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. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

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. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

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. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.

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. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.

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

BACKGROUND: Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, 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. OBJECTIVE: The current review presents the design, development, and identification of the selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. METHODS AND RESULTS: Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure- activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. CONCLUSIONS: Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.

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. The x-ray crystallographic structures of compounds bound to CA IX showed the positions of the bound compounds, whereas computational modeling confirmed that steric clashes prevent the binding of these compounds to other isoforms and thus avoid undesired side effects. Such an approach, based on the Lock-and-Key principle, could be used for the development of enzyme-specific drug candidate compounds.

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 (Kd in the range of 5.0-37 nM). Compounds with triazole or oxadiazole groups attached directly to pyrrolidinone moiety bound all CAs weaker than compounds with more flexible tail groups. Chloro group at the meta position of benzenesulfonamide derivatives increased affinity to all CAs as compared with binding data for nonchlorinated compounds. The compounds have a potential for further development of CA inhibitors with higher selectivity for a particular CA isozyme.

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. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.

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

The Ki of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO2 hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO2 and the inability to determine the Ki below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the Kd below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO2 concentration were developed for the determination of kcat and KM of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor Ki. The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the Kd of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly overexpressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design.

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. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs.

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.

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. Several 2,4-substituted-3,5,6-trifluorobenzenesulfonamides were effective CA XIII inhibitors with high selectivity over off-target CA I and CA II. 3,4-Disubstituted-2,5,6-trifluorobenzenesulfonamides bound CAs with higher affinity than 2,4-disubstituted-3,5,6-trifluorobenzenesulfonamides. Many such fluorinated benzenesulfonamides were found to be nanomolar inhibitors of CA II, CA VII, tumor-associated CA IX and CA XII, and CA XIII. X-ray crystal structures of inhibitors bound in the active sites of several CA isoforms provide structure-activity relationship information for inhibitor binding affinities and selectivity.

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. The binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding-linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer- and pH-independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature-stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.

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. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.

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.