Search

Amyloid fibrils are protein aggregates formed by protein assembly through cross β structures. Inhibition of amyloid fibril formation may contribute to therapy against amyloid-related disorders like Parkinson's, Alzheimer's, and type 2 diabetes. Here we report that several fluorinated sulfonamide compounds, previously shown to inhibit human carbonic anhydrase, also inhibit the fibrillation of different proteins. Using a range of spectroscopic, microscopic and chromatographic techniques, we found that the two fluorinated sulfonamide compounds completely inhibit insulin fibrillation over a period of 16 h and moderately suppress α-synuclein and Aβ fibrillation. In addition, these compounds decreased cell toxicity of insulin incubated under fibrillation-inducing conditions. We ascribe these effects to their ability to maintain insulin in the native monomeric state. Molecular dynamic simulations suggest that these compounds inhibit insulin self-association by interacting with residues at the dimer interface. This highlights the general anti-aggregative properties of aromatic sulfonamides and suggests that sulfonamide compounds which inhibit carbonic anhydrase activity may have potential as therapeutic agents against amyloid-related disorders.

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.

Biophysical journal 119(8), 1513 - 1524 (2020). doi:10.1016/j.bpj.2020.08.037, 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., Published by Soc., Bethesda, Md.

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol-1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pKa values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work.

Numerous chemical compounds have been previously shown to be efficient inhibitors of human CAs. However, the primary-sulfonamide-group-bearing compounds have always stood as exceptionally strong binders. The binding affinity can be significantly increased by introducing electron-withdrawing groups near the sulfonamide group in order to increase the acidity of the amino group. Fluorine substituent is one of the strongest available electron-withdrawing groups that increase the acidity by several pH units. In this chapter, we describe the design and organic synthesis of a series of fluorine-substituted benzenesulfonamides. Furthermore, the compounds possess one or more additional bulky functional groups that determine compound selectivity towards a particular CA isoform. Such compounds have been designed to not fit the active site cavity of every CA except the targeted isoform. In some cases, the fluorine-bearing compounds reached thousand-to-million-fold selectivities and thus are expected to exhibit much smaller side effects than the non-selective compounds.

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.

A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were determined by the thermal shift assay, isothermal titration calorimetry, and stop-flow CO2 hydration assay. All fluorinated benzenesulfonamides exhibited nanomolar binding potency toward tested CAs and fluorinated benzenesulfonamides posessed higher binding potency than non-fluorinated compounds. The crystal structures of 4-[(4,6-dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetrafluorobenzenesulfonamide in complex with CA II and CA XII, and 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfonamide in complex with CA XIII were determined. The observed dissociation constants for several fluorinated compounds reached subnanomolar range for CA I isozyme. The affinity and the selectivity of the compounds towards tested isozymes are presented.

The binding and inhibition strength of a series of benzimidazo[1,2-c][1,2, 3]thiadiazole-7-sulphonamides were determined for recombinant human carbonic anhydrase isoforms I, II, and IX. The inhibition strength was determined by a stop-flow method to measure carbon dioxide hydration. Inhibitor-enzyme binding was determined by two biophysical techniques isothermal titration calorimetry and thermal shift assay. The co-crystal structure was determined by X-ray crystallography. Comparing the results obtained using three different inhibition and binding methods increased the accuracy of compound affinity ranking and the ability to determine compound inhibitory specificity towards a particular carbonic anhydrase isoform. In most cases, all three methods yielded the same results despite using very different approaches to measure the binding and inhibition reactions. Some of the compounds studied are submicromolar inhibitors of the isoform IX, a prominent cancer target.

—A series of benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulfonamides were synthesized and their binding to two carbonic anhy-drase isozymes measured by isothermal titration calorimetry (ITC). Human carbonic anhydrase I (hCAI) and bovine carbonic anhy-drase II (bCAII) bound the inhibitors with observed association constants in the range from 1.1 · 10 6 to 2.6 · 10 7 M 1 . 2007 Elsevier Ltd. All rights reserved. Carbonic anhydrases are zinc-containing metallopro-teins widespread in all life kingdoms. 1 There are 16 car-bonic anhydrase isoforms identified in mammals(humans have 15 isoforms) that differ in their subcellularlocalization and catalytic activity. 2,3 These enzymes areefficient catalysts for reversible hydration of carbondioxide to bicarbonate ðCO 2 þ H 2 O $ HCO 3 þ H þ Þ,and are thus involved in essential physiological pro-cesses such as respiration, pH and CO 2 homeostasis,electrolyte secretion, bone resorption, calcification, bio-synthetic reactions (e.g., lipogenesis, gluconeogenesis,and ureagenesis), tumorigenicity and many other physi-ological or pathological processes.

The carbonic anhydrases (CA) are established as therapeutic targets [1]. There are 12 catalytically active CA isozymes in human body. At least 30 CA sulfonamide inhibitors have been used as drugs to treat glaucoma, epileptic seizures, altitude sickness, and as diuretics. However, most of them exhibit poor selectivity towards target isozymes and result in various side effects.In this work, a class of 4-substituted-2,3,5,6-tetrafluorobenzensulfonamides as inhibitors of CA is reported. Crystal structures of CAII, CAXII and CAXIII bound with the fluorinated compounds were solved and provided structural details of inhibitor binding. The binding affinity to carbonic anhydrases I, II, VII, XII and XIII was measured by isothermal titration calorimetry and the fluorescent thermal shift assay, and inhibition was determined by stopped-flow CO2 hydration assay. The combined use of these methods has provided a detailed picture of protein-ligand interactions. Experimentally obtained binding data usually depends on various factors including temperature, pH, buffer, etc. In this study, we present intrinsic parameters of binding that are independent of experimental conditions. Structure-thermodynamics correlations were studied using intrinsic parameters. All used biophysical methods have confirmed that fluorinated sulfonamides bound stronger to CA than non-fluorinated, because of the presence of electronegative substituents that decrease the pKa of sulfonamide group and this correlates with an increase in the CA inhibitory properties [2]. A large group of fluorinated compounds possessed nanomolar affinity for selected CAs and several of them were selective towards CAI.