biol. Chem. 2006 , 281 , 13199 ), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes ( Nature 2010 , 467 , 460 ). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (1), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (2) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (4m). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.

Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.

A novel sulfanyltriazole was discovered as an HIV-1 non-nucleoside reverse transcriptase inhibitor via HTS using a cell-based assay. Chemical modifications and molecular modeling studies were carried out to establish its SAR and understand its interactions with the enzyme. These modifications led to the identification of sulfanyltriazoles with low nanomolar potency for inhibiting HIV-1 replication and promising activities against selected NNRTI resistant mutants. These novel and potent sulfanyltriazoles could serve as advanced leads for further optimization.