Marion Lanier

Small-Molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell–Derived Mesoderm

Rationale: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. Objective: Our aim was to develop a human embryonic stem cell–based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. Methods and Results: Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell–derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity. Conclusions: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.

Small Molecule-Mediated TGF-β Type II Receptor Degradation Promotes Cardiomyogenesis in Embryonic Stem Cells

The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle, and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a 1,4-dihydropyridine inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC(50) ~0.4-0.8 μM). ITD-1 was used to evaluate TGF-β involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-β inhibitor and reveals an unexpected role for TGF-β signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.

Identification of Novel, Water-Soluble, 2-Amino-N-pyrimidin-4-yl Acetamides as A2A Receptor Antagonists with In Vivo Efficacy

Potent adenosine hA2A receptor antagonists are often accompanied by poor aqueous solubility, which presents issues for drug development. Herein we describe the early exploration of the structure-activity relationships of a lead pyrimidin-4-yl acetamide series to provide potent and selective 2-amino-N-pyrimidin-4-yl acetamides as hA2A receptor antagonists with excellent aqueous solubility. In addition, this series of compounds has demonstrated good bioavailability and in vivo efficacy in a rodent model of Parkinsons disease, despite having reduced potency for the rat A2A receptor versus the human A2A receptor.

A Chemical Biology Approach to Myocardial Regeneration

Heart failure is one of the major causes of death in the Western world because cardiac muscle loss is largely irreversible and can lead to a relentless decline in cardiac function. Novel therapies are needed since the only therapy to effectively replace lost myocytes today is transplantation of the entire heart. The advent of embryonic and induced pluripotent stem cell (ESC/iPSC) technologies offers the unprecedented possibility of devising cell replacement therapies for numerous degenerative disorders. Not only are ESCs and iPSCs a plausible source of cardiomyocytes in vitro for transplantation, they are also useful tools to elucidate the biology of stem cells that reside in the adult heart and define signaling molecules that might enhance the limited regenerative capability of the adult human heart. Here, we review the extracellular factors that control stem cell cardiomyogenesis and describe new approaches that combine embryology with stem cell biology to discover drug-like small molecules that stimulate cardiogenesis and potentially contribute to the development of pharmaceutical strategies for heart muscle regeneration.

2-Amino-N-pyrimidin-4-ylacetamides as A2A Receptor Antagonists: 1. Structure−Activity Relationships and Optimization of Heterocyclic Substituents

Previously we have described a novel series of potent and selective A 2A receptor antagonists (e.g., 1) with excellent aqueous solubility. While these compounds are efficacious A 2A antagonists in vivo, the presence of an unsubstituted furyl moiety was a cause of some concern. In order to avoid the potential metabolic liabilities that could arise from an unsubstituted furyl moiety, an optimization effort was undertaken with the aim of replacing the unsubstituted furan with a more metabolically stable group while maintaining potency and selectivity. Herein, we describe the synthesis and SAR of a range of novel heterocyclic systems and the successful identification of a replacement for the unsubstituted furan moiety with a methylfuran or thiazole moiety while maintaining potency and selectivity.

Lead Optimization of 4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines as A2A Adenosine Receptor Antagonists for the Treatment of Parkinson’s Disease

4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinsons disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

Discovery of NBI-77860/GSK561679, a potent corticotropin-releasing factor (CRF1) receptor antagonist with improved pharmacokinetic properties.

Antagonists of the corticotropin-releasing factor (CRF) neuropeptide may prove effective in treating stress and anxiety related disorders. In an effort to identify antagonists with improved physico-chemical properties a new series of CRF(1) antagonists were designed to substitute the propyl groups at the C7 position of the pyrazolo[1,5-a]pyrimidine core of 1 with heterocycles. Compound (S)-8d was identified as a high affinity ligand with a pK(i) value of 8.2 and a functional CRF(1) antagonist with pIC(50) value of 7.0 in the in vitro CRF ACTH production assay.

Design and synthesis of selective inhibitors of Placental Alkaline Phosphatase

Placental Alkaline Phosphatase (PLAP) is a tissue-restricted isozyme of the Alkaline Phosphatase (AP) superfamily. PLAP is an oncodevelopmental enzyme expressed during pregnancy and in a variety of human cancers, but its biological function remains unknown. We report here a series of catechol compounds with great affinity for the PLAP isozyme and significant selectivity over other members of the AP superfamily. These selective PLAP inhibitors will provide small molecule probes for the study of the pathophysiological role of PLAP.

N-[6-amino-2-(heteroaryl)pyrimidin-4-yl]acetamides as A2A receptor antagonists with improved drug like properties and in vivo efficacy.

In the present article, we report on a strategy to improve the physical properties of a series of small molecule human adenosine 2A (hA2A) antagonists. One of the aromatic rings typical of this series of antagonists is replaced with a series of aliphatic groups, with the aim of disrupting crystal packing of the molecule to lower the melting point and in turn to improve the solubility. Herein, we describe the SAR of a new series of water-soluble 2,4,6-trisubstituted pyrimidines where R1 is an aromatic heterocycle, R2 is a short-chain alkyl amide, and the typical R3 aromatic heterocyclic substituent is replaced with an aliphatic amino substituent. This approach significantly enhanced aqueous solubility and lowered the log P of the system to provide molecules without significant hERG or CYP liabilities and robust in vivo efficacy.

Small molecule corticotropin-releasing factor antagonists

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide neurohormone that plays a major role in the body’s response to stress by modulating the endocrine, autonomic, behavioural and immune systems. The peptide interacts with two known receptors, CRF1 and CRF2, which belong to class B (secretin-like) G-protein-coupled receptors. Over the past ten years, a number of small molecule antagonists have been published in the patent literature. The present review covers recent patent literature (since 2000) where low molecular weight, non-peptide CRF antagonists are disclosed. These can be divided into four main classes of molecules: monocyclic, bicyclic and tricyclic compounds as well as a miscellaneous category, which include compounds that are unlike the traditional small molecule CRF1 antagonists.