Lina Baranauskienė

A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands

Equilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands.

Inhibition and binding studies of carbonic anhydrase isozymes I, II and IX with benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides.

The binding and inhibition strength of a series of benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides were determined for recombinant human carbonic anhydrase isoforms I, II, and IX. The inhibition strength was determined by a stop-flow method to measure carbon dioxide hydration. Inhibitor-enzyme binding was determined by two biophysical techniques – isothermal titration calorimetry and thermal shift assay. The co-crystal structure was determined by X-ray crystallography. Comparing the results obtained using three different inhibition and binding methods increased the accuracy of compound affinity ranking and the ability to determine compound inhibitory specificity towards a particular carbonic anhydrase isoform. In most cases, all three methods yielded the same results despite using very different approaches to measure the binding and inhibition reactions. Some of the compounds studied are submicromolar inhibitors of the isoform IX, a prominent cancer target.

Titration Calorimetry Standards and the Precision of Isothermal Titration Calorimetry Data

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested as possible enthalpy standards. The performances of several commercial calorimeters, including the VP-ITC, ITC200, and Nano ITC-III, were validated using these suggested standard reactions.

Measurement of nanomolar dissociation constants by titration calorimetry and thermal shift assay - radicicol binding to Hsp90 and ethoxzolamide binding to CAII.

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.

Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII

BackgroundHuman carbonic anhydrases (CAs) play crucial role in various physiological processes including carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions, and various pathological processes, especially tumor progression. Therefore, CAs are interesting targets for pharmaceutical research. The structure-activity relationships (SAR) of designed inhibitors require detailed thermodynamic and structural characterization of the binding reaction. Unfortunately, most publications list only the observed thermodynamic parameters that are significantly different from the intrinsic parameters. However, only intrinsic parameters could be used in the rational design and SAR of the novel compounds.ResultsIntrinsic binding parameters for several inhibitors, including ethoxzolamide, trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIII isozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy, entropy, and the heat capacity. They were determined by titration calorimetry and thermal shift assay in a wide pH and temperature range to dissect all linked protonation reaction contributions.ConclusionsPrecise determination of the inhibitor binding thermodynamics enabled correct intrinsic affinity and enthalpy ranking of the compounds and provided the means for SAR analysis of other rationally designed CA inhibitors.

Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases.

A series of [(2-pyrimidinylthio)acetyl]benzenesulfonamides were designed and synthesized. Their binding affinities as inhibitors of several recombinant human carbonic anhydrase (CA) isozymes were determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). A group of compounds containing a chlorine atom in the benzenesulfonamide ring were found to exhibit higher selectivity but lower binding affinity toward tested CAs. The crystal structures of selected compounds in complex with CA II were determined to atomic resolution. Docking studies were performed to compare the binding modes of experimentally determined crystallographic structures with computational prediction of the pyrimidine derivative binding to CA II. Several compounds bound to select CAs with single-digit nanomolar affinities and could be used as leads for inhibitor development toward a select CA isozyme.

Indapamide-like benzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, and XIII.

A series of novel 2-chloro-5-[(1-benzimidazolyl- and 2-benzimidazolylsulfanyl)acetyl]benzene-sulfonamides were designed and synthesized. Their binding to recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII was determined by isothermal titration calorimetry and thermal shift assay. The designed S-alkylated benzimidazole derivatives exhibited stronger binding than the indapamide-like N-alkylated benzimidazoles, with the K(d) reaching about 50-100 nM with drug-targeted hCAs VII and XIII. The cocrystal structures of selected compounds with hCA II were determined by X-ray crystallography, and structural features of the binding event were revealed.

Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.

Intrinsic thermodynamics of sulfonamide inhibitor binding to human carbonic anhydrases I and II

Abstract Human carbonic anhydrase (CA) I and II are cytosolic proteins, where their expression disorders can cause diseases such as glaucoma, edema, epilepsy or cancer. There are numerous inhibitors that target these isozymes, but it is difficult to design compounds that could bind to one of these proteins specifically. The binding of sulfonamide inhibitor to a CA is linked to several protonation reactions, namely, deprotonation of the sulfonamide group, protonation of the active site zinc hydroxide and the compensating protonation–deprotonation of buffer. By performing binding experiments at various pHs and buffers, all those contributions were dissected and the “intrinsic” binding parameters were calculated. Intrinsic thermodynamic binding parameters to CA I and II were determined for such widely studied drugs as acetazolamide, ethoxzolamide, methazolamide, trifluoromethanesulfonamide and dichlorophenamide. The assignment of all contributions should enhance our understanding of the underlying energetics and increase our capability to design more potent and specific CA inhibitors.

Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid.