anti-GST Antibody Research Articles

Detection of Retinoic Acid-Active Chemicals in Diverse Sample Matrices Via a Quantum Dots-Based Nuclear Receptor Fluorescence Probe-Mediated Biosensor.

Developing a sensitive and reliable method for the screening of various endocrine-disrupting chemicals (EDCs) is in high demand and yet remains a significant challenge. Herein, we developed a CdSe/ZnS QDs-based nuclear receptor fluorescence probe (QDs-NRFP)-mediated biosensor for the screening of retinoic acid (RA)-active chemicals (a class of EDCs). The QDs-NRFP can be prepared on the spot via an antigen-antibody immunobinding interaction between the GST tag of the human retinoic acid receptor β ligand-binding domain (GST-hRARβ-LBD) and the CdSe/ZnS QDs-labeled anti-GST tag antibody. It can not only maintain the high binding activity of GST-hRARβ-LBD but also improve the sensitivity due to the high quantum yield of CdSe/ZnS QDs. Based on the indirect competition bioassay, the developed biosensor showed a detection limit of 1.8 ng/L all-trans-retinoic acid binding activity equivalent (atRA-BAE) with a linear range of 7.5-1183.6 ng/L. Compared with many cell-dependent in vitro assays, the QDs-NRFP-mediated biosensor is cell-free and unaffected by the cytotoxic substances in matrices and exhibited obvious superiority in detection time (within 40 min) and accuracy. As a case study, the biosensor was applied to detect RA binding activities in various sample matrices obtained from a wastewater treatment plant (WWTP) and physiological samples and showed satisfactory accuracy and reliability. The developed QDs-NRFP-mediated biosensor is expected to be capable of screening various EDCs with universality based on different nuclear receptor signaling pathways, which will substantially accelerate the assessment of global EDCs.

Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis

Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies.

Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Cofactor Recruitment Assay for PPARα and PPARγ.

Peroxisome proliferator-activated receptors are a family of nuclear hormone receptors that control the expression of genes involved in a variety of physiologic processes, through heterodimerization with retinoid X receptor and complex formation with various cofactors. The specific cofactors recruited to PPAR-RXR complexes in response to different ligands lead to major differences in the transactivation of target genes. We developed a cofactor recruitment assay that is based on an europium-labeled anti-GST antibody and streptavidin-APC leading to a fluorescence resonance energy transfer signal. This assay allows for the determination of unique agonistic profiles in terms of potency and co-activator motif. Hence, it is a valuable drug discovery tool to support hit finding and lead optimization campaigns, enabling the characterization of next generation PPAR agonists.

Utilization of a recombinant defensin from Maize (Zea mays L.) as a potential antimicrobial peptide

The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carcinoma colon cancer) with IC50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.

Production and characterization of a fusion form of hepatitis E virus tORF2 capsid protein in Escherichia coli

Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368–606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.

Expression and functional characterization of the CUB domain-containing protein from the triangle sail mussel (Hyriopsis cumingii) in response to pathogenic infection.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280bp complete cDNA of the HcCDCP contained a 1002bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks.

Molecular characterization and tissue localization of glutathione S-transferase from adult Ancylostoma ceylanicum.

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.

Molecular characterization and immune protection of an AN1-like zinc finger protein of Eimeria tenella.

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

Gluthathione S-transferase-resuscitation-promoting factor B recombinant protein of <em>Mycobacterium tuberculosis</em> induces the production of interferon-γ and interleukin-12 in mice splenocytes

BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guérin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro. METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg/ ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays. RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10. CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.

Tạo dòng, biểu hiện và tái gấp cuộn prion protein (PrPc) chuột dung hợp với GST

Oral vaccine is one of the most potential strategies for gastrointestinal infection due to significant advantages over parenteral vaccines. In order to overcome exsiting hurdles in oral vaccine, namely oral tolerance and antigen dispersion, gut-associated M cell is a target for vaccine delivery. Cellular prion protein (PrPC), an M cell receptor, was experimentally proven to interact with heat shock protein Hsp60. In the previous in sillico studies, two Hsp60-deprived peptides have been predicted to be key-players in this interaction. Therefore, providing PrPC for in vitro binding assay with Hsp60 is desired to confirm the binding activity of the two predicted peptides. In this study, murine PrPC-encoding gene (mPrPc) was cloned into pET-gst to generate recombinant vector namedly pET-gst-mPrPc. Next, the vector was transformed into expression host Escherichia coli (E. coli) BL21 (DE3) for protein expression. SDS-PAGE and Western blot probed with anti-GST antibodies showed the protein expressed in inclusion bodies and hence was subsequently solubilized and refolded. After refolding, GST-mPrPC protein was harvested in soluble form with the refolding efficiency reached 88.33%.

The Plasmodium yoelii microgamete surface antigen (PyMiGS) induces anti-malarial transmission blocking immunity that reduces microgamete motility/release from activated male gametocytes

Malaria transmission-blocking vaccines aim to inhibit the development of malaria parasites in mosquitoes by inducing antibodies targeting surface proteins of sexual stage parasites. We have recently identified PyMiGS, a protein specifically expressed in the osmiophilic body of male gametocytes of Plasmodium yoelii (Py). PyMiGS is translocated to the surface of microgametes, and potent transmission-blocking activity was observed in mosquitoes fed on mice passively immunized with antibodies against PyMiGS. Here we demonstrate using a direct feeding assay that recombinant PyMiGS successfully induces anti-PyMiGS antibodies in mice and that the antibodies block parasite development in mosquitoes. We also show using the membrane-feeding assay that rabbit anti-PyMiGS antibody inhibits parasite development in mosquitoes in a dose-dependent manner without complement involvement. To investigate the mode of action of anti-PyMiGS antibodies against parasite development, we observed exflagellation after mixing Py gametocytes with activation medium containing anti-PyMiGS or anti-GST control antibodies. Whereas most microgametes were released from activated male gametocytes in the control group, a significantly reduced number of microgametes were released in the anti-PyMiGS group, with most of the microgametes left attached to the activated male gametocytes. Moreover, anti-PyMiGS antibodies shortened the duration of the active movement of microgametes after the onset of exflagellation. Taken together, these findings suggest that anti-PyMiGS antibodies bind to the microgamete surface immediately after exflagellation, thereby reducing microgamete motility and inhibiting microgamete release from the activated male gametocytes. These results strongly suggest that PyMiGS orthologues in Plasmodium falciparum and Plasmodium vivax can be promising TBV candidates.

Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli.

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5mM IPTG for 3h at 37°C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16°C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20°C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule.

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling.

The Wnt signaling pathway can be grouped into two classes, the β-catenin-dependent and β-catenin-independent pathways. Wnt5a signaling through a β-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.

Characterization of glutathione S-transferase and its immunodiagnostic potential for detecting Taenia multiceps

Taenia multiceps is a widespread zoonotic tapeworm parasite which infects cloven-hoofed animals around the world. Animal infection with Coenurus cerebralis, the coenurus larvae of T. multiceps (Tm), is often fatal, which is a major cause of economic losses in stockbreeding. This study amplified the glutathione S-transferase (GST) gene from the total RNA of C. cerebralis. The resulting protein, Tm-GST, consisted of 201 amino acids, and had a predicted molecular mass of 23.1kDa. Its amino acid sequence shares 77.61% similarity with Echinococcus granulosus GST. Recombinant Tm-GST (rTm-GST) was expressed in Escherichia coli. The protein reacted with serum from goats infected with T. multiceps. Immunofluorescence signals indicated that Tm-GST was largely localized in the parenchymatous area of adult T. multiceps; in addition, it was also apparent in the coenurus. An enzyme-linked immunosorbent assay based on rTm-GST showed specificity of 92.8% (13/14) and sensitivity of 90% (18/20) in detecting anti-GST antibodies in serum from naturally infected animals. This study suggests that Tm-GST has the potential to be used as a diagnostic antigen for Coenurosis.

Expression and purification of p70ΔCT104 S6 K, a 72 kDa c-terminal truncated p70S6 kinase-GST fusion protein in bacterial expression system

The p70ΔCT104 S6K is a 421 amino acid residue long truncated form of p70S6 kinase, with 104 amino acids residues cleaved from the carboxyl terminal end of the original protein. The p70ΔCT104 S6K was cloned in E. coli DH5α and successfully expressed in E. coli BL21 (DE3) strain. Western blot with rabbit polyclonal anti-GST antibody was used to follow the protein during expression and purification. The protein purification was achieved by affinity chromatography using Glutathione resin-agarose beads, followed by chromatography on a spin concentration column. The purified protein was confirmed by rabbit polyclonal anti-p70S6 kinase antibody. MALDI/MS Peptide mass fingerprinting confirmed identity of the expressed product.

Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.

We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Abstract B23: DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo

Abstract Introduction: Activating mutations in Ras oncogenes play a critical role in the development of colorectal cancer and are an indicator of poor prognosis. Constitutive activation of Ras proteins is also associated with resistance to chemotherapy and radiation in which treatment options are limited. Despite extensive efforts, no drugs have been successfully developed that target these aberrant gene products, in part because of the high affinity of Ras binding to GTP, which is essential for Ras activation. Using a phenotypic screening assay designed to select for Ras inhibitors and follow-up chemical optimization, a novel Ras inhibitor, DC070-547 was identified, which potently and selectively inhibits the growth of colon tumor cells with activated Ras in vitro and in vivo. Methods: Tumor cell growth inhibitory activity of DC070-547 was measured in a panel of human colon tumor cell lines using the CellTiter Glo assay following 72 h of treatment. Ras activation levels were measured by precipitating GTP-bound Ras from human colon tumor cell lysates with GST-Raf1-RBD/GSH Sepharose followed by western blotting using anti-Ras antibody. Isogenic cell lines were established by transfecting Ras wild-type HT-29 colon tumor cells with mutant H-Ras. Disruption of Ras-Raf binding was determined by pre-incubating cell lysates or intact cells in the presence of DC070-547 for 30 or 45 min followed by Ras pull-down with GST-Raf beads and western blotting using anti-Ras or an anti-GST antibody as a loading control. Cell cycle arrest and apoptosis were measured by DNA content and annexin V levels, respectively. Antitumor activity was determined in a mouse xenograft model subcutaneously implanted with mutant K-Ras HCT-116 colon tumor cells. Mice were treated with DC070-547 administered i.p. for 14 days at a dose of 2.5 mg/kg bid in a co-solvent formulation. Results: DC070-547 potently inhibits the growth of mutant K-Ras HCT-116 and other colon tumor cell lines having constitutively activated Ras with IC50 values as low as 2 nM and selectivity indices of approximately 100-fold for HT-29 and other colon tumor cells lacking activated Ras. Among a panel of six colon tumor cell lines, a strong correlation was measured between IC50 values for growth inhibition and the level of Ras activation, suggesting that activated Ras is the primary target. Isogenic cell line pairs involving transfecting Ras wild-type cells with mutant H-Ras confirmed that sensitivity to DC070-547 required activated Ras. DC070-547 also blocked Ras-Raf binding in cell lysates and intact cells at concentrations that inhibit the growth of tumor cells with activated Ras. The mechanism of growth inhibition by DC070-547 appears to involve mitotic arrest and apoptosis. Moreover, cells obtained from normal colon mucosa were essentially refractory to treatment with DC070-547. In a mouse xenograft model involving mutant K-Ras human HCT-116 colon tumors, DC070-547 was well tolerated and caused a sustained inhibition of tumor growth in which there was complete tumor regression in 3 of 7 mice. Conclusions: DC070-547 potently and selectively inhibits the growth of colon tumor cells with constitutively activated Ras by disrupting Ras-effector interactions and represents a first-in-class drug development candidate for the treatment of Ras-driven colorectal cancer. Citation Format: Veronica Ramirez-Alcantara, Adam B. Keeton, Bing Zhu, Kevin J. Lee, Joshua Canzoneri, Ashley S. Lindsey, Luciana Madeira da Silva Barnes, Kristy Berry, Jacob Valiyaveettil, Xi Chen, Michael R. Boyd, Gary Piazza. DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B23.

An HTRF based high-throughput screening for discovering chemical compounds that inhibit the interaction between Trypanosoma brucei Pex5p and Pex14p

The glycosome, a peroxisome-related organelle, is essential for the growth and survival of trypanosomatid protozoa. In glycosome biogenesis, Pex5p recognizes newly synthesized glycosomal matrix proteins via peroxisome-targeting signal type-1 (PTS-1) and transports them into glycosomes through an interaction with Pex14p, a component of the matrix protein import machinery on the glycosomal membrane. Knockdown of the PEX5 or PEX14 with RNAi has been shown to inhibit the growth of Trypanosoma brucei. Thus, compounds that inhibit the interaction of TbPex5p–TbPex14p are expected to become lead compounds in the development of anti-trypanosomal drugs. Here, we report a homogenous time-resolved fluorescence (HTRF) assay for the screening of compounds that inhibit the TbPex5p–TbPex14p interaction. The binding of GST-TbPex14p and TbPex5p-His with or without additional compounds was evaluated by measuring the energy transfer of the HTRF pair, using a terbium-labeled anti GST antibody as the donor and an FITC-labeled anti His antibody as the acceptor. The assay was performed in a 384-well plate platform and exhibits a Z’-factor of 0.85–0.91, while the coefficiency of variation is 1.1–7.7%, suggesting it can be readily adapted to a high-throughput format for the automated screening of chemical libraries. We screened 20,800 compounds and found 11 compounds that inhibited energy transfer. Among them, in a pull-down assay one compound exhibited selective inhibition of TbPex5p–TbPex14p without any HsPex5p–HsPex14p interaction.

Selective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8

ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.