Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX.

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds. Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.

Thermott: A comprehensive online tool for protein-ligand binding constant determination.

The thermal shift assay is one of the most universal techniques to determine protein-ligand affinities ranging from millimolar to picomolar levels in a single ligand dosing experiment. However, the complexity of thermodynamic data analysis leads to an underuse of this technique. We have developed a user-friendly, open-source, free online analysis software to study any protein-ligand interaction thermal shift data and yield a comprehensive thermodynamic characterization of the binding reaction.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

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. 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.