PLBD: protein-ligand binding database of thermodynamic and kinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells.

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells.

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII.

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms.

A standard operating procedure for an enzymatic activity inhibition assay.

This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment.

Experimental Approaches to Identify Selective Picomolar Inhibitors for Carbonic Anhydrase IX.

Background: Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials, mostly due to low affinity and selectivity properties.

Isoform-Selective Enzyme Inhibitors by Exploring Pocket Size According to the Lock-and-Key Principle.

In the design of high-affinity and enzyme isoform-selective inhibitors, we applied an approach of augmenting the substituents attached to the benzenesulfonamide scaffold in three ways, namely, substitutions at the 3,5- or 2,4,6-positions or expansion of the condensed ring system. The increased size of the substituents determined the spatial limitations of the active sites of the 12 catalytically active human carbonic anhydrase (CA) isoforms until no binding was observed because of the inability of the compounds to fit in the active site. This approach led to the discovery of high-affinity and high-selectivity compounds for the anticancer target CA IX and antiobesity target CA VB.

Synthesis and structure-affinity relationship of chlorinated pyrrolidinone-bearing benzenesulfonamides as human carbonic anhydrase inhibitors.

A novel set of pyrrolidinone-based chlorinated benzenesulfonamide derivatives were synthesized and investigated for their binding affinity and selectivity against recombinant human carbonic anhydrases I-XIV using fluorescent thermal shift, p-nitrophenyl acetate hydrolysis and stopped-flow enzymatic inhibition assays. The hydrazones 10-22 prepared from 1-(2-chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid exhibited low nanomolar affinity against cancer-related CA IX (K in the range of 5.0-37 nM).

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors.

Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening.

Picomolar inhibitors of carbonic anhydrase: Importance of inhibition and binding assays.

The K of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO and the inability to determine the K below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K below several nM.

Volume of Hsp90 Protein-Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR.

Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied.

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.

Functionalization of fluorinated benzenesulfonamides and their inhibitory properties toward carbonic anhydrases.

Substituted tri- and tetrafluorobenzenesulfonamides were designed, synthesized, and evaluated as high-affinity and isoform-selective carbonic anhydrase (CA) inhibitors. Their binding affinities for recombinant human CA I, II, VA, VI, VII, XII, and XIII catalytic domains were determined by fluorescent thermal shift assay, isothermal titration calorimetry, and a stopped-flow CO2 hydration assay. Variation of the substituents at the 2-, 3-, and 4-positions yielded compounds with a broad range of binding affinities and isoform selectivities.

Intrinsic binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI.

Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity-purified from human saliva.

Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I, II, VII, XII, and XIII.

The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy.

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.