Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design

Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ΔG) in a single experiment. The product of temperature and change in entropy (TΔS) can be obtained by the subtraction of ΔG from ΔH, and the change in heat capacity (ΔC p ) can be determined as a slope of the temperature dependence of the binding ΔH. Despite the apparent value of ITC in characterization of interactions, it is often forgotten that many protein-ligand binding reactions are linked to protonation-deprotonation reactions or various conformational changes. In such cases, it is important to determine the linked-reaction contributions and obtain the intrinsic values of the changes in Gibbs energy (affinity), enthalpy, and entropy. These energy values can then be used in various SAR-type structure-thermodynamics and combined with structure-kinetics correlations in drug design, when searching for small molecules that would bind the protein target molecule. This manuscript provides a detailed protocol on how to determine the intrinsic values of protein-ligand binding thermodynamics by ITC

Switching the Inhibitor‐Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors

PLBD: protein–ligand binding database of thermodynamic and kinetic intrinsic parameters

We introduce a protein–ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein–ligand crystal structures, enabling structure–thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein–ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein–ligand recognition and could be integrated into small molecule drug design

Ugdymo planavimas ir vertinimas : 4 modulinė programa

Vytauto Didžiojo universitetasŠvietimo akademij

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

Pedagogo kompetencijos : mentoriaus rengimo vadovėlis : dėstytojo knyga ; [sudarė Ona Monkevičienė, Kristina Stankevičienė]

Projektas "Studijų praktikos ir pradedančių dirbti mokytojų globos sistemos tobulinimas, parengiant mentoriaus kompetencijas turinčius pedagogus globėjus"Vytauto Didžiojo universitetasŠvietimo akademij