Development and validation of an in vitro 3D model of NASH with severe fibrotic phenotype.

Nonalcoholic steatohepatitis represents a significant and rapidly growing unmet medical need. The development of novel therapies has been hindered in part, by the limitations of existing preclinical models. There is a strong need for physiologically relevant in vivo and in vitro liver fibrosis models that are characterized by better translational predictability. In this study, we used the InSphero 3D InSightTM three-dimensional (3D) human liver microtissue (3D-hLMT) system prepared by co-culturing primary human hepatocytes with hepatic stellate cells, Kupffer cells and endothelial cells to develop a model of NASH with a severe fibrotic phenotype. In our model, palmitic acid (PA) induced a robust proinflammatory and profibrogenic phenotype in the 3D-hLMT. PA significantly increased several markers of the inflammatory and profibrotic process including gene expression of collagens, α-sma, tissue inhibitor of matrix metalloprotease 1 (timp1) and the stellate cell activation marker pdgfrß as well as secreted CXCL8 (IL8) levels. We also observed TGFß pathway activation, increase in active collagen synthesis and significant overall increase in tissue damage in the 3D-hLMTs. Immunohistochemistry analysis demonstrated the upregulation of collagen, cleaved caspase 3 as well as of the PDGFRß protein. We further validated the model using a phase 3 clinical compound, GS-4997, an apoptosis signal-regulating kinase 1 (ASK-1) inhibitor and showed that GS-4997 significantly decreased PA induced profibrotic and proinflammatory response in the 3D-hLMTs with decreases in apoptosis and stellate cell activation in the microtissues. Taken together we have established and validated an in vitro 3D-hLMT NASH model with severe fibrotic phenotype that can be a powerful tool to investigate experimental compounds for the treatment of NASH.

Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes.

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis.

Discovery of Vibegron: A Potent and Selective ß3 Adrenergic Receptor Agonist for the Treatment of Overactive Bladder.

The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective ß3 adrenergic receptor agonists for the treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human ß3 adrenergic receptor agonists.

A novel class of human ß(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed ß(3)-AR agonists. As observed, many of the ß(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human ß(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional ß(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new ß(3)-AR agonists containing the pyrrolidine moiety.

Heterocyclic acetamide and benzamide derivatives as potent and selective beta3-adrenergic receptor agonists with improved rodent pharmacokinetic profiles.

A series of amide derived beta(3)-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation beta(3)-AR agonists for the treatment of overactive bladder.

Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence.

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for the entry of macrophage-tropic (R5) HIV-1 strains into target cells. Posttranslational sulfation of tyrosine residues in the N-terminal tail of CCR5 is critical for high affinity interaction of the receptor with the HIV-1 envelope glycoprotein gp120 in complex with CD4. Here, we focused on defining precisely the sulfation pattern of the N terminus of CCR5 by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a synthetic peptide that corresponds to amino acids 2-18 of the receptor (CCR5 2-18). Analysis of the reaction products was made with a combination of reversed-phase HPLC, proteolytic cleavage, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found that CCR5 2-18 is sulfated by both TPST isoenzymes leading to a final product with four sulfotyrosine residues. Sulfates were added stepwise to the peptide producing specific intermediates with one, two, or three sulfotyrosines. The pattern of sulfation in these intermediates suggests that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a structural basis for understanding the role of tyrosine sulfation of CCR5 in HIV-1 coreceptor and chemokine receptor function.