Abstract
The binding of sulfonamides to human carbonic anhydrase II (hCAII) is a complex and long-debated example of protein-ligand recognition and interaction. In this study, we investigate the para-substituted n-alkyl and hydroxyethylene-benzenesulfonamides, providing a complete reconstruction of their binding pathway to hCAII by means of large-scale molecular dynamics simulations, density functional calculations, surface plasmon resonance (SPR) measurements, and X-ray crystallography experiments. Our analysis shows that the protein-ligand association rate (kon) dramatically increases with the ligand's hydrophobicity, pointing to the existence of a prebinding stage largely stabilized by a favorable packing of the ligand's apolar moieties with the hCAII "hydrophobic wall". The characterization of the binding pathway allows an unprecedented understanding of the structure-kinetic relationship in hCAII/benzenesulfonamide complexes, depicting a paradigmatic scenario for the multistep binding process in protein-ligand systems.
Highlights
Quantitative NMR measurements1, 2 regarding compound 5 were exclusively performed at 500.2 MHz
Chemical shifts are stated in parts per million and were referenced to tetramethylsilane (TMS) at δ 0.00 ppm, except for Quantitative NMR (qNMR) measurements, where the solvent residual peak for DMSOd6 at δ 2.50 ppm served as reference. qNMR purities for the different purity levels of compound 5 were obtained from the calculation of the observed integral values of the Certified Reference Material (CRM) and the aromatic 2`,6` protons in relation to their molar proportion to each other using the formula described below
TLC was carried out using 0.2 mm aluminium plates coated with silica gel 60 F254 (Macherey-Nagel) and the respective substance
Summary
Roberto Gaspari,† Chris Rechlin,‡ Andreas Heine,‡ Giovanni Bottegoni,† Walter Rocchia,† Daniel Schwarz,§ Jörg Bomke,§ Hans-Dieter Gerber,‡ Gerhard Klebe,*,‡ Andrea Cavalli,*,†,||. Quantitative NMR (qNMR) measurements regarding compound 5 were exclusively performed at 500.2 MHz. As Certified Reference Material (CRM), maleic acid (δ 6.26 ppm), purchased from Sigma Aldrich, was used as internal reference standard. QNMR purities for the different purity levels of compound 5 were obtained from the calculation of the observed integral values of the CRM and the aromatic 2`,6` protons in relation to their molar proportion to each other using the formula described below.. 4-(2-hydroxyethyl)-benzene-1-sulfonamide (5): The commercial compound was chromatographed twice on a prepacked silica gel column utilizing chloroform:ethyl acetate, 10:1 (v/v) as eluent. Calculation of the purity (P) of the sample (analyte) 5 according to qNMR measurements was performed by the following formula: lit.
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