Calcium channel blockers as potential therapeutics for obesity-associated autophagy defects and fatty liver pathologies

Nonalcoholic fatty liver disease (NAFLD), typically associated with overnutrition and obesity, is one of the most common liver diseases both in the US and worldwide. During obesity and NAFLD, lipotoxic injuries to hepatocytes can provoke formation of protein inclusions consisting of SQSTM1/p62 and ubiquitinated proteins. It has been suggested that autophagy deregulation during obesity contributes to protein inclusion formation and progression of other liver pathologies including insulin resistance, steatohepatitis, and hepatocellular carcinoma. To examine how lipotoxicity and obesity affect autophagy, we established an in vitro system where cultured HepG2 cells exhibit prominent accumulation of SQSTM1 and ubiquitinated proteins in insoluble inclusion bodies upon treatment with saturated fatty acids. Using this system and a mouse model of obesity, we have determined that obesity induces chronic elevation of cytosolic calcium levels in hepatocytes, which interferes with the fusion between autophagosomes and lysosomes. Intriguingly, pharmacological inhibition of calcium channels using the FDA-approved drug verapamil successfully restores autophagic flux and suppresses protein inclusions, not only in HepG2 cells but also in mouse liver. Verapamil also reduces hepatic lipid droplet accumulation, insulin resistance and steatohepatitis, suggesting that calcium channel blockers can be used for correction of general NAFLD pathologies. Indeed, there have been a number of clinical observations in which beneficial effects of calcium channel blockers against obesity-associated metabolic pathologies are observed in humans and animal models.     

Calcium channel blockers as potential therapeutics for obesity-associated autophagy defects and fatty liver pathologies

Nonalcoholic fatty liver disease (NAFLD), typically associated with overnutrition and obesity, is one of the most common liver diseases both in the US and worldwide. During obesity and NAFLD, lipotoxic injuries to hepatocytes can provoke formation of protein inclusions consisting of SQSTM1/p62 and ubiquitinated proteins. It has been suggested that autophagy deregulation during obesity contributes to protein inclusion formation and progression of other liver pathologies including insulin resistance, steatohepatitis, and hepatocellular carcinoma. To examine how lipotoxicity and obesity affect autophagy, we established an in vitro system where cultured HepG2 cells exhibit prominent accumulation of SQSTM1 and ubiquitinated proteins in insoluble inclusion bodies upon treatment with saturated fatty acids. Using this system and a mouse model of obesity, we have determined that obesity induces chronic elevation of cytosolic calcium levels in hepatocytes, which interferes with the fusion between autophagosomes and lysosomes. Intriguingly, pharmacological inhibition of calcium channels using the FDA-approved drug verapamil successfully restores autophagic flux and suppresses protein inclusions, not only in HepG2 cells but also in mouse liver. Verapamil also reduces hepatic lipid droplet accumulation, insulin resistance and steatohepatitis, suggesting that calcium channel blockers can be used for correction of general NAFLD pathologies. Indeed, there have been a number of clinical observations in which beneficial effects of calcium channel blockers against obesity-associated metabolic pathologies are observed in humans and animal models.     

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.     

Medicinal mushroom modulators of molecular targets as cancer therapeutics

Empirical approaches to discover anticancer drugs and cancer treatments have made limited progress in the past several decades in finding a cure for cancer. The expanded knowledge of the molecular basis of tumorigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in mushrooms, provided unique opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer. This review focuses on mushroom low-molecular-weight secondary metabolites targeting processes such as apoptosis, angiogenesis, metastasis, cell cycle regulation, and signal transduction cascades. Also discussed in this review are high-molecular-weight polysaccharides or polysaccharide–protein complexes from mushrooms that appear to enhance innate and cell-mediated immune responses, exhibit antitumor activities in animals and humans, and demonstrate the anticancer properties of selenium compounds accumulated in mushrooms.     

Pterostilbene fluorescent probes as potential tools for targeting neurodegeneration in biological applications

Several epidemiological studies suggest that a diet rich in fruit and vegetables reduces the incidence of neurodegenerative diseases. Resveratrol (Res) and its dimethylated metabolite, pterostibene (Ptb), have been largely studied for their neuroprotective action. The clinical use of Res is limited because of its rapid metabolism and its poor bioavailability. Ptb with two methoxy groups and one hydroxyl group has a good membrane permeability, metabolic stability and higher in vivo bioavailability in comparison with Res. The metabolism and pharmacokinetics of Ptb are still sparse, probably due to the lack of tools that allow following the Ptb destiny both in living cells and in vivo. In this contest, we propose two Ptb fluorescent derivatives where Ptb has been functionalised by benzofurazan and rhodamine-B-isothiocyanate, compounds 1 and 2, respectively. Here, we report the synthesis, the optical and structural characterisation of 1 and 2, and, their putative cytotoxicity in two different cell lines.     

Nanostructures as tailored biological probes

A new generation of spectroscopic dyes is gradually becoming available to biological researchers, from an unexpected source: materials chemists who study the synthesis and properties of nano-sized inorganic objects. Research into tailoring the optical properties, surface chemistry and biocompatibility of metallic and semiconductor nanoparticles, exemplified in part by a recent report by Mirkin, Schatz and coworkers, is fulfilling the promise of these nanostructures as customizable substitutes for organic molecular probes.     

Synthetic siderophores as biological probes

Summary Synthetic molecules that mimic the properties of the natural siderophores promise to become powerful tools in the exploration of microbial iron(III)-uptake phenomena. Such molecules can serve as probes to (i) establish the essential structural requirements for biological action, (ii) trace alternative reaction pathways and (iii) compare receptors of different biological origins. In this article a series of synthetic ferrichrome analogs will be described. The strategy adopted for the design and synthesis of these compounds will be outlined and their properties in vitro and in vivo examined. The growth promotion activity of these compounds inArthrobacter flavescens is used to map the ferrichrome receptor surface. Their activities towardsZea mays allow us to trace the plants' reductive iron(III) uptake routes. Potential applications of modified ferrichrome analogs for the isolation of ferrichrome receptors, the generation of fluorescent probes and ultimately new families of antibiotic or antifungal agents, will also be indicated.     

Prenylated xanthones as potential P-glycoprotein modulators

Dimethylallyl (DMA) derivatives of a naturally occurring xanthone (decussatin 1) were prepared. Their activity as potential P-glycoprotein inhibitors was monitored by affinity of direct binding and compared to that of corresponding DMA-flavones. Both classes of compounds exhibited the same structure-activity relationships. Decreasing polarity enhanced the binding affinity for the P-glycoprotein C-terminal cytosolic domain since DMA derivatives were more active, but unsubstituted hydroxyl group close to the carbonyl was required for efficient activity.     

New metal complexes as potential therapeutics

The many activities of metal ions in biology have stimulated the development of metal-based therapeutics. Cisplatin, as one of the leading metal-based drugs, is widely used in treatment of cancer, being especially effective against genitourinary tumors such as testicular. Significant side effects and drug resistance, however, have limited its clinical applications. Biological carriers conjugated to cisplatin analogs have improved specificity for tumor tissue, thereby reducing side effects and drug resistance. Platinum complexes with distinctively different DNA binding modes from that of cisplatin also exhibit promising pharmacological properties. Ruthenium and gold complexes with antitumor activity have also evolved. Other metal-based chemotherapeutic compounds have been investigated for potential medicinal applications, including superoxide dismutase mimics and metal-based NO donors/scavengers. These compounds have the potential to modulate the biological properties of superoxide anion and nitric oxide.     

Design, total synthesis, and biological evaluation of neodysiherbaine A derivative as potential probes

To enable studies to elucidate the detailed biological function of dysiherbaine and neodysiherbaine A, potent and subunit-selective agonists for ionotropic glutamate receptors, the derivative with a hydroxymethyl substituent at the C10 position has been developed. Preliminary biological evaluation of the analogue showed that a C10 hydroxymethyl substituent produced significant alterations in binding affinities for the ionotropic glutamate receptor subtypes.     

Abyssinones and related flavonoids as potential steroidogenesis modulators

Abyssinones and related flavonoids were screened against 3 enzymes (3βHSD, 17βHSD and Aromatase) of steroidogenesis pathway. The virtual screening experiment shows high affinity for flavonones than their respective chalcones. A 4'' ­OH blocked prenylated flavonone 2b (2­(2'', 2''­dimethyl chroman­6''­yl)­7­hydroxy chroman­4­one) had consistent binding affinity to all the three enzymes used in this study showing higher binding affinity to aromatase. A good correlation was observed between cytotoxic data (MCF­7, breast cancer cell line) and docking results indicating flavonone as a better steroidogenesis modulator in hormone dependent cancer.     

Natural and engineered ribonucleases as potential cancer therapeutics

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.     

Novel bifunctional natriuretic peptides as potential therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.     

Berberine as a potential autophagy modulator

Today, pharmacognosy is considered a valuable science in the prevention and treatment of diseases. Among herbals, Berberine is an isoquinoline alkaloid found in the Berberis species. Surprisingly, it shows antimicrobial, antiviral, antidiarrheal, antipyretic, and anti-inflammatory potential. Furthermore, it diminishes drug resistance in cancer therapy and enhances tumor suppression in part through autophagy and cell cycle arrest mechanisms. In the present review, we discuss the effect of berberine on diverse cellular pathways and describe how berberine acts as an autophagy modulator to adjust physiologic and pathologic conditions and diminishes drug resistance in cancer therapy.     

Anthranilic diamides derivatives as potential ryanodine receptor modulators: Synthesis,biological evaluation and structure activity relationship

A series of novel anthranilic diamides derivatives (7a–s) containing halogen, trifluoromethyl group and cyano group were designed, synthesized, and characterized by melting point, ¹H NMR, ¹³C NMR and elemental analyses. The bioactivity revealed that most of them showed moderate to excellent activities against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella). Above all, the larvicidal activity of 7o against oriental armyworm was 100% and 40% at 0.25 and 0.1?mg?L^?1, comparable to that of the standard chlorantraniliprole (100%, 0.25?mg?L^?1 and 20%, 0.1?mg?L^?1). What is more, 7o against diamondback moth displayed 90% insecticidal activity at 0.01?mg?L^?1, superior to chlorantraniliprole (45%, 0.01?mg?L^?1). The experiments 7o on the American cockroach (Periplaneta Americana) heart beating rates (Dorsal vessel) and contractile force were compared with chlorantraniliprole. In addition, 7o could affect the calcium homeostasis in the central neurons of the third larvae of oriental armyworm, which revealed that the ryanodine receptor is the potential target of 7o. The density functional theory (DFT) calculation results revealed the amide bridge, the benzene ring of anthraniloyl moiety and pyrazole ring might play an important role in the insecticidal activity through hydrophobic interactions and π-π conjugations.     

Astrocytes as potential modulators of mercuric chloride neurotoxicity

Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca²⁺-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.     

Developing microcolin A analogs as biological probes

Three microcolin A and B analogs have been synthesized. Their biological activity profiles were evaluated against several cell lines, revealing the existence of a structural determinant whose role in mediating the antiproliferative effect of the microcolins has heretofore gone unrecognized. While previously described as potent immunosuppressive natural products, we found that these microcolin analogs possessed no selective cytotoxicity when comparing immune cell versus nonimmune cell proliferation. In addition, the amino-terminus of microcolin A has been modified to incorporate a biotinylated polyethylene glycol linker. The biological activity of this biotinylated microcolin A analog was determined. This affinity reagent was shown to retain limited biological activity and thus can serve as a useful probe for elucidating the mechanism of action of microcolin A.