Conformational Changes in Alkyl Chains Determine the Thermodynamic and Kinetic Binding Profiles of Carbonic Anhydrase Inhibitors

Abstract

Thermodynamics and kinetics of protein−ligand binding are both important aspects for the design of novel drug molecules. Presently, thermodynamic data are collected with isothermal titration calorimetry, while kinetic data are mostly derived from surface plasmon resonance. The new method of kinITC provides both thermodynamic and kinetic data from calorimetric titration measurements. The present study demonstrates the convenient collection of calorimetric data suitable for both thermodynamic and kinetic analysis for two series of congeneric ligands of human carbonic anhydrase II and correlates these findings with structural data obtained by macromolecular crystallography to shed light on the importance of shape complementarity for thermodynamics and kinetics governing a protein−ligand binding event. The study shows how minute chemical alterations change preferred ligand conformation and can be used to manipulate thermodynamic and kinetic signatures of binding. They give rise to the observation that analogous n-alkyl and nalkyloxy derivatives of identical chain length swap their binding kinetic properties at unchanged binding affinity.

Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase

“…the final thermodynamic minimum 203 . These highly relevant data, which are in total contrast with the entire Matulis "intrinsic" theory, were thereafter validated by interesting work from the groups of Klebe 205,206 and Whitesides [207][208][209] , and are supported by our analysis of the hydrophobicity of the CA active site 204 as well as the report of several classes of inhibitors which anchor to zinc-bound hydroxide/water molecule 128 . Whitesides' group elegantly demonstrated using ITC, crystallography and MD that the differences in binding between homologous sulphonamide ligands stem from changes in the number and organisation of active site localised water molecules rather than (or perhaps in addition to) the release of structured water from the apposed hydrophobic surfaces 207 .…”

“…The values for k on ranged from 0.003 to 31 x 10 6 L À1 M À1 , whereas the k off range was exceedingly low, 0.01-0.05 s À1 . Thus, the inhibitory activity was entirely influenced by the association rate k on as also detailed by the groups of Klebe, Cavalli and Whitesides [203][204][205][206][207][208][209][210] , which depends primarily on hydrophobic effects. That is, another major error in the analysis of Matulis and colleagues is the lack of considering the hydrophobic effects when analysing the kinetics of association of the sulphonamide inhibitor within the active site.…”

A Proof-of-Concept Fragment Screening of a Hit-Validated 96-Compounds Library against Human Carbonic Anhydrase II

“…Notably, the coordination geometry enables a hydrogen bond between Nβ of the hydrazide group and the side chain hydroxy function of Thr199. This entails a different orientation of the phenyl moiety compared to the complex of hCAII with its prototypical inhibitor benzenesulfonamide 10 (BSA) [17], and triggers a flip of the sidechain of Leu198, which was also observed in the complex of hCAII with N-hydroxybenzamide (11) by Di Fiore et al (Figure 3) [27]. Unsurprisingly, sulfonamide 3 can be expected to bind to hCAII, and does so with the conventional geometry.…”

“…Crystals of protein complexes hCAII•1 to hCAII•9 were grown at 18 °C in a mixture of a solution containing 2.70 M ammonium sulfate and 0.1 M TRIS at pH 7.8, which was saturated with parachloromercuribenzoic acid (PCMB) and protein solution (c = 10 mg mL −1 ) in the final expression buffer (TRIS 0.05 M, pH 7.8). Then, 2 µL of each were mixed on a siliconized cover slip (Jena Bioscience) and placed on the well (24-well plate, Hampton Research), with silicon grease as sealant and 0.5 mL of the first solution in the reservoir [17]. Crystals grew within one day.…”

Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020

“…Small Chemicals, Polymers, and Minerals, [158][159][160][161][162][163][164][165][166][167] 9. Binding and Reaction Kinetics, 26,43,[168][169][170][171][172][173][174] 10. Process Development, [175][176][177][178][179][180][181][182][183][184][185][186][187][188] and 11.…”