Genome-wide CRISPR Screening to Identify Drivers of TGF-ß-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to identify hepatic stellate cell (HSC)-derived mediators of transforming growth factor (TGF)-ß-induced liver fibrosis. The functional genomics phenotypic screening platform described here revealed the novel biology of TGF-ß-induced fibrogenesis and potential drug targets for liver fibrosis.

Human induced pluripotent stem cell-derived macrophages ameliorate liver fibrosis.

With an increasing number of patients with degenerative hepatic diseases, such as liver fibrosis, and a limited supply of donor organs, there is an unmet need for therapies that can repair or regenerate damaged liver tissue. Treatment with macrophages that are capable of phagocytosis and anti-inflammatory activities such as secretion of matrix metalloproteinases (MMPs) provide an attractive cellular therapy approach. Human induced pluripotent stem cells (iPSCs) are capable of efficiently generating a large-scale, homogenous population of human macrophages using fully defined feeder- and serum-free differentiation protocol. Human iPSC-macrophages exhibit classical surface cell markers and phagocytic activity similar to peripheral blood-derived macrophages. Moreover, gene and cytokine expression analysis reveal that these macrophages can be efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. M1 macrophages express high level of CD80, TNF-α, and IL-6 while M2 macrophages show elevated expression of CD206, CCL17, and CCL22. Here, we demonstrate that treatment of liver fibrosis with both human iPSC-derived macrophage populations and especially M2 subtype significantly reduces fibrogenic gene expression and disease associated histological markers including Sirius Red, αSMA and desmin in immunodeficient Rag2-/- γc-/- mice model, making this approach a promising cell-based avenue to ameliorate fibrosis.

Dopamine and ghrelin receptor co-expression and interaction in the spinal defecation centers.

BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.

Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

MetAP2 inhibition increases energy expenditure through direct action on brown adipocytes.

Inhibitors of methionine aminopeptidase 2 (MetAP2) have been shown to reduce body weight in obese mice and humans. The target tissue and cellular mechanism of MetAP2 inhibitors, however, have not been extensively examined. Using compounds with diverse chemical scaffolds, we showed that MetAP2 inhibition decreases body weight and fat mass and increases lean mass in the obese mice but not in the lean mice. Obesity is associated with catecholamine resistance and blunted ß-adrenergic receptor signaling activities, which could dampen lipolysis and energy expenditure resulting in weight gain. In the current study, we examined effect of MetAP2 inhibition on brown adipose tissue and brown adipocytes. Norepinephrine increases energy expenditure in brown adipose tissue by providing fatty acid substrate through lipolysis and by increasing expression of uncoupled protein-1 (UCP1). Metabolomic analysis shows that in response to MetAP2 inhibitor treatment, fatty acid metabolites in brown adipose tissue increase transiently and subsequently decrease to basal or below basal levels, suggesting an effect on fatty acid metabolism in this tissue. Treatment of brown adipocytes with MetAP2 inhibitors enhances norepinephrine-induced lipolysis and energy expenditure, and prolongs the activity of norepinephrine to increase ucp1 gene expression and energy expenditure in norepinephrine-desensitized brown adipocytes. In summary, we showed that the anti-obesity activity of MetAP2 inhibitors can be mediated, at least in part, through direct action on brown adipocytes by enhancing ß-adrenergic-signaling-stimulated activities.

CX3CL1-Fc treatment prevents atherosclerosis in Ldlr KO mice.

OBJECTIVE: Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. METHODS: In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. RESULTS: CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. CONCLUSION: These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis.

Chronic fractalkine administration improves glucose tolerance and pancreatic endocrine function.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and ß cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased ß cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered ß cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

Structure-based drug design of novel ASK1 inhibitors using an integrated lead optimization strategy.

Structure-based drug design is an iterative process that is an established means to accelerate lead optimization, and is most powerful when integrated with information from different sources. Herein is described the use of such methods in conjunction with deconstruction and re-optimization of a diverse series of ASK1 chemotypes along with high-throughput screening that lead to the identification of a novel series of efficient ASK1 inhibitors displaying robust MAP3K pathway inhibition.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11ß-hydroxysteroid dehydrogenase type 1: strategies to eliminate reactive metabolites.

N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11ß-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure-activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11ß-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.

A novel antiandrogen, Compound 30, suppresses castration-resistant and MDV3100-resistant prostate cancer growth in vitro and in vivo.

Resistance to antiandrogen drugs, like MDV3100, occurs in patients with castration-resistant prostate cancer (CRPC). Thus, preventing or treating antiandrogen resistance is a major clinical challenge. We identified a novel antiandrogen, Compound 30, and compared its efficacy with MDV3100. We found that Compound 30 inhibits androgen receptor (AR) activity in LNCaP cells, C4-2 cells, as well as MDV3100-resistant cell lines. Compared with MDV3100, Compound 30 treatment induces greater reduction in AR, prostate-specific antigen (PSA), and AR transcriptional activity, and prevents AR nuclear translocation in AR-sensitive LNCaP cells. Compound 30 has antiproliferative effects in LNCaP cells, in castrate-resistant C4-2 cells, and those resistant to MDV3100. Compound 30 was equally as effective as MDV3100 in reducing tumor volume and PSA in vivo. More importantly, Compound 30 is effective at inhibiting AR activity in MDV3100-resistant cell lines and significantly prevented tumor growth and PSA increases in mice bearing MDV3100-resistant xenografts. Together, our data show that Compound 30 strongly inhibited AR activity and suppressed castration-resistant LNCaP growth as well as MDV3100-resistant cell growth in vitro and in vivo. These data provide a preclinical proof-of-principle that Compound 30 could be a promising next generation anti-AR agent, especially in the context of antiandrogen-resistant tumors.

Dose-dependent effects of small-molecule antagonists on the genomic landscape of androgen receptor binding.

BACKGROUND: The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery. RESULTS: Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor. CONCLUSIONS: Our study provides a global and dynamic view of AR's regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics.

From sequence to molecular pathology, and a mechanism driving the neuroendocrine phenotype in prostate cancer.

The current paradigm of cancer care relies on predictive nomograms which integrate detailed histopathology with clinical data. However, when predictions fail, the consequences for patients are often catastrophic, especially in prostate cancer where nomograms influence the decision to therapeutically intervene. We hypothesized that the high dimensional data afforded by massively parallel sequencing (MPS) is not only capable of providing biological insights, but may aid molecular pathology of prostate tumours. We assembled a cohort of six patients with high-risk disease, and performed deep RNA and shallow DNA sequencing in primary tumours and matched metastases where available. Our analysis identified copy number abnormalities, accurately profiled gene expression levels, and detected both differential splicing and expressed fusion genes. We revealed occult and potentially dormant metastases, unambiguously supporting the patients' clinical history, and implicated the REST transcriptional complex in the development of neuroendocrine prostate cancer, validating this finding in a large independent cohort. We massively expand on the number of novel fusion genes described in prostate cancer; provide fresh evidence for the growing link between fusion gene aetiology and gene expression profiles; and show the utility of fusion genes for molecular pathology. Finally, we identified chromothripsis in a patient with chronic prostatitis. Our results provide a strong foundation for further development of MPS-based molecular pathology.

Design of oxobenzimidazoles and oxindoles as novel androgen receptor antagonists.

Oxobenzimidazoles (e.g., 1), a novel series of androgen receptor (AR) antagonists, were discovered through de novo design guided by structure-based drug design. The compounds in this series were reasonably permeable and metabolically stable, but suffered from poor solubility. The incorporation of three dimensional structural features led to improved solubility. In addition, the observation of a 'flipped' binding mode of an oxobenzimidazole analog in an AR ligand binding domain (LBD) model, led to the design and discovery of the novel oxindole series (e.g., 2) that is a potent full antagonist of AR.

Discovery of 3-aryloxy-lactam analogs as potent androgen receptor full antagonists for treating castration resistant prostate cancer.

High throughput cell-based screening led to the identification of 3-aryloxy lactams as potent androgen receptor (AR) antagonists. Refinement of these leads to improve the ADME profile and remove residual agonism led to the discovery of 12, a potent full antagonist with greater oral bioavailability. Improvements in the ADME profile were realized by designing more ligand-efficient molecules with reduced molecular weights and lower lipophilicities.

Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists.

An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.

The development and SAR of pyrrolidine carboxamide 11beta-HSD1 inhibitors.

The design and development of a series of highly selective pyrrolidine carboxamide 11beta-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11beta-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11beta-HSD1 selective inhibitor 42.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11beta-hydroxysteroid dehydrogenase type 1: Discovery of PF-915275.

N-(Pyridin-2-yl) arylsulfonamides are identified as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), an enzyme that catalyzes the reduction of the glucocorticoid cortisone to cortisol. Dysregulation of glucocorticoids has been implicated in the pathogenesis of diabetes and the metabolic syndrome. In this Letter, we present the development of an initial lead to an efficient ligand with improved physiochemical properties using a deletion strategy. This strategy allowed for further optimization of potency leading to the discovery of the clinical candidate PF-915275.

Demonstration of proof of mechanism and pharmacokinetics and pharmacodynamic relationship with 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide (PF-915275), an inhibitor of 11 -hydroxysteroid dehydrogenase type 1, in cynomolgus monkeys.

Glucocorticoids, through activation of the glucocorticoid receptor (GR), regulate hepatic gluconeogenesis. Elevated hepatic expression and activity of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) play a key role in ligand-induced activation of the GR through the production of cortisol. Evidence from genetically modified mice suggests that inhibition of 11betaHSD1 might be a therapeutic approach to treat the metabolic syndrome. We have identified a potent 11betaHSD1 inhibitor, 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide (PF-915275), that is selective for the primate and human enzymes. The objective of this study was to demonstrate target inhibition with PF-915275 and to quantify the relationship between target inhibition and drug exposure in monkeys. We characterized the ability of PF-915275 to inhibit the conversion of prednisone, a synthetic cortisone analog that can be distinguished from the endogenous substrate cortisone, enabling a direct measure of substrate to product conversion without the complication of feedback. Adult cynomolgus monkeys were administered either vehicle or various doses of PF-915275 followed by a 10-mg/kg dose of prednisone. Prednisone conversion to prednisolone and the concentrations of PF-915275 were measured by liquid chromatography/tandem mass spectrometry. PF-915275 dose-dependently inhibited 11betaHSD1-mediated conversion of prednisone to prednisolone, with a maximum of 87% inhibition at a 3-mg/kg dose. An exposure-response relationship was demonstrated, with an estimated EC(50) of 391 nM (total) and 17 nM (free). Insulin levels were also reduced in a dose-related manner. These results should enable the development of a biomarker for evaluating target modulation in humans that will aid in identifying 11betaHSD1 inhibitors to treat diabetes and other related metabolic diseases.