scholarly journals Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3975
Author(s):  
Simone Gastaldi ◽  
Valentina Boscaro ◽  
Eleonora Gianquinto ◽  
Christina F. Sandall ◽  
Marta Giorgis ◽  
...  
Keyword(s):  
Acrylic Acid ◽  
Ligand Binding ◽  
Atpase Activity ◽  
Binding Sites ◽  
Computational Simulations ◽  
Complete Model ◽  
Human Macrophages ◽  
Chemical Modulation ◽  
Inflammasome Inhibitors

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1β release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1β release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein–ligand binding that might explain the activity of the compounds.

scholarly journals Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

2021 ◽  
Vol 22 (20) ◽  
pp. 11142
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Yi-Zong Lee ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Receptor Interaction ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

scholarly journals Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of thefadRegulon

2016 ◽  
Vol 82 (9) ◽  
pp. 2819-2832 ◽  
Author(s):  
Rongsui Gao ◽  
Jingxia Lin ◽  
Han Zhang ◽  
Youjun Feng
Keyword(s):  
Transcriptional Regulation ◽  
Fatty Acid ◽  
Ligand Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Regulatory Proteins ◽  
Content Type ◽  
Ligand Binding Sites

ABSTRACTRecently, our group along with others reported that theVibrioFadR regulatory protein is unusual in that, unlike the prototypicalfadRproduct ofEscherichia coli, which has only one ligand-binding site,VibrioFadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of thevc2105gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of thevc2105gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates thevc2105promoter from the FadR regulator. The expression level of theVibrio cholerae vc2105gene was elevated 2- to 3-fold in afadRnull mutant strain, validating that FadR is a repressor for thevc2105gene. The β-galactosidase activity of avc2105-lacZtranscriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike thefadDgene, a member of theVibrio fadregulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression ofctxAB(cholera toxin A/B) andtcpA(toxin coregulated pilus A). Given that the transcriptional regulation ofvc2105fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of theVibrio fadregulon.IMPORTANCETheVibrioFadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterizedin vitroandin vivo. An auxiliaryfadgene (vc2105) fromVibriowas proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Furtherin vitroandin vivoexperiments revealed that theVibrioFadR is a repressor for thevc2105gene. UnlikefadD, a member of theVibrio fadregulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxABandtcpA). Therefore, since transcriptional regulation ofvc2105fits the criteria forfadgenes, it seems likely thatvc2105acts as a new auxiliary member of theVibrio fadregulon.

scholarly journals Effect of oxidized glutathione and temperature on inositol 1,4,5-trisphosphate binding in permeabilized hepatocytes

1995 ◽  
Vol 310 (1) ◽  
pp. 185-192 ◽  
Author(s):  
D C Renard-Rooney ◽  
S K Joseph ◽  
M B Seitz ◽  
A P Thomas
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Oxidized Glutathione ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Temperature Dependent ◽  
Inositol 1,4,5 Trisphosphate ◽  
Number Of Binding Sites

The effect of oxidized glutathione (GSSG) on inositol 1,4,5-trisphosphate (IP3) binding and the activity of IP3-gated Ca2+ channels was examined in permeabilized hepatocytes. The permeability properties of the channel were measured by using Mn2+ quenching of compartmentalized fura-2 at 37 degrees C and at 4 degrees C for comparison with IP3-binding measurements. GSSG (2 mM) increased the IP3-sensitivity of Mn2+ quenching, consistent with previous studies based on Ca(2+)-release measurements [Renard, Seitz and Thomas (1992) Biochem. J. 284, 507-512]. Measurements of [3H]IP3 binding were made at 4 degrees C after preincubation of permeabilized hepatocytes at 37 degrees C in the absence or presence of GSSG. Under these conditions GSSG stimulated IP3 binding by increasing the number of binding sites without changing the Kd. This effect was observed in the absence or presence of Ca2+, but was abolished when the preincubation with GSSG was carried out at 4 degrees C. Thimerosal also stimulated [3H]IP3 binding, but this effect was mediated both by an increase in the maximum number of binding sites and by a decrease in the Kd. The effects of thimerosal and GSSG were not additive. Further analysis of the effect of GSSG revealed that preincubation of permeabilized hepatocytes at 37 degrees C results in a progressive loss of [3H]IP3-binding sites that can be prevented and reversed by inclusion of GSSG. A parallel loss of IP3-sensitive Mn(2+)-quenchable stores was observed after incubation at 37 degrees C, and this could also be reversed by adding back GSSG. The loss of IP3 binding was not the result of IP3-receptor proteolysis, as judged by Western blotting of immunoreactive protein. The sensitivity of [3H]IP3 binding in permeabilized hepatocytes to varied ratios of GSSG and GSH suggests that the IP3 receptor responds to an oxidized redox environment such as that found in the lumen of the endoplasmic reticulum. GSSG had no direct effect on the ligand-binding activity of detergent-solubilized and partially purified IP3 receptors. We conclude that GSSG exerts an indirect effect on the IP3 receptors in permeabilized hepatocytes by preventing a temperature-dependent loss of IP3-binding sites. We suggest that the hepatic IP3 receptors may interact with a thiol-disulphide oxidoreductase that utilizes GSSG as a substrate and prevents inappropriate unfolding of the ligand-binding domain occurring after incubation of the receptor at 37 degrees C in vitro.

Responsiveness of gill Na+/K+-ATPase to cortisol is related to gill corticosteroid receptor concentration in juvenile rainbow trout

1999 ◽  
Vol 202 (8) ◽  
pp. 987-995 ◽  
Author(s):  
J.M. Shrimpton ◽  
S.D. Mccormick
Keyword(s):  
Rainbow Trout ◽  
Atpase Activity ◽  
Binding Sites ◽  
Seasonal Changes ◽  
Radioligand Binding ◽  
Tissue Response ◽  
Juvenile Rainbow Trout ◽  
Receptor Concentration ◽  
Cr Concentration

A positive relationship between receptor concentration and tissue responsiveness is an often-assumed and rarely tested principle in endocrinology. In salmonids, seasonal changes in levels of plasma cortisol and gill corticosteroid receptors (CRs) during the spring indicate a potential role for this hormone in the parr-smolt transformation. It is not known whether these seasonal changes result in alterations in gill responsiveness to cortisol. The relationship between CR concentration and tissue responsiveness was, therefore, examined in the gills of juvenile rainbow trout (Oncorhynchus mykiss). Gill CR concentration (Bmax) and affinity (Kd) were assessed using a radioligand binding assay with the synthetic glucocorticoid triamcinolone acetonide. Gill responsiveness to cortisol was quantified by measuring in vitro Na+/K+-ATPase activity. Gill CR concentration was manipulated by stress or hormonal treatments. Repeated handling stresses resulted in a significant reduction in CR numbers. The decrease in CR Bmax corresponded to a reduction in gill responsiveness to cortisol. Triiodothyronine, but not growth hormone, treatment was found to increase CR Bmax significantly. The increase in CR numbers was correlated with a marked increase in gill responsiveness to cortisol. A significant positive linear relationship exists between the in vitro gill Na+/K+-ATPase activity response to cortisol and CR Bmax (r2=0.614, P<0.001). We have demonstrated that binding sites for cortisol in the gills of rainbow trout have high affinity, high specificity and saturable binding and that the number of binding sites is correlated with the tissue response to cortisol.

scholarly journals Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells.

1980 ◽  
Vol 28 (3) ◽  
pp. 223-230 ◽  
Author(s):  
N Nakamura ◽  
R E Hurst ◽  
S S West ◽  
J M Menter ◽  
J F Golden ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Constant ◽  
Binding Constants ◽  
Cooperative Binding ◽  
Binding Parameters ◽  
Binding Studies ◽  
Mastocytoma Cells ◽  
Biochemical Analyses

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.

scholarly journals Structure-based functional study of a peptide of an ecdysozoan superfamily: reveling a common molecular architecture and receptor-interacting residues

2020 ◽  
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
Yi-Zong Lee ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Functional Study ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

ABSTRACTA neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs and increased significantly after animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr53) and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substituted and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, G73) are located at either end of the sequence, which are sterically close to each other and presumably constitutes the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily revealing a molecular architecture, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

2020 ◽  
Author(s):  
Samuel C. Gill ◽  
David Mobley
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Sites ◽  
Degrees Of Freedom ◽  
Dynamics Simulation ◽  
Hiv Integrase ◽  
Binding Modes ◽  
System A ◽  
Multiple Binding ◽  
Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

Sign in / Sign up

Export Citation Format

Share Document