Novel 2-indolinones containing a sulfonamide moiety as selective inhibitors of candida β-carbonic anhydrase enzyme

Abstract Inhibition of the β-carbonic anhydrase (CA, EC 4.2.1.1) from pathogenic Candida glabrata (CgNce103) by 1H-indole-2,3-dione 3-[N-(4-sulfamoylphenyl)thiosemicarbazones] 4a–m was investigated. All the compounds were found to be potent inhibitors of CgNce103, with inhibition constants in the range of 6.4-63.9 nM. The 5,7-dichloro substituted derivative 4l showed the most effective inhibition (KI of 6.4 nM) as well as the highest selectivity for inhibiting CgNce103 over the cytosolic human (h) isoforms hCA I and II. A possible binding interaction of compound 4l within the active site of CgNce103 has been proposed based on docking studies.


Introduction
Candida species are yeasts that normally live on human skin, mucous membranes, and the gastrointestinal tract without causing infections. However, in immunocompromised patients these microorganisms can cause fungal infections of the mouth or throat, mucous membranes or vagina (candidiadis) or it may enter the blood stream to cause more serious candidemia 1,2 . While many Candida species such as are responsible for these infections, Candida glabrata infections are becoming more frequent 3 . The development of drug resistance against the clinically used antifungals is a very important medical problem. Compared to other Candida strains, C. glabrata infections are more difficult to treat because of the rapid development of drug resistance against many classical antifungal agents 4,5 . The C. glabrata Carbonic Anhydrase CgNce103 enzyme may constitute a novel target for new classes of antifungals.
CgNce103 is involved in the virulence and pathogenesis of C. glabrata due to its effects on the CO 2 /HCO 3 À concentrations and as such it constitutes a novel target for antifungal agents with a novel mechanism of action 31-33 . Supuran et al. have evaluated several sulfonamides, sulfamates, sulfamides N-mono, and N,N-disubstituted dithiocarbamates against CgNce103 in their search for structurally novel inhibitors [34][35][36][37][38] . The results indicated that several of their compounds are potent CgNce103 inhibitors 34,35 . As such, the development of selective and potent CgNce103 inhibitors may result in new antifungal drugs and could overcome the problem of developing resistance against currently used antifungal drugs.
In a recent study, we described the synthesis of novel 1Hindole-2,3-dione 3-thiosemicarbazone derivatives (compounds 4a-m) carrying a sulfamoyl group at 4-position of the phenyl ring, molecular modeling studies for target enzymes and the effects of the synthesised compounds on tumor-associated hCA IX and XII enzymes 39 . All of the tested compounds showed selective inhibition in the low nanomolar range for hCA IX and XII enzymes over cytosolic isoforms hCA I and II enzymes. In this new study, these compounds were investigated for their inhibitions against Candida species CgNce103. Molecular modeling studies were conducted to rationalise the obtained inhibition values.

CgNe103 enzyme inhibition assays
An Applied Photophysics (Leatherhead, UK) stopped-flow instrument has been used for assaying the CA-catalysed CO 2 hydration activity 40 . Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM TRIS (pH 8.3) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations 36,41-47 .

Molecular docking studies
Three-dimensional structures for compounds 4a-m were generated in their lowest energy conformation (C¼N double bond in Z isomer) using the MOE software package (v2018.0101, Chemical Computing Group Inc., Montreal, QC). The sulfonamide moiety was given a negative charge (R-SO 2 NH À ), because this moiety binds to the active site Zn 2þ -ion. Subsequently, a steepest-descent energy minimisation protocol was applied using the MMFF94x force field. All ligands were docked into the active site of the CgNce103 homology model described in a previous study using the ChemScore scoring function (50 dockings per ligand; active site defined as all amino acids within 12 Å of centroid with coordinates x: 24.937, y: À20.463, z: À9.641) in the GOLD suite software package (v5.6.2, CCDC, Cambridge, UK) 36 .

CgNce103 enzyme inhibition assays
Compounds 4a-m and acetazolamide were tested in CgNce103 enzyme inhibition assays. The compounds inhibited CgNce103 with K I values in the range of 6.4-63.9 nM (Table 1). Compounds 4g (R ¼ 5-I), 4h, 4j, 4k and 4l (R ¼ 5,7-diCl) showed the best inhibitory effects against CgNce103 with K I values 19.8, 19.8, 15.0, 12.8, and 6.4 nM, respectively. Among them, compound 4l has the highest activity and the best selectivity for CgNce103 over hCA I and II. The K I values of compound 4l for hCA I and II are respectively 87-fold and 122-fold higher compared to CgNce103.
The unsubstituted compound 4a showed the highest measured K I value (K I : 63.9 nM), while the lowest K I value was measured for the 5,7-dichloro substituted compound 4l (K I : 6.4 nM). As such, there is only approximately 10-fold difference in the highest and lowest measured K I value amongst compounds 4a-m. This narrow activity window makes it rather difficult to suggest structure-activity relationships for these compounds. Together with the fact that the compounds only differ in their substituents on the 5 and 7 positions, we expect that the binding interactions of the compounds with the CgNce103 active site is very similar.

Molecular docking studies
Docking studies were performed to unravel putative ligand-enzyme binding interactions for this series of compounds. To this end, three-dimensional structures for compounds 4a-m were generated in their lowest energy conformation (C¼N double bond in Z isomer) and docked into the active site of the CgNce103 homology model as previously described 36 . In short, the crystal structure of Saccharomyces cerevisiae CA Nce103 (pdb code: 3eyx; 2.04 Å), which shows 52.3% sequence identity to CgNce103, was used as a template to construct the homology model for CgNce103 using the MOE software package 36 . The CgNce103 sequence was obtained from the National Center for Biotechnology Information (NCBI; GenBank: CAG59355.1; 219 amino acids). The template backbone was fixed during the homology model construction, The homology model with the highest contact score was selected and a steepest-descent energy minimisation protocol was applied using the AMBER12:EHT force field.  To this end, all heavy atoms of the active site residues, the zinc ion, the zinc-binding residues, and the protein backbone were fixed and the other parts were minimised using a controlled release of position restraints. The minimised structure was used in the docking studies.
The docked pose of compound 4l, the compound with the lowest measured K I value, shows an interaction of the anionic sulfonamide moiety (R-SO 2 NH -) with the active site zinc ion ( Figure  1). The phenyl group adjacent to this sulfonamide moiety forms hydrophobic interactions (edge-to-face p-p stacking) with the aromatic side chain of Phe93. The thiosemicarbazone group forms hydrogen bonds with the side chain of Asn97, while the ligand's carbonyl group forms a hydrogen bond to the side chain of Thr116. All other molecules of the tested series adopt very similar poses as described for compound 4l, as the substituents points toward the solvent and do not form an interaction with the protein.

Conclusions
In this study, novel 1H-indole-2,3-dione 3-thiosemicarbazone derivatives 4a-m carrying a sulfamoyl group at the 4-position of the phenyl ring were synthesised and tested against CgNce103 of the opportunistic pathogen C. glabrata. The compounds showed K I values in the range 6.4-63.9 nM against CgNce103 and between 2-fold and 120-fold higher K I values for the wide-spread human carbonic anhydrase isoforms I and II. Compound 4l has the highest activity and the best selectivity for CgNce103 over hCA I and II. The selectivity rates of 4l for CgNce103 over hCA I and II were found to be 4-fold and 111-fold higher than acetazolamide, respectively. Docking studies have suggested a possible binding pose for these compounds in the active site of CgNce103. These compounds may have the potential to serve as leads for developing new antifungal drugs with a novel mechanism of action which could overcome the problem of developing resistance against the classical antifungal agents.

Disclosure statement
No potential conflict of interest was reported by the authors.