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Ligands and binding

Johannes A Eble
The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record...
February 15, 2018: Journal of Visualized Experiments: JoVE
Sandra Claes, Thomas D'huys, Anneleen Van Hout, Dominique Schols, Tom Van Loy
G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor's function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12)...
February 20, 2018: Journal of Visualized Experiments: JoVE
Toshiaki Teratani, Kengo Tomita, Takahiro Suzuki, Hirotaka Furuhashi, Rie Irie, Makoto Nishikawa, Junji Yamamoto, Toshifumi Hibi, Soichiro Miura, Tohru Minamino, Yuichi Oike, Ryota Hokari, Takanori Kanai
Incidence of nonalcoholic steatohepatitis (NASH), which is considered a hepatic manifestation of metabolic syndrome, has been increasing worldwide with the rise in obesity; however, its pathological mechanism is poorly understood. Here, we demonstrate that the hepatic expression of aortic carboxypeptidase-like protein (ACLP), a glycosylated, secreted protein, increases in NASH in humans and mice. Furthermore, we elucidate that ACLP is a ligand, unrelated to WNT proteins, that activates the canonical WNT pathway and exacerbates NASH pathology...
March 19, 2018: Journal of Clinical Investigation
Po-Chia Chen, Pawel Masiewicz, Vladimir Rybin, Dmitri I Svergun, Janosch Hennig
We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information of biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pair-wise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly-uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl)...
March 19, 2018: ACS Combinatorial Science
Marta Sánchez-Soto, Verònica Casadó-Anguera, Hideaki Yano, Brian Joseph Bender, Ning-Sheng Cai, Estefanía Moreno, Enric I Canela, Antoni Cortés, Jens Meiler, Vicent Casadó, Sergi Ferré
The poor norepinephrine innervation and high density of Gi/o-coupled α2A - and α2C -adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D2 -like receptor ligands, such as the D3 receptor agonist 7-OH-PIPAT and the D4 receptor agonist RO-105824, to α2 -adrenoceptors in cortical and striatal tissue, which express α2A -adrenoceptors and both α2A - and α2C -adrenoceptors, respectively...
March 18, 2018: Molecular Neurobiology
Sreejesh Sreedharan, Alessandro Sinopoli, Paul J Jarman, Darren Robinson, Christopher Clemmet, Paul A Scattergood, Craig R Rice, Carl G W Smythe, James A Thomas, Paul I P Elliott
Two new biscyclometalated complexes [Ir(ptzR)2(dppz)]+ (dppz = dipyridophenazene; ptzRH = 4-phenyl-1-benzyl-1,2,3-triazole (1+) and 4-phenyl-1-propyl-1,2,3-triazole (2+)) have been prepared. The hexafluorophosphate salts of these complexes have been fully characterized and, in one case, the X-ray structure of a nitrate salt was obtained. The DNA binding properties of the chloride salts of the complexes were investigated, as well as their cellular uptake by A2780 and MCF7 cell lines. Both complexes display an increase in the intensity of phosphorescence upon titration with duplex DNA, indicating the intercalation of the dppz ligand and, given that they are monocations, the complexes exhibit appreciable DNA binding affinity...
March 19, 2018: Dalton Transactions: An International Journal of Inorganic Chemistry
Ava Yuan Xue, Antonella Di Pizio, Anat Levit, Tali Yarnitzky, Osnat Penn, Tal Pupko, Masha Y Niv
The 25 human bitter taste receptors (hT2Rs) recognize thousands of structurally and chemically diverse bitter substances. The binding modes of human bitter taste receptors hT2R10 and hT2R46, which are responsible for strychnine recognition, were previously established using site-directed mutagenesis, functional assays, and molecular modeling. Here we construct a phylogenetic tree and reconstruct ancestral sequences of the T2R10 and T2R46 clades. We next analyze the binding sites in view of experimental data to predict their ability to recognize strychnine...
2018: Frontiers in Molecular Biosciences
Hua Zhu, Yue Huang, Heling Su, Yili Ma, Yiming Tao, D Joshua Liao, Yongming Liu, Zhenbo Feng
Since the early 1990s, multiple human estrogen receptor-α (hER-α) splice variants have been identified, of which the majority contain ≥1 deleted exon, and some are expressed as proteins with modified functions from the wild-type 66 kDa hER-α (ER-α66). In the present study, a novel hER-α splice variant, ER-α30, was identified and cloned from clinical breast cancer tissue. The ER-α30 sequence lacked a ligand-binding domain and a ligand-dependent transcriptional activation domain but retained the N-terminal transcriptional activation domain, the DNA-binding domain and a partial hinge domain, and possesses a unique 10-amino-acid domain...
April 2018: Oncology Letters
Lina Badimon, Esther Peña, Gemma Arderiu, Teresa Padró, Mark Slevin, Gemma Vilahur, Gemma Chiva-Blanch
C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands...
2018: Frontiers in Immunology
Mei-Yi Lu, Syuan-Shao Lu, Shiann-Luen Chang, Fang Liao
CCR6 is a G protein-coupled receptor (GPCR) that recognizes a single chemokine ligand, CCL20 and is primarily expressed by leukocytes. Upon ligand binding, CCR6 activates Gαi heterotrimeric G proteins to induce various potential cellular outcomes through context-specific cell signaling. It is well known that differential phosphorylation of Ser and Thr residues in the C-terminal domains or intracellular loops of GPCRs can generate barcodes that regulate GPCR function by regulating the recruitment of β-arrestins...
2018: Frontiers in Immunology
Aiko Machiya, Sho Tsukamoto, Satoshi Ohte, Mai Kuratani, Mai Fujimoto, Keigo Kumagai, Kenji Osawa, Naoto Suda, Alex N Bullock, Takenobu Katagiri
Various substitution mutations in ALK2, a transmembrane serine/threonine kinase receptor for bone morphogenetic proteins (BMPs), have been identified in patients with genetic disorders such as fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG) and heart defects. In this study, we characterized the ALK2 mutants R258G, G328V and F246Y, which were identified in patients with severe FOP, DIPG and unusual hereditary skeletal dysplasia, respectively. Both R258G and G328V were gain-of-function mutations, but F246Y was equivalent to wild-type ALK2...
March 15, 2018: Bone
Happy Agarwal, Soumya Menon, S Venkat Kumar, S Rajeshkumar
A large array of diseases caused by bacterial pathogens and origination of multidrug resistance in their gene provokes the need of developing new vectors or novel drug molecules for effective drug delivery and thus, better treatment of disease. The nanoparticle has emerged as a novel drug molecule in last decade and has been used in various industrial fields like cosmetics, healthcare, agricultural, pharmaceuticals due to their high optical, electronic, medicinal properties. Use of nanoparticles as an antibacterial agent remain in current studies with metal nanoparticles like silver, gold, copper, iron and metal oxide nanoparticles like zinc oxide, copper oxide, titanium oxide and iron oxide nanoparticles...
March 15, 2018: Chemico-biological Interactions
Keppanan Ravindran, Sivaperumal Sivaramakrishnan, Mubasher Hussain, Chandra Kanta Dash, Bamisope Steve Bamisile, Muhammad Qasim, Wang Liande
Entomopathogenic fungi are rich sources of bioactive secondary metabolites that possess insecticidal properties. The present study reported a novel approach for the identification of insecticidal compounds produced by Lecanicillium lecanii 09 and to assess their toxicity against the diamondback moth Plutella xylostella L. The cyclic peptides groups of toxic substances were separated from L. lecanii 09 through submerged liquid state fermentation. The most abundant toxic metabolite, Bassianolide was purified by high-performance liquid chromatography (HPLC) and its molecular weight and purity were determined by Liquid chromatography - mass spectroscopy (LC-MS), Fourier transformed infrared spectroscopy (FT-IR), and H1 nuclear magnetic resonance (NMR) respectively...
March 15, 2018: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
Atsushi Hirano, Kazuki Iwashita, Shun Sakuraba, Kentaro Shiraki, Tsutomu Arakawa, Tomoshi Kameda
Ion-exchange chromatography and multimodal ion-exchange chromatography are widely used for the separation of small molecules, peptides and proteins. Salts generally attenuate the electrostatic interactions between charged moieties of solutes and those of resins through electrostatic screening. However, little is known about how salts affect the interaction between the uncharged moieties of the solutes, such as aromatic moieties, and the charged moieties of the resins. In this study, we used alkyl gallates as model aromatic solutes to investigate the interaction mechanism of aromatic moieties with multimodal and conventional ion-exchange resins...
March 6, 2018: Journal of Chromatography. A
Justin Weinberg, Shaojie Zhang, Allison Kirkby, Enosh Shachar, Giorgio Carta, Todd Przybycien
We have proposed chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) as a strategy to increase the resin selectivity and robustness by providing the ligand with a steric repulsion barrier against non-specific binding. Here, we report on robustness and selectivity benefits for Repligen CaptivA PriMAB resin with ligands modified with 5.2 kDa and 21.5 kDa PEG chains, respectively. PEGylation of ProA ligands allowed the resin to retain a higher percentage of static binding capacity relative to the unmodified resin upon digestion with chymotrypsin, a representative serine protease...
February 24, 2018: Journal of Chromatography. A
Justin Weinberg, Shaojie Zhang, Gillian Crews, Giorgio Carta, Todd Przybycien
Chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) has been proposed as a strategy to increase the process selectivity and resin robustness by providing the ligand with a steric repulsion barrier against non-specific binding. This article comprises a comprehensive study of IgG adsorption and transport in Repligen CaptivA PriMAB resin with PEGylated ProA ligands that are modified using 5.2 and 21.5 kDa PEG chains. We studied the impact of the molecular weight of the PEG as well as the extent of PEGylation for the 5...
March 9, 2018: Journal of Chromatography. A
João Crispim Encarnação, Tim Schulte, Adnane Achour, Hanna Björkelund, Karl Andersson
Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracer®, originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria...
March 17, 2018: Applied Microbiology and Biotechnology
Tian Liu, Dongliang Zhang, Yuan Zhang, Xiangsheng Xu, Bo Zhou, Liang Fang, Yun Zhang, Yingjun Su, Boquan Jin, Ran Zhuang, Shuzhong Guo
BACKGROUND/AIMS: Regulatory T cells (Tregs) play key roles in maintaining peripheral tolerance and preventing autoimmune disease. Treg modulation could be helpful in treating malignancies, autoimmune disease, and allergies, as well as to facilitate organ transplantation. Signals transduced by co-stimulatory molecules are essential for Treg differentiation, homeostasis, and function. One well-known active receptor, CD226, also known as DNAM-1 or PTA1, is an adhesion molecule that interacts primarily with CD155 and is involved in Treg differentiation and immune tolerance to transplanted tissue...
March 13, 2018: Cellular Physiology and Biochemistry
Peter Christ, Sharon R Hill, Joachim Schachtner, Frank Hauser, Rickard Ignell
Mosquito blood feeding transiently inhibits sugar- and host seeking through neuropeptide signaling. Short neuropeptide F (sNPF) is one of the neuromodulators involved in this regulation. Here, we identified the genes for the sNPF precursor and the sNPF receptor in the southern house mosquito, Culex quinquefasciatus. Comparative analyses are made with the genes of the sNPF precursor and receptor from two other important vectors, Aedes aegypti and Anopheles coluzzii. We functionally characterized the receptors in all three species using endogenous neuropeptides, and quantified their transcript expression following a blood meal and a sugar meal...
March 14, 2018: Peptides
Cláudia S Nóbrega, Bart Devreese, Sofia R Pauleta
The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be - 170 mV, + 133 mV and + 210 mV, respectively, at pH 7...
March 14, 2018: Biochimica et Biophysica Acta
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