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Key Concepts in Assay Development, Screening and the Properties of Lead and Candidate Compounds
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
One method of overcoming the issues associated with the development of artificial substrates in assay systems is to use label free methods that obviate the requirement for the alteration of molecules in assays that could interfere with binding modes with the protein target and substrate of interest. Many of these label free techniques (e.g., biacore and isothermal titration calorimetry (Shoji et al. 2017)) are very powerful in that they allow the quantitative characterisation of interactions. The main disadvantage with these techniques is that they usually are not amenable to screening large numbers of compounds and can also consume large amounts of protein. However, strides are being made to increase their throughput and reduce reagent consumption.
Kinetic Thinking: Back to the Future
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Initial analysis of the stopped-flow data was performed by fitting an exponential function to each reaction profile, although some measurements deviated from pseudo-first-order conditions (Figure 10.9b). The concentration dependence showed indications of an initial decrease in kobs with increasing ligand concentration, indicative of conformational selection. Note the decrease in kobs occurs in the region where [peptide] < [recoverin] but increases when [peptide] > [recoverin]. The latter behavior suggests k−3 < k+0 (Figure 2.15). An attempt to fit an induced-fit mechanism failed to account for the initial decrease in kobs at low [peptide]. Given the diagnostic behavior that occurs in the region where the profiles should deviate from a single exponential profile, the authors [66] also carried out a global fit to the models across the full concentration range with either the recoverin or the peptide in molar excess. This analysis likewise showed a better fit to the conformational-selection model than an induced-fit model. Isothermal titration calorimetry was used to determine the overall equilibrium constant and place limits on the fits of the kinetic data. The best-fit values for Equation 10.14 were determined as in Table 10.2.
Primary Amyloidosis: Conformational Intermediates in Immunoglobulin Light Chain Proteins Upon Interaction with Congo Red under Physiological Conditions
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
Mary T. Walsh, Violet Roskens, Lawreen Heller Connors, Martha Skinner
CR was added to each LC in phosphate buffered saline, pH 7.4. Immediately after CR addition LC conformation was monitored for 8 hours at 37°C by continuously recording far UV circular dichroic spectra from 250 to 200 nm on an Aviv 62DS CD Spectropolarimeter (Aviv Biomedical, Inc., Lakewood, New Jersey) equipped with a thermoelectric temperature controller (5). Time-dependent changes in β-sheet (217 nm, LC interior), and β-turn/ random coil / loops (206.5 nm, LC surface) were measured for 10 hours at 37°C on separate samples. LC thermal unfolding / stability at 217 nm for β-sheet, and 206.5 nm for β-turn/ random coil / loops were measured from 5-95°C. Thermal stability studies were performed on CR-containing LC samples prepared at 25°C 7 days prior to performing the study, thus ensuring that CR-induced conformational changes were complete. LC concentration = 0.4 mg/ml (6); CR concentration=114 uM; pathlength of cuvette = 0.05 cm. Isothermal titration calorimetry confirmed the binding of CR to each LC.
Ranking mAb–excipient interactions in biologics formulations by NMR spectroscopy and computational approaches
Published in mAbs, 2023
Chunting Zhang, Steven T. Gossert, Jonathan Williams, Michael Little, Marilia Barros, Barton Dear, Bradley Falk, Ankit D. Kanthe, Robert Garmise, Luciano Mueller, Andrew Ilott, Anuji Abraham
Systematic screening is conducted to choose the right excipients for a given protein, but the mechanisms by which the excipients provide stability to the protein are not fully understood. Understanding why some excipients are better stabilizers of proteins can help with developing robust biopharmaceutical formulations in an accelerated manner. Moreover, having an analytical tool to quantify and rank the factors leading to their stability simplifies the excipient selection process, making it systematic and practical. However, few studies that provide a mechanistic understanding of the stabilizing effect of excipients to maintain protein stability have been published, and no direct evidence for protein–excipient interactions was identified.19–22 Preferential exclusion by carbohydrates is one of the most prevalent mechanisms by which protein can be stabilized, adding beneficial effects on aggregation and the conformational stability of the protein.19–21 Using isothermal titration calorimetry (ITC), Kim et al. identified proteins with a high binding affinity to carbohydrates, probably due to the hydrogen bond formation between the protein-binding sites with the carbohydrate molecules.19 Souillac et al. used Fourier transform infra-red (FTIR) spectroscopic studies to show that, in the presence of carbohydrates, the secondary structure was replenished by hydrogen bonds formed between the polar groups on the surface of the protein and carbohydrate moieties during the lyophilization process.22
Computational and experimental validation of morin as adenosine deaminase inhibitor
Published in Journal of Receptors and Signal Transduction, 2018
K. G. Arun, C. S. Sharanya, C. Sadasivan
The thermodynamics of morin and ADA binding has been investigated by isothermal titration calorimetric (ITC) analysis. The calorimetric titration experiment was conducted at the temperature of 298.15 K using Microcal VP-ITC isothermal titration calorimeter (Northampton, MA, USA), as described in the manufacturer’s instruction manual. ADA (0.003 mM) and morin (0.02 mM) were prepared in 50 mM potassium phosphate buffer of pH 7.5. The degased ligand solution was injected by using a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 ml of ADA solution. Volume of ligand solution for the first injection was 3 µL and subsequently 10 µL were injected each time. A time interval of 180 s was set between two successive injections. A total of 29 injections were made. The reference power was adjusted as 10 µcal and the stirring speed was set at 307 rpm. The binding constants (K), change in enthalpy (ΔH) and change in entropy (ΔS) were determined. The Binding free energy (ΔG) was calculated from Gibbs equation,
Designing of enzyme inhibitors based on active site specificity: lessons from methyl gallate and its lipoxygenase inhibitory profile
Published in Journal of Receptors and Signal Transduction, 2018
Sharanya C. S., Arun K. G., Vijaytha V., Sabu A., Haridas M.
Thermodynamic parameters like binding constant, free energy change, and entropy of system were analyzed through isothermal titration calorimetry. For ITC 0.01 mM protein and 0.2 mM, purified compound solutions were prepared. Temperature and reference power were set as 25 °C and 15 µcal. The stirrer speed was maintained at 309 rpm. The calorimetric titrations were performed at the temperature of 298.15 K. Time was set at 10 s for each injection. A time interval of 180 s was also set between injections to allow the peak resulting from each injection to return to the baseline. Total 29 injections were made. The volume of the 1st injection was set as 3 µL to avoid inaccuracy. The heat changes between the LOX and compound were recorded. The final data were fitted by a nonlinear least squares method with ORIGIN software from the Microcal. The binding constant (K), entropy change (ΔS), binding free energy (ΔG) and enthalpy change (ΔH), were calculated using the ORIGIN.