Intrinsic thermodynamics of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding to carbonic anhydrases by isothermal titration calorimetry.

Para substituted tetrafluorobenzenesulfonamides bind to carbonic anhydrases (CAs) extremely tightly and exhibit some of the strongest known protein-small ligand interactions, reaching an intrinsic affinity of 2 pM as determined by displacement isothermal titration calorimetry (ITC). The enthalpy and entropy of binding to five CA isoforms were measured by ITC in two buffers of different protonation enthalpies. The pKa values of compound sulfonamide groups were measured potentiometrically and spectrophotometrically, and enthalpies of protonation were measured by ITC in order to evaluate the proton linkage contributions to the observed binding thermodynamics. Intrinsic means the affinity of a sulfonamide anion for the Zn bound water form of CAs. Fluorination of the benzene ring significantly enhanced the observed affinities as it increased the fraction of deprotonated ligand while having little impact on intrinsic affinities. Intrinsic enthalpy contributions to the binding affinity were dominant over entropy and were more exothermic for CA I than for other CA isoforms. Thermodynamic measurements together with the X-ray crystallographic structures of protein-ligand complexes enabled analysis of structure-activity relationships in this enzyme ligand system.

Intrinsic thermodynamics of sulfonamide inhibitor binding to human carbonic anhydrases I and II.

Human carbonic anhydrase (CA) I and II are cytosolic proteins, where their expression disorders can cause diseases such as glaucoma, edema, epilepsy or cancer. There are numerous inhibitors that target these isozymes, but it is difficult to design compounds that could bind to one of these proteins specifically. The binding of sulfonamide inhibitor to a CA is linked to several protonation reactions, namely, deprotonation of the sulfonamide group, protonation of the active site zinc hydroxide and the compensating protonation-deprotonation of buffer. By performing binding experiments at various pHs and buffers, all those contributions were dissected and the "intrinsic" binding parameters were calculated. Intrinsic thermodynamic binding parameters to CA I and II were determined for such widely studied drugs as acetazolamide, ethoxzolamide, methazolamide, trifluoromethanesulfonamide and dichlorophenamide. The assignment of all contributions should enhance our understanding of the underlying energetics and increase our capability to design more potent and specific CA inhibitors.

4-amino-substituted benzenesulfonamides as inhibitors of human carbonic anhydrases.

A series of N-aryl-ß-alanine derivatives and diazobenzenesulfonamides containing aliphatic rings were designed, synthesized, and their binding to carbonic anhydrases (CA) I, II, VI, VII, XII, and XIII was studied by the fluorescent thermal shift assay and isothermal titration calorimetry. The results showed that 4-substituted diazobenzenesulfonamides were more potent CA binders than N-aryl-ß-alanine derivatives. Most of the N-aryl-ß-alanine derivatives showed better affinity for CA II while diazobenzenesulfonamides possessed nanomolar affinities towards CA I isozyme. X-ray crystallographic structures showed the modes of binding of both compound groups.

Saccharin sulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII.

A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1-10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1-10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.

Characterization of human carbonic anhydrase XII stability and inhibitor binding.

Human carbonic anhydrase isozyme XII is a transmembrane protein that is overexpressed in many human cancers. Therefore CA XII is an anticancer drug target. However, there are few compounds that specifically target CA XII. The design of specific inhibitors against CA XII relies on the detailed understanding of the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. To characterize the thermodynamic parameters of the binding of known sulfonamides, namely ethoxzolamide, acetazolamide and trifluoromethanesulfonamide, we used isothermal titration calorimetry and fluorescent thermal shift assay. The binding of these sulfonamides to CA XII was buffer and pH-dependent. Dissection of protonation-deprotonation reactions of both the water molecule bound to the CA XII active site and the sulfonamide group of the inhibitor yielded the intrinsic thermodynamic parameters of binding, such as binding enthalpy, entropy and Gibbs free energy. Thermal shift assay was also used to determine CA XII stabilities at various pH and in the presence of buffers and salts.

Discovery and validation of SIRT2 inhibitors based on tenovin-6: use of a ¹H-NMR method to assess deacetylase activity.

The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a ¹H-NMR-based method is developed and used to validate further this class of sirtuin inhibitors. A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented.