Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

Abstract

The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhydrase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contributions to the binding affinity of ethoxzolamide to CA VB. FTSA is a high-throughput assay that measures protein thermal stabilization by added ligands. FTSA enabled determination of extremely high affinity of several compounds binding to CA VB. CA VB is one of two isoforms that are expressed in mitochondria, participate in carbon metabolism and pH homeostasis and are implicated in diseases such as obesity. Therefore CA VB is a drug target. Here a series of para-substituted tetrafluoro benzenesulfonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measurements such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is necessary to obtain the intrinsic parameters that in turn could be correlated with the chemical structure of the inhibitors. Intrinsic dissociation constants of the inhibitors were estimated and they reached 1 pM, one of the strongest binding reactions observed between any protein-ligand binding.

Identification and Analysis of Long Non-coding RNAs in Leuciscus waleckii Adapted to Highly Alkaline Conditions

“…The carbon anhydrase (CA) gene family plays an important role in CO 2 emission and acid-base regulation in fish (Gilmour and Perry, 2009;Fehsenfeld and Weihrauch, 2013). CA-VB is widely distributed in the tissues of humans and mice and participates in carbon metabolism and pH homeostasis (Kasiliauskait et al, 2015). Previous studies have also found that CA and CAHZ genes are differentially expressed in populations of L. waleckii living in a highly alkaline environment (Xu et al, 2013b;Chen et al, 2019); these findings concur with our present data.…”

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…Solid curve is a fit of the model, solid line is intrinsic Gibbs energy of binding, dashed line is a fraction of ligand, dotted line is a fraction of protein. Experiments were performed in universal buffer made of 50 mM sodium phosphate, 50 mM sodium acetate, and 25 mM sodium borate containing 100 mM NaCl and up to 2% DMSO as previously described in: CA I and CA II (Morkūnaitė et al ., 2015), CA IV (Mickevičiūtė et al ., 2017), CA VB (Kasiliauskaitė et al ., 2016), CA VI (Kazokaitė et al ., 2015), CA VII (Pilipuitytė and Matulis, 2015), CA IX (Linkuvienė et al ., 2016 b ), CA XII (Jogaitė et al ., 2013), CA XIII (Baranauskienė and Matulis, 2012), and CA XIV (Juozapaitienė et al ., 2016). All panels are drawn at the same scale to help visualize the differences in affinities of binding.…”

Introduction of Intrinsic Kinetics of Protein–Ligand Interactions and Their Implications for Drug Design

“…To better understand the interaction mechanism and its pH dependency, we have here exploited our previous observation that it is possible to determine the intrinsic binding thermodynamic constants for CA inhibitors by analyzing the pH-dependency of these interactions − and, for the first time here, determine the intrinsic rate constants by using surface plasmon resonance (SPR) biosensor technology. Although SPR-based kinetic analysis of interactions between drug-like compounds and target proteins has become a key method for drug discovery, and has been a subject of several reviews, − it is yet not used to its full potential for characterizing the details of molecular interactions.…”