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.

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.

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.

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.

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.

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.

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.

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.