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The Evolution of Anticancer Therapies
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Analogous to Lipinski’s “Rule of Five”, it has been proposed that ideal fragments for FBDD should follow a “rule of three” (i.e., molecular weight < 300, ClogP < 3, the number of hydrogen bond donors and acceptors < 3). Although such fragments have relatively low affinity for their targets, they have high water solubility so they can be screened at higher concentrations. However, the low binding affinities pose significant challenges for screening, and so many biophysical techniques have been investigated to address this issue such as NMR, Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), and Microscale Thermophoresis (MST). Once a fragment (or a combination of fragments) has been identified, protein X-ray crystallography can then be used to obtain structural models of the protein-fragment complex. This information is then used to guide the synthesis of higher-affinity ligands.
Experimental Strategies
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
A method that does not require immobilization is microscale thermophoresis, which uses the measurement of the differential mobility of bound and free molecules when a sample is locally heated with an infrared laser. The mobility is monitored using extrinsic or intrinsic fluorescence of one of the reactants. By changing the concentration of one of the components, an estimate of Kd can be made [371,372]. Ultimate sensitivity is achieved in single-molecule assays, as discussed in Section 6.8, but these measurements are usually performed using custom-built instruments, which are optimized for the task at hand.
Microbiota-derived butyrate is an endogenous HIF prolyl hydroxylase inhibitor
Published in Gut Microbes, 2021
Ruth X. Wang, Morkos A. Henen, J. Scott Lee, Beat Vögeli, Sean P. Colgan
Microscale thermophoresis (MST) was performed on a NanoTemper Monolith NT.115 Pico instrument (NanoTemper Technologies GmbH) at 25°C using auto-detect Pico Red at 20% excitation power. His-tagged PHD2181-402 was fluorescently labeled by incubating 100 nd generation dye (100 nM) for 30 min. The reaction mixture was centrifuged for 10 min at 4 °C and 15,000 g speed. 25 nM of the protein and 16 two-fold dilution series of butyrate were loaded into sixteen standard capillaries (NanoTemper Technologies GmbH; the highest concentration was 75 mM). In the competition assay with 2-OG, a fixed concentration of 500 nM was added. The sigmoidal curves obtained were analyzed to extract the KD value using NanoTemper Technologies GmbH analysis software.
Rational optimization of a monoclonal antibody improves the aggregation propensity and enhances the CMC properties along the entire pharmaceutical process chain
Published in mAbs, 2020
Joschka Bauer, Sven Mathias, Sebastian Kube, Kerstin Otte, Patrick Garidel, Martin Gamer, Michaela Blech, Simon Fischer, Anne R Karow-Zwick
The superior developability of the in silico optimized variant produced by stable cell lines fit well with previous findings on the transiently expressed analog.6 Particularly striking for the consistent trend observed for transiently and stably produced material, the colloidal stability of the in silico optimized variant was likewise increased as indicated by a higher initial monomer content and decelerated monomer loss over time.6 Furthermore, the herein determined biological activity confirmed previously conducted microscale thermophoresis and SPR binding experiments.6 The good interplay between both studies qualifies transient material for an early screening of in silico-designed antibody variants concerning biological activity combined with manufacturability, paving the way for routine testing of in silico optimized sequences using transient expression systems early in the process chain.12,36,49 Subsequently, only the most promising candidates move into full cell line development campaigns. As a consequence, the successful combination of in silico tools with robust bioprocess development platforms will be key for future advancement within the inherently complex field of biopharmaceutical development, helping to bring innovative therapeutics to patients more quickly.
Testing for drug-human serum albumin binding using fluorescent probes and other methods
Published in Expert Opinion on Drug Discovery, 2018
Michael Ronzetti, Bolormaa Baljinnyam, Adam Yasgar, Anton Simeonov
Microscale thermophoresis (MST) has emerged as a simple and rapid biophysical approach to identify and quantify binding events in free solution. It is based on the simultaneous measurement of two distinct effects. The first is the temperature-related intensity change (TRIC) which describes the binding-dependent fluorescence intensity change of fluorophores as a function of temperature [76]. The second is the thermophoretic movement, or the directed motion of a molecule in a temperature gradient. Both of these effects are highly sensitive to binding-induced changes of molecular properties such as size, charge, shape, and hydration shell [77]. When using a titration approach, where a fluorescently labeled small molecule or protein is held constant and the binding partner is titrated, MST will allow the researcher to measure the affinity constant for a binding equilibrium. Protein fluorescence can be monitored directly through the native fluorescence of aromatic amino acids or with an attached fluorophore.