Targeting Immune-Fibroblast Crosstalk in Myocardial Infarction and Cardiac Fibrosis.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1ß) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1ß signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1ß and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.

Malonyl CoA Decarboxylase Inhibition Improves Cardiac Function Post-Myocardial Infarction.

Alterations in cardiac energy metabolism after a myocardial infarction contribute to the severity of heart failure (HF). Although fatty acid oxidation can be impaired in HF, it is unclear if stimulating fatty acid oxidation is a desirable approach to treat HF. Both immediate and chronic malonyl coenzyme A decarboxylase inhibition, which decreases fatty acid oxidation, improved cardiac function through enhancing cardiac efficiency in a post-myocardial infarction rat that underwent permanent left anterior descending coronary artery ligation. The beneficial effects of MCD inhibition were attributed to a decrease in proton production due to an improved coupling between glycolysis and glucose oxidation.

Potent and Orally Bioavailable GPR142 Agonists as Novel Insulin Secretagogues for the Treatment of Type 2 Diabetes.

GPR142 is a G protein-coupled receptor that is predominantly expressed in pancreatic ß-cells. GPR142 agonists stimulate insulin secretion in the presence of high glucose concentration, so that they could be novel insulin secretagogues with reduced or no risk of hypoglycemia. We report here the optimization of HTS hit compound 1 toward a proof of concept compound 33, which showed potent glucose lowering effects during an oral glucose tolerance test in mice and monkeys.

Aminopyrazole-Phenylalanine Based GPR142 Agonists: Discovery of Tool Compound and in Vivo Efficacy Studies.

Herein, we report the lead optimization of amrinone-phenylalanine based GPR142 agonists. Structure-activity relationship studies led to the discovery of aminopyrazole-phenylalanine carboxylic acid 22, which exhibited good agonistic activity, high target selectivity, desirable pharmacokinetic properties, and no cytochrome P450 or hERG liability. Compound 22, together with its orally bioavailable ethyl ester prodrug 23, were found to be suitable for in vivo proof-of-concept studies. Compound 23 displayed good efficacy in a mouse oral glucose tolerance test (OGTT). Compound 22 showed GPR142 dependent stimulation of insulin secretion in isolated mouse islets and demonstrated a statistically significant glucose lowering effect in a mouse model bearing transplanted human islets.

Discovery and optimization of a novel series of GPR142 agonists for the treatment of type 2 diabetes mellitus.

The discovery and initial optimization of a series of phenylalanine based agonists for GPR142 is described. The structure-activity-relationship around the major areas of the molecule was explored to give agonists 90 times more potent than the initial HTS hit in a human GPR142 inositol phosphate accumulation assay. Removal of CYP inhibition by exploration of the pyridine A-ring is also described.

Phenylalanine derivatives as GPR142 agonists for the treatment of type II diabetes.

GPR142 is a novel GPCR that is predominantly expressed in pancreatic ß-cells. GPR142 agonists potentiate glucose-dependent insulin secretion, and therefore can reduce the risk of hypoglycemia. Optimization of our lead pyridinone-phenylalanine series led to a proof-of-concept compound 22, which showed in vivo efficacy in mice with dose-dependent increase in insulin secretion and a decrease in glucose levels.

GPR35 is a target of the loop diuretic drugs bumetanide and furosemide.

We report that the loop diuretic drugs bumetanide and furosemide used in the treatment of hypertension are GPR35 agonists. We utilized calcium flux, inositol phosphate accumulation, and dynamic redistribution assays to examine the pharmacology of these compounds on the human, mouse and rat GPR35. While potent on human GPR35, neither bumetanide nor furosemide were active against mouse or rat GPR35. Furthermore, the Na(+)-Cl(-) cotransporter inhibi- tors chlorothiazide and hydrochlorothiazide were inactive against GPR35 in all three species. We also demonstrate that GPR35 is expressed in human skin where it has been shown that loop diuretics inhibit histamine-induced flare and itch response. These findings suggest that GPR35 may play an important role in skin cell biology and be a potential target for the treatment of a variety of immune disorders.

High throughput screening for orphan and liganded GPCRs.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.

Prevention of polyurethane valve cusp calcification with covalently attached bisphosphonate diethylamino moieties.

OBJECTIVE: Calcification of polyurethane prosthetic valve leaflets causes a major functional impairment. Previously we showed that polyurethane heart valves modified with covalently linked bisphosphonate groups were resistant to calcification in vivo. However, we also found that the highly polar anionic bisphosphonate groups on the polyurethane surface attracted sodium counter ion adsorption, and thereby increased the elastomer's water absorption to 20% of total weight. In this study we address the increased water absorption by investigating the hypothesis that covalently attaching cationic diethylamino groups to the bisphosphonate-modified polyurethane will reduce water absorption. Thus we evaluated the mechanical and in vivo anticalcification properties of heart-valve leaflets composed of this modified polymer. METHODS: Diethylamino and bisphosphonate groups (DBP) were appended to the polyurethane Biospan's hard segment using previously published bromoalkylation methodology. Water absorption and biaxial mechanical and uniaxial failure testing were used to determine the mechanical properties of the DBP-modified polymer. Rat subdermal implants (60 days) and extended (150 days) single pulmonary leaflet replacements in juvenile sheep provided in vivo assessments of the bisphosphonate-modified polyurethane. RESULTS: The water absorption properties of the DBP-modified polymers and unmodified polyurethanes were 1.86 and 2.3 %, respectively. Biaxial mechanical tests showed the DBP-modified polymer was more compliant than the unmodified control material, but all polymeric material had similar uniaxial failure properties. In both rat subdermal and sheep circulatory implants, the DBP-modified polyurethane resisted calcification, as assessed by scanning electron microscopy, with complete calcification inhibition in prosthetic sheep valve leaflet replacements. CONCLUSION: DBP polyurethane possesses physical (water absorption) and biomechanical properties comparable to unmodified polyurethane and can resist intrinsic heart-valve leaflet calcification in blood-stream implants.

Thyrotoxicosis induced by alpha-interferon therapy in chronic viral hepatitis.

BACKGROUND AND OBJECTIVE: It has been well documented that treatment of chronic hepatitis B and C infection with interferon alpha (IFN-alpha) can lead to the induction of thyroid autoantibodies and hypothyroidism. Thyrotoxicosis, however, is less frequently observed and less well characterized. PATIENTS AND METHODS: We reviewed the medical records of patients who developed thyrotoxicosis while receiving IFN-alpha for either chronic hepatitis B or C infection at Westmead Hospital between 1996 and March 2001. RESULTS: Ten patients (four males and six females) were found to have biochemical thyrotoxicosis. The patients could be divided into two groups with different characteristics. The first group consisted of six patients who had clinical manifestations consistent with Graves' disease, with either diffuse uptake in thyroid scintigraphy and/or positive thyroid-stimulating antibodies. They all required prolonged treatment with antithyroid medications. The second group included three patients who had transient thyrotoxicosis, with progression to hypothyroidism after resolution of thyrotoxicosis. CONCLUSION: Our study suggests that IFN therapy may provoke two different forms of thyrotoxicosis: a Graves' disease picture or a biphasic thyroiditis pattern. The two entities should be differentiated, as they have different implications for treatment.