Search

Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca 2+ -ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins., (© 2024. The Author(s).)

As the global health crisis due to evolution of mutations in SARS-CoV-2 continues, it is important to develop several effective antivirals to control the disease. Targeting papain-like protease (PLpro) of SARS-CoV-2 for drug development is a promising strategy due to its dual role in promoting viral replication and dysregulating host immune responses. Here, we screened a library of compounds to find potential inhibitors of PLpro. We find aurintricarboxylic acid (ATA) inhibits PLpro with K i and IC 50 values of 16 μM and 30 μM, respectively. The binding of ATA to PLpro was further characterized using isothermal titration calorimetry, differential scanning fluorimetry, dynamic light scattering and circular dichroism spectrometry. In vitro assays showed the antiviral potential of ATA with IC 50 of 50 μM. In vivo efficacy was studied in Syrian hamsters and the results are being discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Cardiac hypertrophy is regarded as a compensation mechanism to overcome the increased workload. Aurintricarboxylic acid (ATA) is a derivative of quinomethanes and a selective inhibitor of TWEAK/Fn14 pathway. In this study, we investigated the effect of ATA on isoproterenol (ISO)-induced pathological cardiac hypertrophy., Methods: Cardiac hypertrophy in H9C2 cells was induced using ISO 20 μM dissolved in PBS. H9C2 cells were treated with ATA (5 µM, 10 µM, 20 µM) followed by ISO stimulation for 24 h. Male SD rats were injected ISO (5 mg/kg/day, s.c) for 21 days and followed by treatment with ATA (10 mg/kg, i.p.) for 14 days. Cardiac functions were assessed. After sacrifice, hearts were subjected to histopathological and western blot analysis., Results: In in-vitro results, upon ATA treatment, ICC results showed significant decrease in TWEAK and ANP expression. In in-vivo results, echocardiography showed significant restoration of cardiac function in ATA treated rats. Histopathological analysis showed a significant decrease in left ventricular wall thickness, cardiomyocytes width and reduced fibrosis in ATA treated rats. Western blotting showed decreased expression of the cardiac hypertrophy maker ANP, inflammatory markers including TWEAK and apoptotic markers after ATA treatment., Conclusion: These findings suggested that the TWEAK/Fn14 pathway could be a potential target for therapeutic exploration in ISO induced cardiac hypertrophy. ATA, as an inhibitor of this pathway, exerted significant cardioprotective effect against ISO-induced cardiac hypertrophy in rats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pathologic expansions of DNA nucleotide tandem repeats may generate toxic RNA that triggers disease phenotypes. RNA toxicity is the hallmark of multiple expansion repeat disorders, including myotonic dystrophy type 1 (DM1). To date, there are no available disease-modifying therapies for DM1. Our aim was to use drug repositioning to ameliorate the phenotype of affected individuals in a nematode model of DM1. As the RNA interference pathway plays a key role in mediating RNA toxicity, we investigated the effect of aurintricarboxylic acid. We demonstrated that by perturbing the RNA interference machinery using aurintricarboxylic acid, we could annihilate the RNA toxicity and ameliorate the phenotype. As our approach targets a universal disease mechanism, it is potentially relevant for more expansion repeat disorders.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death ( p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19., (© 2021 The Author(s).)

Endothelial cell (EC) dysfunction is the underlying cause for the development of several pathologies, and the interdependency between the pancreatic β-cells and ECs has been established in the pathophysiology of diabetes. ECs release several factors that govern the expression of genes involved in the proliferation, physiology, and survival of the β-cells. Of the known factors that collapse this intricately balanced system, endothelial dysfunction is the crucial condition that manifests as the causative factor for micro and macrovascular diseases. Our earlier studies demonstrated that activation of nuclear factor erythroid-related factor (Nrf2) renders protection to the ECs experiencing ER stress. In this study, using a co-culture system, the crosstalk between pancreatic cells under ER stress and ECs and the effect of a novel Nrf2 activator Rosolic Acid (RA), on the crosstalk was investigated. ECs pre-treated with different concentrations RA and co-cultured with thapsigargin-induced ER stressed pancreatic β-cells showed increased levels of Nrf2 and its downstream targets such as heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase-1 (NQO-1), and reduction of ER stress evinced by the decreased levels of glucose-regulated protein (GRP) 78 and C/ERB homologous protein (CHOP). The sensitization of ECs using RA, offered protection to pancreatic cells against ER stress as displayed by increased intracellular insulin and upregulated expression of cell survival and proliferative genes BCl2 and PDX-1. In addition, RA treatment resulted in elevated levels of various angiogenic factors, while inflammatory (TNF-α and IL-1β) and apoptotic markers (CXCL10 and CCL2) decreased. RA treatment normalized the levels of 115 proteins of the 277, which were differentially regulated as revealed by proteomic studies of ER stressed pancreatic β-cells in co-culture conditions. These findings clearly indicate the role of small molecule activators of Nrf2 not only in restoring the functioning of pancreatic cells but also in increasing the cell mass. Further, the study impinges on the strategies that can be developed to balance the pancreatic microenvironment, leading to the restoration of β-cell mass and their normophysiology in diabetic patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC ® 1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases., Competing Interests: We declare that we have no competing financial interests and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interests of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, the manuscript., (Copyright © 2021 Karan Naresh Amin et al.)

Objective: Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model., Design: Cartilage explants were subjected to blunt impact (0.59 J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level., Results: Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma., Conclusions: Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration-response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC 50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC 50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [ 3 H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration-response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (T con s ) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor T cons with cosalane ex vivo before transplantation. Cosalane did not result in T con toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor T con trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor T con s to produce inflammatory cytokines. Furthermore, cosalane-treated T con s retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor T con s before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

We identify three submicromolar inhibitors with new chemical scaffolds for cystathionine γ-lyase (CSE) by a tandem-well-based high-throughput assay. NSC4056, the most potent inhibitor with an IC 50 of 0.6 μM, which is also known as aurintricarboxylic acid, selectively binds to Arg and Tyr residues of CSE active site and preferably inhibits the CSE activity in cells rather than cystathionine β-synthase (CBS), the other H 2 S-generating enzyme. Moreover, NSC4056 effectively rescues hypotension in hemorrhagic shock rats.

CC-chemokine receptor 7 (CCR7) is a G protein-coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available β-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 μM, 5.72 μM and 1.18 mM, respectively., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild-type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild-type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-l-NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Rapidly-dividing cancer cells have higher requirement for iron compared to non-transformed cells, making iron chelating a potential anticancer strategy. In the present study we compared the anticancer activity of uncommon iron chelator aurintricarboxylic acid (ATA) with the known deferoxamine (DFO)., Materials and Methods: We investigated the impact of ATA and DFO on the viability and proliferation of MCF-7 cancer cells. Moreover we performed enzymatic activity assays and computational analysis of the ATA and DFO effects on pro-oncogenic phosphatases PTP1B and SHP2., Results: ATA and DFO decrease the viability and proliferation of breast cancer cells, but only ATA considerably reduces the activity of PTP1B and SHP2 phosphatases. Our studies indicated that ATA strongly inactivates and binds in the PTP1B and SHP2 active site, interacting with arginine residue essential for enzyme activity., Conclusion: We confirmed that iron chelating can be considered as a potential strategy for the adjunctive treatment of breast cancer., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aims: Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis., Methods and Results: We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs., Conclusions: Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA., Competing Interests: The authors have declared that no competing interests exist.

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability., Competing Interests: The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

The increasing threats of antibiotic resistance urge the need for developing new strategies against bacterial infections. Targeting eukaryotic-like Ser/Thr phosphatase Stp1-mediated virulence regulation represents a promising approach for combating staphylococcal infection yet to be explored. Here, we report the 2.32-Å resolution crystal structure of Stp1. Stp1 binds an unexpected fourth metal ion, which is important for Stp1's enzymatic activity as demonstrated by amino acid substitution studies. Inspired by the structural details of Stp1, we identified a potent and selective Stp1 inhibitor, aurintricarboxylic acid (ATA). Transcriptome analysis and biochemical studies supported Stp1 as the target of ATA inhibition within the pathogen, preventing upregulation of virulence genes. Notably, ATA did not affect in vitro growth of Staphylococcus aureus, while simultaneously attenuating staphylococcal virulence in mice. Our findings demonstrate that ATA is a potent anti-virulence compound against staphylococcal infection, laying the foundation for further developing new scaffolds for Stp1-targeted small molecules., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

Bis-naphthalene macrocycles, which bind with high affinity and selectivity to abasic sites in DNA, efficiently inhibit their cleavage by APE1 (IC50 = 55-60 nM in the kinetic assay with a model THF substrate). These results demonstrate that substrate masking by non-covalent abasic-site ligands is an efficient strategy for inhibition of APE1.

YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from -2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue.

Background: The membrane attack complex (MAC) is a key player in the pathogenesis of age-related macular degeneration (AMD) and is a putative activator of the NLRP3 inflammasome. Amyloid beta (Aβ), a component of drusen deposits, has also been implicated in inflammasome activation by our work and those of others. However, the interactions of MAC and Aβ are still poorly understood, especially their roles in aging and retinal degenerative pathologies. Since inflammasome activation may represent a key cellular pathway underlying age-related chronic inflammation in the eye, the purpose of this study is to identify the effects associated with MAC and inflammasome activation in the retinal pigment epithelium (RPE)/choroid and to evaluate the therapeutic merits of MAC suppression., Methods: Adult Long-Evans rats were divided into treatment and control groups. Treatment groups received oral aurin tricarboxylic acid complex (ATAC), a MAC inhibitor, in drinking-water, and control groups received drinking-water alone (No ATAC). Groups were sacrificed at 7.5 or 11.5 months, after approximately 40 days of ATAC treatment. To study age-related changes of Aβ and MAC in RPE/choroid, naive animals were sacrificed at 2.5, 7.5, and 11.5 months. Eye tissues underwent immunohistochemistry and western blot analysis for MAC, Aβ, NF-κB activation, as well as cleaved caspase-1 and IL-18. Vitreal samples were collected and assessed by multiplex assays for secreted levels of IL-18 and IL-1β. Statistical analyses were performed, and significance level was set at p ≤ 0.05., Results: In vivo studies demonstrated an age-dependent increase in MAC, Aβ, and NF-κB activation in the RPE/choroid. Systemic ATAC resulted in a prominent reduction in MAC formation and a concomitant reduction in inflammasome activation measured by cleaved caspase-1 and secreted levels of IL-18 and IL-1β, but not in NF-κB activation. In vitro studies demonstrated Aβ-induced MAC formation on RPE cells., Conclusions: Age-dependent increases in Aβ and MAC are present in the rodent outer retina. Our results suggest that suppressing MAC formation and subsequent inflammasome activation in the RPE/choroid may reduce chronic low-grade inflammation associated with IL-18 and IL-1β in the outer retina.

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5' end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a Kd= 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5'-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z' of 0.83 and was successfully used to screen a library of known bioactives., (© 2014 Society for Laboratory Automation and Screening.)

Great efforts have been undertaken in the last decades to develop new oximes to reactivate acetylcholinesterase inhibited by organophosphorus compounds (OP). So far, a broad-spectrum oxime effective against structurally diverse OP is still missing, and alternative approaches, e.g. stoichiometric and catalytic scavengers, are under investigation. Fresh frozen plasma (FFP) has been used in human OP pesticide poisoning which prompted us to investigate the in vitro kinetics of OP nerve agent degradation by FFP. Degradation was rapid and calcium-dependent with the G-type nerve agents tabun, sarin, soman and cyclosarin with half-lives from 5 to 28 min. Substantially longer and calcium-independent degradation half-lives of 23-33 h were determined with the V-type nerve agents CVX, VR and VX. However, at all the tested conditions, the degradation of V-type nerve agents was several-fold faster than spontaneous hydrolysis. Albumin did not accelerate the degradation of nerve agents. In conclusion, the fast degradation of G-type nerve agents by FFP might be a promising tool, but would require transfusion shortly after poisoning. FFP does not seem to be suitable for detoxifying relevant agent concentrations in case of human poisoning by V-type nerve agents.

Objectives: Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage., Design and Methods: We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera., Results: We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes., Conclusions: We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.

Purpose: Immunocytochemical and genetic data implicate a significant role for the activation of complement in the pathology of AMD. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and RPE relative to individuals homozygous for the wild-type allele. An R95X polymorphism in C9, a protein necessary for the final assembly of MAC, is partially protective against the formation of choroidal neovascularization (CNV) in AMD patients. Aurintricarboxylic Acid (ATA) is a small molecule inhibitor of MAC. Our hypothesis was that attenuation of the formation of MAC on ocular tissues by ATA may protect mice against laser-induced CNV., Methods: The ability of ATA to inhibit human complement-mediated cell lysis, inhibit formation of human MAC, and inhibit formation of tubes by endothelial cells was examined in vitro. Subsequently, the Bruch's membrane of adult mice was damaged using an argon laser, followed by intravitreal injection of ATA. One week later, choroidal flat mounts from these mice were stained for the presence of MAC, endothelial cells, and macrophages., Results: ATA protects cells from human complement-mediated lysis, attenuates assembly of the MAC, and inhibits tube formation by endothelial cells in vitro. ATA also attenuates CNV, MAC deposition, and macrophage infiltration in a murine model of exudative AMD., Conclusions: ATA warrants further study as a potential drug for the treatment of exudative and nonexudative AMD.

Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4(mut)GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4(mut)GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4(mut)GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart., (© 2013.)

Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.

Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Overactivation of CD4(+) T cells, especially the Th1 and Th17 subpopulations, is thought to be the direct cause of this disease. Aurintricarboxylic acid (ATA), an inhibitor of protein-nucleic acid interaction, has been reported to block with the JAK/STAT signaling pathway that is critical for Th cell differentiation. In this study, we discovered that ATA treatment significantly reduces the clinical score of EAE, but it does not directly inhibit the differentiation of Th1 and Th17 cells in vitro. ATA was found to block the chemotaxis and accumulation of dendritic cells in the spleen of EAE mice before the onset of the disease and to reduce the percentage of Th1 and Th17 cells in the spleen. Further study revealed that ATA also blocks the infiltration of pathogenic T cells into the CNS and blocks the onset of passive EAE. ATA was found to inhibit the functions of many chemokine receptors. By blocking chemokine-mediated migration of dendritic cells and pathogenic T cells, ATA alleviates the pathogenesis of EAE and might be used to treat autoimmune diseases, including multiple sclerosis.

Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 μg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 μg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Complement plays a vital role in both the innate and adaptive immune systems. It recognizes a target, opsonizes it, generates anaphylatoxins, and directly kills cells through the membrane attack complex (MAC). This final function, which assembles C5b-9(n) on viable cell surfaces, can kill host cells through bystander lysis. Here we identify for the first time compounds that can inhibit bystander lysis while not interfering with the other essential functions of complement. We show that aurin tricarboxylic acid (ATA), aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block the addition of C9 to C5b-8 so that the MAC cannot form. These molecules inhibit hemolysis of human, rat, and mouse red cells with a half maximal inhibitory concentration (IC(50)) in the nanomolar range. When given orally to Alzheimer disease type B6SJL-Tg mice, they inhibit MAC formation in serum and improve memory retention. On autopsy, they show no evidence of harm to any organ. Aurin tricarboxylic acid, aurin quadracarboxylic acid, and aurin hexacarboxylic acid may be effective therapeutic agents in Alzheimer disease and other degenerative disorders where self damage from the MAC occurs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA methyltransferase 1 (DNMT1) is an emerging target for the treatment of cancer, brain disorders, and other diseases. Currently, there are only a few DNMT1 inhibitors with potential application as therapeutic agents or research tools. 5,5-Methylenedisalicylic acid is a novel scaffold previously identified by virtual screening with detectable although weak inhibitory activity of DNMT1 in biochemical assays. Herein, we report enzyme inhibition of a structurally related compound, trimethylaurintricarboxylic acid (NSC97317) that showed a low micromolar inhibition of DNMT1 (IC(50) = 4.79 μM). Docking studies of the new inhibitor with the catalytic domain of DNMT1 suggest that NSC97317 can bind into the catalytic site. Interactions with amino acid residues that participate in the mechanism of DNA methylation contribute to the binding recognition. In addition, NSC97317 had a good match with a structure-based pharmacophore model recently developed for inhibitors of DNMT1. Trimethylaurintricarboxylic acid can be a valuable biochemical tool to study DNMT1 inhibition in cancer and other diseases related to DNA methylation.

c-Met [the hepatocyte growth factor (HGF) receptor] is a receptor tyrosine kinase playing a role in various biological events. Overexpression of the receptor has been observed in a number of cancers, correlating with increased metastatic tendency and poor prognosis. Additionally, activating mutations in c-Met kinase domain have been reported in a subset of familial cancers causing resistance to treatment. Receptor trafficking, relying on the integrity of the microtubule network, plays an important role in activation of downstream targets and initiation of signalling events. Aurintricarboxylic acid (ATA) is a triphenylmethane derivative that has been reported to inhibit microtubule motor proteins kinesins. Additional reported properties of this inhibitor include inhibition of protein tyrosine phosphatases, nucleases and members of the Jak family. Here we demonstrate that ATA prevents HGF-induced c-Met phosphorylation, internalisation, subsequent receptor trafficking and degradation. In addition, ATA prevented HGF-induced downstream signalling which also affected cellular function, as assayed by collective cell migration of A549 cells. Surprisingly, the inhibitory effect of ATA on HGF-induced phosphorylation and signalling in vivo was associated with an increase in basal c-Met kinase activity in vitro. It is concluded that the inhibitory effects of ATA on c-Met in vivo is an allosteric effect mediated through the kinase domain of the receptor. As the currently tested adenosine triphosphate competitive tyrosine kinase inhibitors (TKIs) may lead to tumor resistance (McDermott U, et al., Cancer Res 2010;70:1625-34), our findings suggest that novel anti-c-Met therapies could be developed in the future for cancer treatment., (Copyright © 2011 UICC.)

DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel anticancer drugs and other diseases. Molecular modeling and experimental approaches are being used to identify and develop inhibitors of human DNMTs. Most of the computational efforts conducted so far with DNMT1 employ homology models of the enzyme. Recently, a crystallographic structure of the methyltransferase domain of human DNMT1 bound to unmethylated DNA was published. Following on our previous computational and experimental studies with DNMTs, we herein present molecular dynamics of the crystal structure of human DNMT1. Docking studies of established DNMT1 inhibitors with the crystal structure gave rise to a structure-based pharmacophore model that suggests key interactions of the inhibitors with the catalytic binding site. Results had a good agreement with the docking and pharmacophore models previously developed using a homology model of the catalytic domain of DNMT1. The docking protocol was able to distinguish active DNMT1 inhibitors from, for example, experimentally known inactive DNMT1 inhibitors. As part of our efforts to identify novel inhibitors of DNMT1, we conducted the experimental characterization of aurintricarboxylic acid (ATA) that in preliminary docking studies showed promising activity. ATA had a submicromolar inhibition (IC(50)=0.68 μM) against DNMT1. ATA was also evaluated for Dnmt3a inhibition showing an IC(50)=1.4 μM. This chapter illustrates the synergy from integrating molecular modeling and experimental methods to further advance the discovery of novel candidates for epigenetic therapies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.

Activation of the vascular endothelium and increased adhesion of circulating leukocytes to the activated endothelium are important events in inflammation and coagulation. Aurintricarboxylic acid (ATA), a triphenylmethyl dye compound, is known to inhibit platelet adhesion by interfering with the binding of von Willebrand factor to platelet glycoprotein Ib. However, the effect of ATA on the inflammatory response of endothelial cells has not yet been investigated. Here, we investigated the functional role and molecular mechanism of ATA on the activation of human endothelial cells. ATA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1), and endothelial cell selectin (E-selectin) was upregulated on human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-α or lipopolysaccharide (LPS). We also observed the inhibitory effect of ATA on LPS-induced mRNA expression of ICAM-1 and E-selectin. Furthermore, ATA inhibited the binding of leukocytes to activated HUVECs. ATA significantly inhibited the nuclear translocation of nuclear factor-κB (NF-κB) and degradation of IκB on activated HUVECs, suggesting that ATA inhibits NF-κB signaling. Finally, three NF-κB inhibitors effectively inhibited the expressions of ICAM-1 and E-selectin on activated endothelial cells. The present data suggest that ATA exerts beneficial effect in various inflammation conditions through inhibition of adhesion molecule expression in activated endothelial cells and the resulting inhibition of leukocytes tissue accumulation.

In this paper, the nature of the interactions between bovine serum albumin (BSA) and aurintricarboxylic acid (ATA) has been investigated by measuring steady state and time-resolved fluorescence, circular dichroism (CD), FT-IR and fluorescence anisotropy in protein environment under physiological conditions. From the analysis of the steady state and time-resolved fluorescence quenching of BSA in aqueous solution in presence of ATA it has been inferred that the nature of the quenching originates from the combined effect of static and dynamic modes. From the determination of the thermodynamic parameters obtained from temperature-dependent changes in K(b) (binding constant) it was apparent that the combined effect of hydrophobic association and electrostatic attraction is responsible for the interaction of ATA with BSA. The effect of ATA on the conformation of BSA has been examined by analyzing CD spectrum. Though the observed results demonstrate some conformational changes in BSA in presence of ATA but the secondary structure of BSA, predominantly of α-helix, is found to retain its identity. Molecular docking of ATA with BSA also indicates that ATA docks through hydrophobic interaction., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Cosalane and related compounds are a peculiar group of anti-HIV agents with activities against a broad range of viral targets, such as viral entry and reverse transcriptase (RT). Cosalane and its analogues having anionic pharmacophore inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The alkenyldiarylmethanes (ADAMs), characterized by the lack of the steroidal moiety of cosalane, are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that have potential value in the treatment of HIV infection. In this article, the structural modifications, structure-activity relationship (SAR) studies and/or crystallographic studies of cosalane related derivatives as potent antiviral agents were reviewed, which will be beneficial to the discovery of next generation cosalane derivatives with improved antiviral potency, metabolic stability and bioavailability.

Background: Aurintricarboxylic acid (ATA) and ethacrynic acid (ECA) have been reported to exhibit antiviral activity against vaccinia virus infections in cell culture by inhibiting early and late gene transcription, respectively. The purpose of this work was to determine if these inhibitors would effectively treat vaccinia virus infections in mice, which has not previously been studied., Methods: ECA was investigated by cell culture plaque reduction assay for the inhibition of cowpox and vaccinia virus infections to clarify issues regarding its potency and selectivity. Mice infected intranasally with vaccinia virus were treated by intraperitoneal route twice daily for 5 days with ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) or once daily for 2 days with cidofovir (100 mg/kg/day)., Results: ECA caused 50% inhibition of virus plaque formation at 20-79 muM in four cultured cell lines, with 50% cytotoxicity at 84-173 muM, giving low (1.3-4.2) selectivity index values. Preliminary toxicity tests in uninfected mice indicated that ATA and ECA were both overtly toxic at 100 mg/kg/day. No protection from mortality was afforded by treatment of vaccinia virus infections with ATA or ECA, but 100% survival was achieved in the cidofovir group. ATA- and ECA-treated mice died significantly sooner than placebo-treated animals, indicating that these compounds exacerbated the infection., Conclusions: Both ATA and ECA lack antiviral potency and selectivity in cell culture. The compounds were ineffective in treating mice at intraperitoneal doses of

Objectives: Enterovirus 71 (EV71) causes serious diseases in humans. The aim of this study was to examine the effects of aurintricarboxylic acid (ATA) on EV71 replication and to explore the underlying mechanism., Methods: To measure the activity of ATA in inhibiting the cytopathic effect (CPE) of EV71, a cell-based neutralization (inhibition of virus-induced CPE) assay was performed. The effect of ATA was further confirmed using plaque reduction and viral yield reduction assays. A time of addition assay was performed to identify the mechanisms of ATA's anti-EV71 activity. We examined the effects of ATA on the following key steps involved in virus replication: (i) translation of the internal ribosomal entry site (IRES)-mediated viral polyprotein; (ii) the proteolytic activity of viral proteases 2A and/or 3C; and (iii) the viral 3D RNA-dependent RNA polymerase (RdRp) activity., Results: In this study, ATA was found to be a potent inhibitor of the replication of EV71. In the antiviral neutralization assay, ATA exhibited inhibitory activity against EV71 (TW/4643/98) and EV71 (TW/2231/98). Plaque assay further demonstrated that ATA inhibited EV71 replication with an EC(50) (effective concentration at which 50% of plaques were removed) of 2.9 microM. Studies on the mechanism of action revealed that ATA targets the early stage of the viral life cycle after viral entry. ATA was able to inhibit the RdRp activity of EV71, while neither the IRES-mediated translation of viral polyprotein nor the viral 3C protease activity was affected., Conclusions: Overall, the findings in this study suggest that ATA is able to effectively inhibit EV71 replication through interfering with the viral 3D polymerase.

Background: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies., Methodology/principal Findings: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus., Conclusions/significance: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I (K(d)=9.019 microM)), RNase A (K(d)=2.33 microM) reverse transcriptase (K(d)=0.255 microM) and Taq polymerase (K(d)=81.97 microM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells., Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations., Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC(50)] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with > or =fivefold decreased potency compared with continuous polymerization conditions. The IC(50) value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects., Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K(d)=3.62+/-2.1 x 10(-8)M) or the RNA corresponding sequence (K(d)=2.7+/-0.82 x 10(-8) M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.