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Pharmacokinetic Aspects of the in Vivo, Noninvasivestudy of Neuroreceptors in Man
Published in William C. Eckelman, Lelio G. Colombetti, Receptor-Binding Radiotracers, 2017
Although the dissociation rate constant is likely an important determinant of the rate at which the receptor-bound ligand clears from the brain, it does not totally describe the process. Comparison of in vitro dissociation rate constants and in vivo clearance rates of receptor-bound ligand demonstrates that while in vitro dissociation halftimes are on the order of 10 min,15 the in vivo clearance half-times can approach values on the order of 10 hr for the ligands with affinities approaching 1010M-1 (Kuhar, unpublished). The probable reason for this discrepancy is that the phenomena of “rebinding” occurs due to the high local concentrations of neuroreceptors.16 The ligand dissociates from and reassociates with the receptors many times before it finally escapes from the region of high local receptor concentration and is transported back into the blood compartment and cleared from the brain. Thus clearance rates should depend not only on the intrinsic molecular dissociation rate constant but also on the local receptor concentration. This matter will be taken up again in a subsequent section.
Overview of Techniques for Direct Measurement of Receptor-Ligand Interactions
Published in John C. Matthews, Fundamentals of Receptor, Enzyme, and Transport Kinetics, 2017
Determination of the association and dissociation rate constants will also provide information about the length of time necessary for the receptor and ligand interaction to come to equilibrium. If both the association and dissociation rate constants are small numbers, it means that both reactions are slow. If both rate constants are large numbers, it means that both reactions are fast. Receptor-ligand interactions can be qualitatively classified as fast or slow, based upon the length of time necessary for the system to reach equilibrium. Ultimately this is dependent upon the relative magnitudes of the rate constants. Since the affinity constant is the ratio of the rate constants it is possible to have fast or slow receptor-ligand interactions with the same KD value.
Enzyme Kinetics
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
The kinetics of a cooperative system are likely to be complex. Any induced change in the affinity of a ligand can arise by a change in its association or dissociation rate constant, or both. In modeling possible reaction schemes, it is important to keep the mechanism in thermodynamic balance. For example, in the case of a heterotropic interaction, if the affinity of ligand A for a protein is increased 10-fold in the presence of bound ligand B, then it follows that the affinity of ligand B must increase 10-fold when A is bound. This is evident from the following thermodynamic box:
Kinetic analysis of ternary and binary binding modes of the bispecific antibody emicizumab
Published in mAbs, 2023
Stefanie Mak, Agnes Marszal, Nena Matscheko, Ulrich Rant
First, the kinetics of binary interactions were measured by loading either FIX or FX onto the Y-structure, while leaving the other arm unmodified. Sensorgrams are shown in Figure 3(a,b). The real-time signals could be fitted well globally with single-exponential functions, as expected for one-to-one interactions. Association and dissociation rates are summarized in the rate-scale plot in Figure 3(e) and Table 1. Emicizumab binds FIX and FX in a very dissimilar manner, clearly favoring FX. It associates 5 times faster and remains bound 20 times longer to FX than to FIX. Taken together, this results in a 100-fold higher affinity for FX (Kd = 56 nM) compared to FIX (Kd = 5.5 µM). These results confirm immunoassay data by Kitazawa et al., which were inconsistent with SPR data obtained by the same group.15
HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure
Published in mAbs, 2023
Saw Yen Ow, Eugene A. Kapp, Vesna Tomasetig, Anton Zalewski, Jason Simmonds, Con Panousis, Michael J. Wilson, Andrew D. Nash, Matthias Pelzing
Binding kinetics were measured using a Biacore® 8 K+ SPR Biosensor (Cytiva) docked with a Series S CM5 sensor chip (Cytiva). Anti-human IgG Fc (Thermo Fisher, Catalog No.: H10500) was immobilized using standard amine coupling chemistry to ~15,000 response units. Garadacimab was captured on active flow cells at the beginning of each cycle to a surface density of ~250 response units. Flow cell 1, in which no garadacimab was captured, was used as a reference. Recombinant β-FXIIa was injected over both flow cells for 350 seconds at concentrations between 0.02 nM and 10 nM and dissociation was monitored for 1200 seconds. A buffer blank was used for referencing purposes. After each cycle, the surface was regenerated with a 45-seconds injection of 100 mM H3PO4. The analysis was performed at 37°C at a flow rate of 30 µL/min in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v/v surfactant P20 supplemented with 0.1% bovine serum albumin and adjusted to pH 7.4. Biacore Insight Evaluation software v3.0.12.15655 (Cytiva) was used to fit double-referenced sensorgrams to a 1:1 kinetics model including a term for mass transport limitation.39 The Rmax value was fitted locally to account for slight deviations in the level of garadacimab captured. The association rate (ka), dissociation rate (kd), and equilibrium (KD) were fitted globally.
Phosphorus containing analogues of SAHA as inhibitors of HDACs
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Michael D. Pun, Hsin-Hua Wu, Feyisola P. Olatunji, Britany N. Kesic, John W. Peters, Clifford E. Berkman
The time course enzyme inhibition assay using compound 2 showed optimal inhibition at 8 h for HeLa cell Lysate and HDAC 8, and 4 h for HDAC 3. These results suggest that our series of phosphoryl compounds are slow binding inhibitors. These types of inhibitors also express tight binding qualities such that the molecules have low dissociation rates and long drug target residence time29. In vitro, this strong binding quality can disrupt cell viability due to the inhibitors ability to shutdown the enzyme for a long period of time. If enzyme synthesis time in targeted cells cannot overcome the inhibition time, cell viability can be affected30. The advantage of slow and tight binding inhibitors for in vivo biomedical purposes stems from the decreased off target toxicity of the compound. Because of the decreased systemic circulation time and increased inhibitor residence time a lower concentration of the compound is available in the blood stream to bind to non-targeted protein31. There are several known examples of slow binding as FDA-approved drugs32. There are also several know types of HDAC inhibitors that exhibit slow binding kinetics33.