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Radioautographic Localization of Estrogen Receptors in the Rat Uterus: A Tool for the Study of Classical and Nontraditional Mechanisms of Hormone Action
Published in Louis P. Pertschuk, Sin Hang Lee, Localization of Putative Steroid Receptors, 2019
Andrei N. Tchernitchin, Miguel A. Mena, Angel Rodriguez, Mauricio Maturana
For a more accurate characterization of the eosinophil estrogen receptors, uterine cryostat sections were incubated with different concentrations of 3H-estradiol (with or without excess of unlabeled steroid) and processed by the wet radioautographic technique.143aFigure 3A shows the resulting saturation analysis and Figure 3B shows a plot of bound steroid (as radioautographic grains) divided by total hormone concentration (which nearly equals free) as a function of bound. Data suggest the existence of two specific binding components, the higher-affinity component has a dissociation constant of about 0.4 nM. This value is similar to the previously reported Kd = 0.56 nM in biochemical studies that considered the existence of only one binding component in eosinophils.36,37
Gonadotropins and Sex Hormones
Published in Istvan Berczi, Pituitary Function and Immunity, 2019
Specific, high affinity estrogen receptors have been found in thymuses of rat, mouse, man, and cattle. The cytoplasmic receptors bound estradiol and similar estrogenic compounds, such as diethylstilbestrol, but this receptor had no affinity to other steroid hormones: progesterone, testosterone, or cortisol. A dissociation constant of 2 × 10−10M was reported by several investigators. Autoradiographic studies showed that the radioactive label was confined in single cells, which may be identical with thymic reticuloepithelial cells.31,36
Pharmacokinetic-Pharmacodynamic Relationships of Cardiovascular Drugs
Published in Hartmut Derendorf, Günther Hochhaus, Handbook of Pharmacokinetic/Pharmacodynamic Correlation, 2019
where Kd is the equilibrium dissociation constant of the receptor-drug complex. If RT equals the total concentration of receptors ([RT] = [R] + [RC]), then we can solve for [R] the concentration of receptors not bound to any drug, and substitute in Equation 3 to obtain
Evolution of the drug-target residence time model
Published in Expert Opinion on Drug Discovery, 2021
As originally formulated [7], the drug-target residence time model posits that the duration of in vivo PD is determined by the lifetime, or residence time (τR), of the binary drug-target complex. If we consider a typical in vitro experiment, in which a fixed concentration of drug molecules is mixed with a fixed concentration of target molecules, in a fixed volume of solution, the formation of a binary drug-target complex – in the simplest model of interaction – is determined by two kinetic processes: the rate of binding or association of the free drug with target (quantified by the rate constant kon) and the rate of binary complex dissociation (quantified by the rate constant koff). When the system reaches equilibrium, the strength of binding (i.e. the affinity) of the drug for its target is commonly quantified by the thermodynamic equilibrium dissociation constant, Kd (in Molar units) which is defined as the ratio of koff (in units of s−1) over kon (in units of M−1 s−1), as shown in Figure 2(A). Note that the rate of drug binding to its target is determined both by the value of kon and by the concentration of drug present. The rate of binary complex dissociation, in contrast, depends only on koff. The Kd value thus quantifies how tightly the drug binds to its intended target, but it does not give any information on the lifetime of the complex under nonequilibrium conditions.
Identification and characterization of M6903, an antagonistic anti–TIM-3 monoclonal antibody
Published in OncoImmunology, 2020
Dong Zhang, Feng Jiang, Rinat Zaynagetdinov, Hui Huang, Vanita D. Sood, Hong Wang, Xinyan Zhao, Molly H. Jenkins, Qingyong Ji, Youbin Wang, David P. Nannemann, Djordje Musil, John Wesolowski, Andrea Paoletti, Tin Bartholomew, Melissa G. Derner, Qi An, Christel Iffland, Joern-Peter Halle
To determine the affinity of M6903 to TIM-3 orthologues from various species via SPR, goat anti-human-IgG Fc polyclonal antibody (Jackson Labs, 109-005-098) was immobilized on CM5 chips (GE Healthcare, BR-1006-68). Flow rate throughout was 30 μL/minute. Anti-TIM-3 antibodies were captured at 0.5 and 1.0 µg/mL for 120 seconds. TIM-3 ECD orthologues from different species were either purchased (Human Novoprotein C356, marmoset Novoprotein NP10506, murine Sino Biologicals 51152), or in the case of ECD from cynomolgus monkey, expressed in Expi293 F cells (ThermoFisher, A14527), and purified by immobilized metal affinity chromatography followed by size exclusion chromatography. ECDs were diluted from 0–100 nM. Association was measured for 180 seconds, followed by dissociation in buffer (HEPES buffered saline with EDTA and P20, GE Healthcare, BR-1006-69) for 600 or 900 seconds. Biacore T200 software version 1.0 was used to fit sensorgrams to a 1:1 Langmuir binding model to derive association (ka) and dissociation (kd) kinetic constants. The equilibrium dissociation constant (KD) was determined as the ratio of kinetic constants.
N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and biological evaluation
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Wei-Yi Li, Wei-Wei Ni, Ya-Xi Ye, Hai-Lian Fang, Xing-Ming Pan, Jie-Ling He, Tian-Li Zhou, Juan Yi, Shan-Shan Liu, Mi Zhou, Zhu-Ping Xiao, Hai-Liang Zhu
The binding kinetics of selected compounds were assayed via surface plasmon resonance (SPR) using an OPEN SPR instrument (Nicoya Lifesciences, Kitchener, Canada). First, urease dissolved (50 µg/mL) in PBS buffer (1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4-12H2O, pH 7.4), was immobilised to a CM5 chip using a standard amine coupling procedure22. Then, SPR measurements were carried out in PBS, and stock solutions were diluted in the same buffer. Data were collected with OpenSPR control software. Experiments were performed by monitoring the refractive index changes as a function of time under constant flow rate of 20 µL/min. The relative amount of inhibitor bound to the urease was determined by measuring the net increase in refractive index over time compared to control running buffer. There is an inline subtraction of reference surface during the run. This change is usually reported in response units (RU). Sensograms were processed and analysed using TraceDrawer software. The binding curves were fit to determine the equilibrium dissociation constant (KD).