Steroids interfere with human carbonic anhydrase activity by using alternative binding mechanisms

Abstract Bile acids have been shown to inhibit human (h) carbonic anhydrases (CA, EC 4.2.1.1) along the gastrointestinal tract, including hCA II. The elucidation of the hormonal inhibition mechanism of the bile acid cholate to hCA II was provided in 2014 by X-ray crystallography. Herein, we extend the inhibition study to a wealth of steroids against four relevant hCA isoforms. Steroids displaying pendants and functional groups of the carboxylate, phenolic or sulfonate types appended at the tetracyclic ring were shown to inhibit the cytosolic CA II and the tumor-associated, transmembrane CA IX in a medium micromolar range (38.9–89.9 µM). Docking studies displayed the different chemotypes CA inhibition mechanisms. Molecular dynamics (MD) gave insights on the stability over time of hyocholic acid binding to CA II.


Introduction
Steroids encompass a great variety of structurally related compounds that are widely distributed in the animal and plant kingdom 1 . The common chemical feature among the diverse classes is constituted by a perhydrocyclopentanophenanthrene nucleus. Steroids include crucial compounds for life as cholesterol, bile acids, and sex hormones that play several physiological responses mediated by both genomic and non-genomic actions 2 . A variety of natural and semi-synthetic steroids are used in therapy as antiinflammatory, immunosuppressive, anabolic, and contraceptive agents, as well as for the prevention of coronary disease, and for the management of diabesity and declared AIDS. Remarkably, steroids are a wealthy source of therapeutic agents for specific forms of cancer 3 : indeed, they act as aromatase and sulfatase modulators against breast cancer, and as 5a-reductase and CYP17 inhibitors to treat benign prostatic hyperplasia and advanced prostate cancer, respectively. Additionally, semi-synthetic steroidal derivatives are very relevant for the discovery of chemical probes for exploring molecular mechanisms of action of understudied biological targets and pathways [4][5][6] . In this paper, we aim to determine the activity of a set of major naturally-occurring steroids (Figures 1 and 2) as carbonic anhydrase inhibitors (CA, EC 4.2.1.1).
Carbonic anhydrases consist in a superfamily of zinc enzymes which catalyze the reversible hydration of CO 2 into HCO 3 and protons by a metal hydroxide nucleophilic mechanism 7,8 . Seven genetically distinct CA families (a-, b-, c-, d-, f-. gand h-CAs.) are known to date [7][8][9][10] .The 15 different a-CA isoforms isolated in humans (h) feature catalytic activity, sub-cellular localization and organ/tissue distribution. Among the catalytically active isoforms, some are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), others are membrane bound (CA IV, CA IX, CA XII, CA XIV and CA XV), two of them are mitochondrial (CA VA and CA VB), and one isozyme is secreted in saliva (CA VI) 7 . A variety of human pathophysiological processes shows abnormal levels or activities of these enzymes, and this makes CA isozymes valuable targets for many pharmacological applications such as antiglaucoma drugs, diuretics, antiobesity, anticonvulsant and/or antitumor agents/ diagnostic tools 7 . Several hCAs along the gastrointestinal tract, including hCA II, have been shown to be inhibited by the 5bsteroids bile acids 11 , the primary end-products of cholesterol catabolism, resulting in a damage of the gastric mucosa. The damage produced mainly by primary bile acids and their conjugates has been related with gastric mucosal CAs inhibition in rats and humans 12 . The structural evidence of the hormonal inhibition mechanism of the bile acid cholate to hCA II has been given in 2014 13 . The carboxylate was found to bind to the zinc ion in a bivalent manner displacing the zinc-bound solvent molecule.
Herein, we extend the inhibition study to a wealth of steroids against 4 relevant hCA isoforms. Beyond understanding the interference of the CA activity by steroids, the knowledge of the CA inhibition profiles of several such derivatives could be of interest and drive the design of new non-sulfonamide-like compounds, that can be easily transported across the cellular membrane.

Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO 2 hydration activity 16 . 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 Hepes (pH 7.4) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed 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 uncatalyzed 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-deionized 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 non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [17][18][19] , and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier 20,21 .
Computational studies 4E3H 26 and 4PXX 13 crystal structures was prepared according to the Protein Preparation module in Maestro -Schr€ odinger suite, assigning bond orders, adding hydrogens, deleting water molecules, and optimizing H-bonding networks 22 . Finally, energy minimization with a root mean square deviation (RMSD) value of 0.30 was applied using an Optimized Potentials for Liquid Simulation (OPLS_2005) force field. 3 D ligand structures were prepared by Maestro 22a and evaluated for their ionization states at pH 7.4 ± 0.5 with Epik 22b OPLS-2005 force field in Macromodel 22e was used for energy minimization for a maximum number of 2500 conjugate gradient iteration and setting a convergence criterion of 0.05 kcal mol À1 Å À1 . The docking grid was generated using Glide 22f with default settings, with the center located on the center of mass of the cocrystallized ligand. Ligands were docked employing the standard precision mode (SP) retaining the best five poses of each molecule as output. The top ranked binding pose of each compound was then analyzed in terms of coordination, hydrogen bond interactions and hydrophobic contacts. The best scored binding pose of 9 to the CA II active site was submitted to a MD simulation using Desmond 23 and the OPLS2005 force field. Specifically, the system was solvated in an orthorhombic box using TIP4PEW water molecules, extended 15 Å away from any protein atom. Then, it was neutralized adding a concentration of 0.15 M chlorine and sodium ions. The simulation protocol included a starting relaxation step and a final production phase of 10 ns. In particular, the relaxation step comprised the following: (a) a stage of 100 ps at 10 K retaining the harmonic restraints on the solute heavy atoms (force constant of 50.0 kcal mol À1 Å À2 ) using the NPT ensemble with Brownian dynamics; (b) a stage of 12 ps at 10 K with harmonic restraints on the solute heavy atoms (force constant of 50.0 kcal mol À1 Å À2 ), using the NVT ensemble and Berendsen thermostat; (c) a stage of 12 ps at 10 K and 1 atm, retaining the harmonic restraints and using the NPT ensemble and Berendsen thermostat and barostat; (f) a stage of 12 ps at 300 K and 1 atm, retaining the harmonic restraints and using the NPT ensemble and Berendsen thermostat and barostat; (g) a final 24 ps stage at 300 K and 1 atm without harmonic restraints, using the NPT Berendsen thermostat and barostat. The final production phase of MD was run using a canonical the NPT Berendsen ensemble at temperature 300 K. During the MD simulation, a time step of 2 fs was used while constraining the bond lengths of hydrogen atoms with the M-SHAKE algorithm. The atomic coordinates of the system were saved every 50 ps along the MD trajectory. The occupancy of intermolecular hydrogen bonds and hydrophobic contacts was calculated along the production phase of the MD simulation with the Simulation Interaction Diagram tools implemented in Maestro. MD snapshots were clustered with the script Cheminformatics -Clustering of Conformers from Schrodinger, using the average linkage clustering method based on the RMSD matrix between the conformers Cartesian coordinatesnon hydrogen atoms only.

Biological activity
Among the twelve catalytically active hCAs, the isoforms chosen for our studies involved the cytosolic hCA I and II (involved in a host of physiologic processes) 7 , the membrane-bound hCA IV (involved in glaucoma, retinitis pigmentosa, stroke and rheumatoid arthritis) 24,25 and the tumor-associated hCA IX (abundant in hypoxic tumors and recently validated as antitumor target) 26, 27 .
Inhibition data of steroids 1-22 against hCA I, II, IV and IX were measured by a stopped flow CO 2 hydrase assay and are shown in Table 1 16 . Acetazolamide, a clinically used sulfonamide inhibitor, was used as standard.
The data of Table 1 show that a basic requirement to address a though weak inhibitory efficacy to steroidal derivatives is the presence of a functional moiety that plays the role of zinc-binding or anchoring group to the metal-coordination center, i.e. carboxylates, sulfonates and phenols. Unlike this latter, it should be stressed that analog derivatives bearing OH moieties of the aliphatic type do not exhibit any inhibitory efficacy.
Nevertheless, the cytosolic hCA I and the membrane-bound hCA IV are inhibited by none of the assayed derivatives below 100 mM, except steroids 5, 9 and 19 which feebly affect hCA I activity with inhibition constants (K I s) of 95.9, 83.3, 87.8 mM, respectively.
The ubiquitous hCA II and tumor-associated hCA IX were comparably inhibited by carboxylic acids 1-6, 8-10, sulfonic acid 7 and phenols 19, 20 in the micromolar range spanning between 38.9 and 89.9 mM. Tauroursodeoxycholic acid (7) stands out as the most efficient hCA IX inhibitor, being instead its action the least efficient against hCA II. Repositioning of the alcoholic moieties mainly located at the outer edge of the molecular structures has been shown to slightly alter the weak inhibition profiles. The lengthening of the carboxyalkyl chain of ursodeoxycholic acid (5) by a glycine unit as in 6 does not affect the derivatives efficacy against both considered isoforms. Reduction of the enone system at ring A of testosterone (13) to 5a-steroids androstanolone (16) and androsterone (14) does not interfere with the hCA II and IX inhibitory efficacy.

Computational studies
According to the binding mechanism of the different class of CAI (phenols, sulfonates, and carboxylates) herein studied [28][29][30][31][32][33][34][35] , docking simulations were carried out on representative steroids with hCA II, namely 7, 9, 19. Sulfonate (7) and compounds bearing a phenol group (19,20) act as zinc-bound nucleophile anchoring group, whereas carboxylates (1-6, 8-10) bind directly to the catalytic Zn ion (zinc binders). It was found that the phenolic OH of a-estradiol (19) is H-bonded to the zinc-bound hydroxide ion, that is in turn stabilized by three other H-bonds, acting as donor to Thr199 and Thr200 side chain OH and as acceptor with the Thr199 backbone NH. Noteworthy, the phenolic portion of a-estradiol anchors to the nucleophile locating more externally than cocrystalized simple ligands such as hydroquinone (PDB code 4E3H), owing to the steric hindrance lined by the tetrahydrophenantrenic core. This latter is involved in p-p and p-alkyl interactions with Leu198, Thr200, Phe131, Val121, His94, Gln92, Asn67, Asn62, and Leu60 side chain. Furthermore, the docked pose features an Hbond between the alcoholic moiety of 19 and the Asn67 side chain carbonyl group. The terminal sulfonate group of tauroursodeoxycholic acid (7) anchors to the pseudo-tetracoordinated zincbound water molecule by a five H-bonds network involving the ligand, the nucleophile and the enzyme (Thr199 and Thr200). Further H-bonds stabilize the docked pose. The carboxy amidic moiety acts as acceptor by the amidic NH 2 of Gln92, the alcoholic function in C 7 position acts as donor to Ile91 and Phe70 backbone   carboxy and NH group respectively and the C 3 -OH donates a H-bonds to Glu69 side chain. Like 19, the tetrahydrophenantrenic ring of 7 was involved in Van der Waals interactions with Val143, Val207, Trp209, Leu198, Thr199, Thr200, His94, His119, Gln92, Phe131, Val121, Ile91, Glu69, and Arg58 residues (Figure 3).
According to the X-ray solved structure (PDB code 4PXX) of cholic acid (2) in complex with hCA II 13 , the carboxylic group of the docked hyocholic acid (9) directly coordinates the zinc ion in a bidentate manner, and showing a very good agreement with the positioning of the crystallographic complex ligand. Thorough analogies between cholic acid (2) crystallography and hyocholic acid (9) docking with hCA II were found. As in the X-ray solved structure, the coordination is further sustained by a H-bond occurring between the backbone NH of Thr199 and the carboxylate group. The alcoholic function in C 7 accepts an H-bond from Gln92 side chain NH 2 , and further stabilization of the pose comes from hydrophobic interactions taking place between the tetrahydrophenantrenic core and His94, His96, His119, Thr199, Thr200, Leu198, Pro201, Val121, Gln92, Phe131 and Ile91 residues. Unlike cholic acid (2), the hydroxy group at C 3 position of 9 is not in H-bond contact with Glu69.
The statistical stability of the docked pose of hyocholic acid (9) as well as the stability of the H-bonds network involving the hydroxy moieties has been studied by analyzing the ligand conformation and the positional changes upon 10 ns molecular dynamics (MD) simulation of 9 docked to hCA II, as starting point. The blue line in Figure 2(B) shows how the protein C-alphas evolve over the 10 ns MD period and it indicates that the system is equilibrated. The green line, representing the aligned-ligand RMSD (only heavy atoms), gives insights onto how compound 9 is stable with respect to the enzyme binding pocket. The bidentate coordination is stably maintained (Figure 4(A)). The H-bond that is established either directly or mediated by a water molecule between the C 7 -OH and the Gln92 side chain NH 2 displayed a 60% stability over 10 ns, whereas analog interaction taking place between the C 3 -OH and Glu69 side chain is maintained for 4 ns (40% of the overall dynamics simulation time) (Figure 4(B)). The inspection of the MD trajectory points out interesting conformational transitions taking place in between 3 and 7 ns of the overall 10 ns time scale. Cluster analysis may help in identifying the relevant internal fluctuations which occur within the ligand structure during the simulation (Figures 5 and 6). Four main conformer families were found which substantially shares the zinc binding mode and differ for the orientation of the methyl group on C21 (orientation A and B according to the side -His64 or Thr200 -towards which the methyl group points to) and the hydrophobic or hydrophilic portion of hCA II occupied by the polycyclic scaffold (Figures 5 and 6). The percentage (about 60%) of time spent by 9 in the docking found conformation testifies the stability of the pose.

Conclusions
Bile acids are known to inhibit hCAs along the gastrointestinal tract, including hCA II 11 . The structural elucidation of the hormonal inhibition mechanism of the bile acid cholate to hCA II was furnished in 2014. The present study reports inhibition studies of physio-pathologically relevant CAs with a set of selected steroids. Steroids displaying pendants and functional groups of the carboxylate, phenolic or sulfonate types appended at the tetracyclic ring were shown to inhibit the cytosolic CA II and the tumor-associated, transmembrane CA IX in a medium micromolar range. Since the aforementioned functional groups are known to exert a CA inhibitory action by different binding mechanism, computational studies on representative derivatives were undertaken. A 10 ns MD gave insights on the stability of hyocholic acid (9) binding to CA II. Beyond understanding the interference of the CA activity by steroids, the knowledge of the CA inhibition profiles of such derivatives could be of interest and drive the design of novel steroidal non-sulfonamide-like CA inhibitors.

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

Funding
Ente Cassa di Risparmio di Firenze, Italy, is gratefully acknowledged for a grant to A.N (ECR 2016.0774). The Ph.D. fellowship for A.B was funded by University of Florence.