Search

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFβ signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFβ signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFβ/SMAD signaling.

This report describes the development of a non-genetic cell-surface modification method, in which a self-assembling small molecule is combined with Halo-tag proteins. Cell-surface functionalization with cancer-linked extracellular proteins led to enhanced cell motility, angiogenesis, and immune shielding of the cells, paving the way for translational opportunities for cell therapy.

Immune potentiators, termed adjuvant, trigger early innate immune responses to ensure the generation of robust and long‐lasting adaptive immune responses of vaccines. Here we present study that takes advantage of a self‐assembling small molecule library for the development of a novel vaccine adjuvant. Cell‐based screening of the library and subsequent structural optimization led to the discovery of a simple, chemically tractable deoxycholate derivative (molecule 6 , also named cholicamide) whose well‐defined nano‐assembly potently elicits innate immune responses in macrophages and dendritic cells. Functional and mechanistic analyses indicate that the virus‐like assembly is engulfed inside cells and stimulates the innate immune response through toll‐like receptor 7 (TLR7), an endosomal TLR that detects single‐stranded viral RNA. As an influenza vaccine adjuvant in mice, molecule 6 was as potent as Alum, a clinically used adjuvant. The studies described here paves the way for a new approach to discovering and designing self‐assembling small‐molecule adjuvants against pathogens, including emerging viruses., 自己集合性ワクチンアジュバントの発見. 京都大学プレスリリース. 2020-10-07., Vaccine ingredients could be hiding in small molecule libraries. 京都大学プレスリリース. 2020-10-07.

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl− conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

Most pancreatic ductal adenocarcinoma (PDAC) develops from pancreatic epithelial cells bearing activating mutant KRAS genes through precancerous lesions, i.e. acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). During pancreatic tumorigenesis, Hes1 expression starts with the transition from acinar cells to ADM, and continues during PanIN and PDAC formation, but the role of Hes1 in pancreatic tumorigenesis is not fully elucidated. Here we show that Hes1 plays an essential role in the initiation and progression of KRAS-driven pancreatic tumorigenesis. In vitro, activation of MAPK signaling due to EGF or mutant KRAS activation induced sustained Hes1 expression in pancreatic acinar cells. In vivo, acinar cell-specific activation of mutant KRAS by Elastase1-CreERT2;KrasG12D induced ADM/PanIN formation with Hes1 expression in mice, and genetic ablation of Hes1 in these mice dramatically suppressed PanIN formation. Gene expression analysis and lineage tracing revealed that Hes1 regulates acinar-to-ductal reprogramming-related genes and, in a Hes1-deficient state, mutant Kras-induced ADM could not progress into PanIN, but re-differentiated into acinar cells. In the Elastase1-CreERT2;KrasG12D;Trp53R172H mouse PDAC model, genetic ablation of Hes1 completely blocked PDAC formation by keeping PanIN lesions in low-grade conditions, in addition to reducing the occurrence of PanIN. Together, these findings indicate that mutant KRAS-induced Hes1 plays an essential role in PDAC initiation and progression by regulating acinar-to-ductal reprogramming-related genes.

Lactone-vitamin D3 is a major metabolite of vitamin D3, a lipophilic vitamin biosynthesized in numerous life forms by sunlight exposure. Although lactone-vitamin D3 was discovered 40 years ago, its biological role remains largely unknown. Chemical biological analysis of its photoaffinity probe identified the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a mitochondrial enzyme that catalyzes β-oxidation of long-chain fatty acids, as its selective binding protein. Intriguingly, the interaction of lactone-vitamin D3 with HADHA does not affect the HADHA enzymatic activity but instead limits biosynthesis of carnitine, an endogenous metabolite required for the transport of fatty acids into the mitochondria for β-oxidation. Lactone-vitamin D3 dissociates the protein-protein interaction of HADHA with trimethyllysine dioxygenase (TMLD), thereby impairing the TMLD enzyme activity essential in carnitine biosynthesis. These findings suggest a heretofore undescribed role of lactone-vitamin D3 in lipid β-oxidation and carnitine biosynthesis, and possibly in sunlight-dependent shifts of lipid metabolism in animals.

Fusicoccins (FCs) exhibit various cellular activities in mammalian cells, but details of the mechanism of action are not fully understood. In this study, we synthesized two pairs of model derivatives of FCs differing only in the presence and absence of a 12-hydroxyl group and evaluated their binding to a 14-3-3 protein together with various mode 1 and mode 3 phosphopeptide ligands. Our results demonstrate that the 12-hydroxyl group hampers binding to 14-3-3 with mode 1 phospholigands, presumably due to steric repulsion with the i+2 residue. Furthermore, cell-based evaluations showed that only non-substituted FCs exhibit significant cytotoxicity and all 12-hydroxyl derivatives were inactive, demonstrating a clear correlation with their ability to form ternary complexes with 14-3-3 and a mode 1 ligand. These results suggest that binding to 14-3-3 and a partner protein(s) possessing a mode 1 sequence plays a role in the mechanism of action of 12-non-substituted FCs.

Among numerous studies on synthetic approaches to and the biological activities of vitamin D analogues, we herein focused on falecalcitriol, an analogue of calcitriol (1α,25-dihydroxyvitamin D3), in which a 26,26,26,27,27,27-hexafluoroisopropanol unit has been introduced into the side chain. Falecalcitriol was designed to escape from the metabolism of CYP24A1 and has been used as a drug to treat secondary hyperparathyroidism since 2001. Its metabolite, the 23-hydroxy form, retains biological activity and resistants to further metabolism. Recent developments in synthetic methodologies for introducing the hexafluoroisopropanol unit into the vitamin D CD-ring side chain were described herein.

ATP7A encodes a copper-transporting P-type ATPase and is one of 23 genes in which mutations produce distal hereditary motor neuropathy (dHMN), a group of diseases characterized by length-dependent axonal degeneration of motor neurons. We have generated induced pluripotent stem cell (iPSC)-derived motor neurons from a patient with the p.T994I ATP7A gene mutation as an in vitro model for X-linked dHMN (dHMNX). Patient motor neurons show a marked reduction of ATP7A protein levels in the soma when compared to control motor neurons and failed to upregulate expression of ATP7A under copper-loading conditions. These results recapitulate previous findings obtained in dHMNX patient fibroblasts and in primary cells from a rodent model of dHMNX, indicating that patient iPSC-derived motor neurons will be an important resource for studying the role of copper in the pathogenic processes that lead to axonal degeneration in dHMNX., Summary: The authors describe a neuronal model to investigate how mutations in the copper transporter ATP7A cause axonal degeneration in the peripheral nervous system.

Background and purpose A second-generation antihistamine, terfenadine, is known to induce arrhythmia by blocking hERG channels. In this study, we have shown that terfenadine also inhibits the activity of G-protein-gated inwardly rectifying K+ (GIRK) channels, which regulate the excitability of neurons and cardiomyocytes. To clarify the underlying mechanism(s), we examined the effects of several antihistamines on GIRK channels and identified the structural determinant for the inhibition. Experimental approach Electrophysiological recordings were made in Xenopus oocytes and rat atrial myocytes to analyse the effects of antihistamines on various GIRK subunits (Kir 3.x). Mutagenesis analyses identified the residues critical for inhibition by terfenadine and the regulation of ion selectivity. The potential docking site of terfenadine was analysed by molecular docking. Key results GIRK channels containing Kir 3.1 subunits heterologously expressed in oocytes and native GIRK channels in atrial myocytes were inhibited by terfenadine and other non-sedating antihistamines. In Kir 3.1 subunits, mutation of Phe137, located in the centre of the pore helix, to the corresponding Ser in Kir 3.2 subunits reduced the inhibition by terfenadine. Introduction of an amino acid with a large side chain in Kir 3.2 subunits at Ser148 increased the inhibition. When this residue was mutated to a non-polar amino acid, the channel became permeable to Na+ . Phosphoinositide-mediated activity was also decreased by terfenadine. Conclusion and implications The Phe137 residue in Kir 3.1 subunits is critical for inhibition by terfenadine. This study provides novel insights into the regulation of GIRK channels by the pore helix and information for drug design.

A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.

Summary Understanding the molecular mechanisms that regulate β cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances β cell mass. Over time, islet β cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of β cells through increasing intracellular cAMP. DPD prevented β cell dedifferentiation, which together highly contributed to the increased β cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of β cells. Drd2 overexpression sensitized the dopamine responsiveness of β cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5′-N-ethylcarboxamidoadenosine, a previously identified promoter of β cell proliferation, acted with DPD to increase the number of β cells. In humans, dopamine also modulates β cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling., Graphical Abstract, Highlights • Dopamine inhibits dedifferentiation and proliferation, and promotes apoptosis of β cells • Dopamine confers its action through binding to DRD2 and decreasing cAMP • Dopamine-DRD2 signal also functions through interaction with adenosine-ADORA2A signal • Dopamine-DRD2-cAMP signal is a potential target for β cell regeneration, In this article, Kume and colleagues report on the identification of a dopamine D2 receptor (DRD2) antagonist as a promoter to increase β cell mass. Blockade of dopamine-DRD2 signal prevents dedifferentiation, increases proliferation, and decreases apoptosis of β cells. Dopamine-DRD2 signaling also negatively regulates other signaling pathways such as adenosine-ADORA2A signaling through heterodimer formation.

Myogenesis-promoting chemicals are an important source of new pharmaceuticals for the treatment of skeletal muscle atrophy that impairs quality of life. This report presents a robust and quantitative bioluminescence-based assay for screening myogenesis-promoting compounds in chemical libraries. The assay system consists of two stable C2C12 myoblast cell lines, each of which expresses either an N-terminal or a C-terminal split luciferase fragment fused to a naturally split DnaE intein as an indicator for cell fusion. Cell fusion during myogenesis induces bioluminescence in the cytosol because of the reconstitution of luciferases. The luminescence intensity quantitatively represents the progress in the cell fusion and therefore indicates the extent of myogenesis. We applied this assay system to a high-throughput screening of myogenesis-promoting compouns in 1191 pharmacologically proven bioactive small molecules, which revealed two chemical compounds as myogenesis-promoting compounds: Imatinib and Doxazosin mesylate. The assay system enabled a robust and quantitative evaluation of the extent of myogenesis through simple luminescence measurements, and is expected to be widely applicable for high-throughput screening of cell fusion-promoting and inhibiting molecules.

This report describes the design and evaluation of phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P), which selectively eliminates tumor-forming proliferative stem cells, including human induced pluripotent stem cells (hiPSCs) and neural stem cells, from iPSC-derived neural cell mixtures. Results of the present study demonstrate that simple phosphorylation of an anticancer drug can provide a safe, cost-effective, and chemically-defined tool for decontaminating hiPSC-derived neuron.

Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment-induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water-soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan-sulfate-bound syndecans, two classes of receptors that are important for extracellular matrix-mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan-4 and integrin β1 on the cell surface, leading to the activation of small GTPase Rac-1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster-inducing molecules may provide a starting point for the design of new synthetic tools for cell-based therapy.

SummaryA small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy.

Sterol regulatory element-binding proteins (SREBPs) are transcriptional factors that control lipid and cholesterol metabolism. Activation of SREBPs in response to a decrease in cellular sterols results in acceleration of the synthesis of fatty acids, triglycerides, and cholesterol. Aberrant SREBP activity has been linked to metabolic disease states, such as obesity, fatty liver, insulin resistance, hyperlipidemia, and atherosclerosis. Thus, inhibition of SREBP activation is a potential therapeutic approach to treating metabolic disorders. This review focuses on direct or indirect small-molecule inhibitors of SREBP activation.

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy.

SummaryHuman pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identified two hit compounds that selectively and partially downregulate SOD1 expression in a dose-dependent manner, without any detectable cellular toxicity. Western blot analysis showed that one hit compound significantly decreased the level of endogenous SOD1 protein in H4 cells, with no reduction in expression of β-actin. The assay developed here provides a powerful strategy for discovering novel lead molecules for treating familial SOD1-mediated ALS.

Received: July 9, 2010 , Accepted: July 30, 2010 , Published: August 6, 2010 // The membrane-anchored matrix metalloproteinase-regulator RECK is often downregulated in various types of cancers; the levels of residual RECK in resected tumors often correlate with better prognosis. Forced expression of RECK in cancer cells suppresses tumor angiogenesis, invasion, and metastasis in xenograft models. RECK is therefore a promising marker for benignancy and a potential effector in cancer therapy. We established a cell line containing two transgene systems: (1) the secreted alkaline phosphatase (SEAP) gene fused to Reck promoter and (2) the HRAS12V oncogene driven by the Tet-off promoter system. This cell line exhibits transformed phenotype in regular medium and flat morphology with increased SEAP activity in the presence of doxycycline, allowing the assessment of RECK-inducing activity of chemicals in the contexts of both transformed and untransformed cells. Our pilot experiments with 880 known bioactive compounds detected 34 compounds that activate RECK promoter; among these, 10 were authentic anticancer drugs. Four selected compounds up-regulated endogenous RECK protein in several human cancer cell lines. The top-ranking compound, disulfiram, strongly suppressed spontaneous lung-metastasis of human fibrosarcoma cells in nude mice. Our data demonstrate the value of this screen in discovering effective cancer therapeutics.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family that is expressed primarily on sensory neurons. This chemosensor is activated through covalent modification of multiple cysteine residues with a wide range of reactive compounds including allyl isothiocyanate (AITC), a spicy component of wasabi. The present study reports on potent and selective agonists of TRPA1, discovered through screening 1657 electrophilic molecules. In an effort to validate the mode of action of hit molecules, we noted a new TRPA1-selective agonist, JT010 (molecule 1), which opens the TRPA1 channel by covalently and site-selectively binding to Cys621 (EC50 = 0.65 nM). The results suggest that a single modification of Cys621 is sufficient to open the TRPA1 channel. The TRPA1-selective probe described herein might be useful for further mechanistic studies of TRPA1 activation.

SummaryInsulin-like growth factor 2 (IGF2) is a potent mitogen whose deregulation plays a role in developing liver, breast, and prostate cancers. Here, we take a small-molecule approach to investigate molecular pathways that modulate IGF2 signaling, by using chromeceptin, a synthetic molecule that selectively impairs the viability and growth of IGF2-overexpressing hepatocellular carcinoma cells. Affinity purification revealed that chromeceptin binds to multifunctional protein 2 (MFP-2), a seemingly multifunctional enzyme implicated in peroxisomal β-oxidation. The small molecule-protein interaction stimulates the expression of IGF binding protein 1 (IGFBP-1) and suppressor of cytokine signaling-3 (SOCS-3), two cellular attenuators of the IGF signals, through activation of signal transducers and activators of transcription 6 (STAT6). The results underline the importance of STATs in IGF/insulin regulation, and they implicate a new pathway for STAT6 activation that is amenable to small-molecule intervention.

An important step in the postgenomic drug discovery is the construction of high quality chemical libraries that generate bioactive molecules at high rates. Here we report a cell-based approach to composing a focused library of biologically active compounds. A collection of bioactive non-cytotoxic chemicals was identified from a divergent library through the effects on the insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic and sensitive morphological alterations in cultured mammalian cells. The resulting focused library amply contained unique compounds with a broad range of pharmacological effects, including glucose-uptake enhancement, cytokine inhibition, osteogenesis stimulation, and selective suppression of cancer cells. Adipogenesis profiling of organic compounds generates a focused chemical library for multiple biological effects that are seemingly unrelated to adipogenesis, just as genetic screens with the morphology of fly eyes identify oncogenes and neurodegenerative genes.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

One of the current obstacles to stem cell therapy is the tumorigenic potential of residual undifferentiated stem cells. The present study reports rediscovery of a synthetic derivative of okadaic acid, a marine polyether toxin, as a reagent that selectively induces the death of human pluripotent stem cells. Cell-based screening of 333 cytotoxic compounds identified methyl 27-deoxy-27-oxookadaate (molecule 1) as a substrate of two ATP-binding cassette (ABC) transporters, ABCB1 (MDR1) and ABCG2 (BCRP), whose expression is repressed in human embryonic stem cells and induced pluripotent stem cells. The results demonstrate that selective elimination of human pluripotent stem cells can be achieved by designing cytotoxic small molecules with appropriate ABC-transporter selectivity.

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)–mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment. Cancer Res; 73(14); 4559–70. ©2013 AACR.

Natural products provide a rich source of biological tools, but elucidating their molecular targets remains challenging. Here we report a cell morphological profiling of a natural product library, which permitted the identification of bisebromoamide and miuraenamide A as actin filament stabilizers. Automated high-content image analysis showed that these two structurally distinct marine natural products induce morphological changes in HeLa cells similar to those induced by known actin-stabilizing compounds. Bisebromoamide and miuraenamide A stabilized actin filaments in vitro, and fluorescein-conjugated bisebromoamide localized specifically to actin filaments in cells. Cell morphological profiling was also used to identify actin-stabilizing or -destabilizing natural products from marine sponge extracts, leading to the isolation of pectenotoxin-2 and lyngbyabellin C. Overall, the results demonstrate that high-content imaging of nuclei and cell shapes offers a sensitive and convenient method for detecting and isolating molecules that target actin.

A designed hybrid (1) of neocarzinostatin chromophore model and netropsin-type minor groove binder was synthesized and its improved ability of DNA cleavage has been demonstrated.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.

Elevated serum levels of insulin-like growth factor 1 (IGF1) have been found in prostate cancer patients, and IGF1-related signal transduction is thought to be an important factor in the development of prostate cancers. The goals of this project are to discover small organic molecules that suppress ICE-activated prostate cancers by cell-based screening and to analyze their action mechanisms. In the first year of funding, we discovered, from our collection of synthetic compounds, the drug-like compound we call 125B11 that suppress IGE1-dependent growth of prostate cancer cells but not serum-dependent growth. During this period of funding, we analyzed the mechanism of action of 125B11 to gain molecular insights into how IGE1 stimulates the growth of prostate cancer cells. DNA microarray and cell biological experiments indicated that 125B11 modulates the function of sterol regulatory element binding protein (SREBP), a transcription factor that activates specific genes involved in cholesterol synthesis, endocytosis of low density lipoproteins, and the synthesis of both saturated and unsaturated fatty acids. Chemical genetics of 125B11 may reveal a novel crosstalk between fatty acid metabolism and prostate cancer progression.

External control of gene expression by small synthetic molecules represents a challenge in chemistry. Here we discuss the synthetic aspects of small molecule transcription factor mimics including a molecule we call STF1 (1). STF1 stimulates the transcription of a reporter gene controlled by specific DNA elements and behaves just as a naturally occurring transcription factor. However, STF1 had limited cell permeability, even though the hairpin-polyamide-FITC conjugate and the wrenchnolol (2) are cell permeable as separate compounds. Systematic synthetic exploration of STF1 analogs (STFs), by optimizing the physical properties of the molecule, is expected to increase its cell permeability for biological studies.

Activation domains are functional modules that enable sequence-specific DNA binding proteins to stimulate transcription. The structural basis for the function of activation domains is poorly understood. A combination of nuclear magnetic resonance (NMR) and biochemical experiments revealed that the minimal acidic activation domain of the herpes simplex virus VP16 protein undergoes an induced transition from random coil to α helix upon binding to its target protein, hTAF II 31 (a human TFIID TATA box – binding protein-associated factor). Identification of the two hydrophobic residues that make nonpolar contacts suggests a general recognition motif of acidic activation domains for hTAF II 31.

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.