Abstract
Zinc-containing metalloenzyme carbonic anhydrase (CA) binds primary sulfonamides with extremely high, up to picomolar, affinity by forming a coordination bond between the negatively charged amino group and the zinc ion and making hydrogen bonds and hydrophobic contacts with other parts of the inhibitor molecule. However, N-methyl-substituted, secondary or tertiary sulfonamides bind CA with much lower affinity. In search for an explanation for this diminished affinity, a series of secondary sulfonamides were synthesized and, together with analogous primary sulfonamides, the affinities for 12 recombinant catalytically active human CA isoforms were determined by the fluorescent thermal shift assay, stopped-flow assay of the inhibition of enzymatic activity and isothermal titration calorimetry. The binding profile of secondary sulfonamides as a function of pH showed the same U-shape dependence seen for primary sulfonamides. This dependence demonstrated that there were protein binding-linked protonation reactions that should be dissected for the estimation of the intrinsic binding constants to perform structure-thermodynamics analysis. X-ray crystallographic structures of secondary sulfonamides and computational modeling dissected the atomic contributions to the binding energetics.
Graphic abstract
Secondary sulfonamides bind to carbonic anhydrases via coordination bond between the negatively charged nitrogen of alkylated amino group and Zn(ii) in the active site of CA. The binding reaction is linked to deprotonation of the amino group and protonation of the Zn(ii)-bound hydroxide. To perform the structure-thermodynamics analysis, contributions of these linked reactions must be subtracted to determine the intrinsic energetics. In this aspect, the secondary sulfonamides are similar to primary sulfonamides as CA inhibitors.
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Acknowledgements
This research was supported by Grant S-MIP-17-87 from the Research Council of Lithuania. The authors thank the local contacts at the EMBL beamlines Dr. G. Bourenkov and Dr. M. Cianci for the help with P13 and P14 EMBL beamline operations at the PETRAIII storage ring (DESY, Hamburg). This research was funded by the European Social Fund under the No 09.3.3-LMT-K-712-09-0070 “Development of Competences of Scientists, other Researchers and Students through Practical Research Activities” measure. Access to beamlines was in part supported from the European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. 283570 and by iNEXT funded by the Horizon 2020 programme of the European Commission [Grant Number 653706].
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Baronas, D., Dudutienė, V., Paketurytė, V. et al. Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases. Eur Biophys J 50, 993–1011 (2021). https://doi.org/10.1007/s00249-021-01561-1
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DOI: https://doi.org/10.1007/s00249-021-01561-1