Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus

The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics.


Introduction
Thioredoxin reductases (TrxRs) belong to a family of flavoproteins with redox-active cysteine residues that link the pyridine nucleotides with sulfur-containing substrates (thioredoxin proteins) [1]. Each monomer in this homodimeric protein includes an NADPH binding site, a FAD prosthetic group, and an active site containing a redox-active disulfide [2]. TrxRs get their name because they catalyze the reduction of oxidized small protein thioredoxin (Trx), which belongs to a group of small (10-12 kDa) redox-active proteins with a conserved Trp-Cys-Gly-Pro-Cys-Lys catalytic motif [3]. Reduction of the substrate disulfide occurs when electrons are transferred from NADPH via FAD to the sulfur-containing active site of TrxR.
The Trx system-collectively comprised of Trx, TrxR and NADPH-plays an essential role in various physiological processes, including regulating cellular viability and function, redox homeostasis, reducing nucleotides to deoxyribonucleotides, and helping to detoxify the body from oxidants and free radicals [4]. Therefore, it is important as a clinical goal to target the inhibition of TrxR in pathogenic microbes, since it is involved in the above-mentioned physiological/pathological processes.  The same nine compounds were then used to run MIC assays against a panel of four other bacterial species (Table 1). Interestingly, some of the compounds showed activity against both S. aureus and E. faecalis, with compound CSD 3 5376 (therein called 5376) having the best MIC of 8 µ g/mL against S. aureus bacteria. Compound 1882 had activity against both S. aureus and E. faecalis, although the MIC values demonstrated only a modicum of efficacy against both species. (MIC values of 32 μg/mL for each species). Additional work could be done in the future to assess the efficacy of 1882 against drug-resistant strains of enterococci that include vancomycin-resistant enterococcal species. We chose compound 5376 as a lead compound because the MIC value was less than 10 μg/mL against the ATCC S. aureus strain.

Ability of Analogues of 5376 to Kill Certain Strains of S. aureus
As a further test, we chose two original compounds, 5376 and 8973, as well as five analogs of the 5376 compound (587084, 5741518, 2082-0182, 2083-1665, and 2083-1773) to perform MIC assays against five well-researched S. aureus strains and four other clinical isolates of S. aureus (Table 2). Oxacillin and vancomycin-two commercially-available anti-staphylococcal drugs-were used as positive controls, and showed good inhibition of bacterial growth with MIC values of 4 and 1 µ g/mL, respectively, for all strains. Compound 5376 showed activity against all S. aureus strains that were tested with an MIC50 of 4 μg/mL and an MIC90 of 8 μg/mL. Minimum bactericial concentration (MBC)    (Table 2). Oxacillin and vancomycin-two commercially-available anti-staphylococcal drugs-were used as positive controls, and showed good inhibition of bacterial growth with MIC values of 4 and 1 µg/mL, respectively, for all strains. Compound 5376 showed activity against all S. aureus strains that were tested with an MIC 50 of 4 µg/mL and an MIC 90 of 8 µg/mL. Minimum bactericial concentration (MBC) assays demonstrated that compound 5376 was bactericidal, reducing viable counts by 2 to 2.5 logs. None of the 5376 analogs had activity against any of the S. aureus strains. Substituted groups in both phenyl X and phenyl Y did not improve the antibacterial activity (Table 3). We found that substituting bromine with a methyl group (compound 5741518) or a hydrogen (compound 5870804) at the same para-position of phenyl X eliminated activity. Other substitutions that eliminated activity were: (1) changing the carboxylate group of phenyl Y to a carboxylate ester (compound 2083-1665) and (2) moving bromine to the meta-position instead of the ortho-position of phenyl X (compound 2083-1773). assays demonstrated that compound 5376 was bactericidal, reducing viable counts by 2 to 2.5 logs. None of the 5376 analogs had activity against any of the S. aureus strains.   Substituted groups in both phenyl X and phenyl Y did not improve the antibacterial activity (Table 3). We found that substituting bromine with a methyl group (compound 5741518) or a hydrogen (compound 5870804) at the same para-position of phenyl X eliminated activity. Other substitutions that eliminated activity were: (1) changing the carboxylate group of phenyl Y to a carboxylate ester (compound 2083-1665) and (2) moving bromine to the meta-position instead of the ortho-position of phenyl X (compound 2083-1773).

Molecular Modeling
Docking was performed into two different crystal structures: the structure for S. aureus TrxR (PDB file 4GCM) and the structure for M. tuberculosis TrxR (PDB file 2A87) [7]. Ligand interactions with specific residues of the protein binding site help elucidate the molecular basis of inhibition by our lead compound 5376 with both structures (Figure 2A,B). Based on the molecular model of the compound docked in the M. tuberculosis crystal structure (2A87), the guanidinium group of Arg186 forms an ionic bond with the carboxylate end of the compound (Figure 3). Both Arg127 and Arg292

Molecular Modeling
Docking was performed into two different crystal structures: the structure for S. aureus TrxR (PDB file 4GCM) and the structure for M. tuberculosis TrxR (PDB file 2A87) [7]. Ligand interactions with specific residues of the protein binding site help elucidate the molecular basis of inhibition by our lead compound 5376 with both structures (Figure 2A,B). Based on the molecular model of the compound docked in the M. tuberculosis crystal structure (2A87), the guanidinium group of Arg186 forms an ionic bond with the carboxylate end of the compound (Figure 3). Both Arg127 and Arg292 interact with 5376 via their backbone, while Asn266 is a side chain donor. Interestingly, the 5376 compound docked

Compound 5376 Inhibits M. tuberculosis TrxR Activity and Binding of M. tuberculosis Thioredoxin C (TrxC) to M. tuberculosis TrxR
A DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) reduction assay was used to measure the inhibitory capability of compound 5376 in vitro. In this assay, thioredoxin (TrxC) is used as a substrate for TrxR. If a compound binds in place of TrxC, it should prevent the production of TNB (1,3,5-trinitrobenzene) in a dose-dependent manner. To test if compound 5376 bound only the NADPH/TrxC binding site, we examined rates of TrxR-catalyzed NADP reduction at various concentrations of TrxC and 5376. Data fit a competitive inhibition model that assumes that both TrxC and 5376 compete for the same binding site on TrxR ( Figure 4A). In this model, 5376 is a competitive inhibitor of TrxC, with a Ki (5376) of 1.26 ± 0.11 µ M and a Km (TrxC) of 29.7 ± 6.5 µ M. Competitive inhibition was also clear when reciprocal data were fit by linear regression on a Lineweaver-Burk plot ( Figure 4B).

Compound 5376 Inhibits M. tuberculosis TrxR Activity and Binding of M. tuberculosis Thioredoxin C (TrxC) to M. tuberculosis TrxR
A DTNB (5,5 -dithiobis(2-nitrobenzoic acid)) reduction assay was used to measure the inhibitory capability of compound 5376 in vitro. In this assay, thioredoxin (TrxC) is used as a substrate for TrxR. If a compound binds in place of TrxC, it should prevent the production of TNB (1,3,5-trinitrobenzene) in a dose-dependent manner. To test if compound 5376 bound only the NADPH/TrxC binding site, we examined rates of TrxR-catalyzed NADP reduction at various concentrations of TrxC and 5376. Data fit a competitive inhibition model that assumes that both TrxC and 5376 compete for the same binding site on TrxR ( Figure 4A). In this model, 5376 is a competitive inhibitor of TrxC, with a K i (5376) of 1.26 ± 0.11 µM and a K m (TrxC) of 29.7 ± 6.5 µM. Competitive inhibition was also clear when reciprocal data were fit by linear regression on a Lineweaver-Burk plot ( Figure 4B).

Compound 5376 Inhibits M. tuberculosis TrxR Activity and Binding of M. tuberculosis Thioredoxin C (TrxC) to M. tuberculosis TrxR
A DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) reduction assay was used to measure the inhibitory capability of compound 5376 in vitro. In this assay, thioredoxin (TrxC) is used as a substrate for TrxR. If a compound binds in place of TrxC, it should prevent the production of TNB (1,3,5-trinitrobenzene) in a dose-dependent manner. To test if compound 5376 bound only the NADPH/TrxC binding site, we examined rates of TrxR-catalyzed NADP reduction at various concentrations of TrxC and 5376. Data fit a competitive inhibition model that assumes that both TrxC and 5376 compete for the same binding site on TrxR ( Figure 4A). In this model, 5376 is a competitive inhibitor of TrxC, with a Ki (5376) of 1.26 ± 0.11 µ M and a Km (TrxC) of 29.7 ± 6.5 µ M. Competitive inhibition was also clear when reciprocal data were fit by linear regression on a Lineweaver-Burk plot ( Figure 4B).    Interestingly, other research groups have also shown that the Trx system is a novel drug target. A class of benzoisoselenazol compounds were shown to be potent inhibitors of the thioredoxin reductase of Bacillus anthracis, the causative agent for anthrax [11]. Biochemical studies found that ebselen and its analogs inhibited the activity of TrxR at a subnanomolar concentration. This study also found that these compounds exhibited a high-barrier against resistance development in Bacillus subtilis, S. aureus, Bacillus cereus, and M. tuberculosis. Another study proposed that the mechanism by which ebselen inhibits is by covalent modification of the enzyme's cysteine residues [12].
In addition, auranofin-an orally bioavailable FDA-approved anti-rheumatic drug-was shown to have potent bactericidal activities against both replicating and non-replicating M. tuberculosis based on a cell-based screen for bactericidal compounds [15]. Auranofin was also active against other Gram-positive bacteria, including B. subtilis, E. faecalis, E. faecium, and S. aureus. Their biochemical study also showed that auranofin inhibited the bacterial TrxR. They hypothesized that auranofin decreases the reducing capacity of target bacteria, thereby sensitizing them to oxidative stress from the host's immune system.
The efficacy of auranofin and five of its analogs were further evaluated in vitro against H. pylori. Auranofin completely inhibited bacterial growth at 1.2 μM and inhibited the activity purified H. pylori TrxR in a cell-free assay (IC50 ~88 nM). However, the compounds were not as toxic against HEK-293T human embryonic kidney cells. Interestingly, they discovered a synergy between auranofin and amoxicillin, suggesting that H. pylori infections can be treated by targeting both the reductive enzyme TrxR and cell wall synthesis.
We have identified a new class of antibiotics built on a pyrazolone scaffold that inhibits the growth of drug resistant S. aureus strains, potentially by inhibiting the S. aureus TrxR protein. Future work will try to confirm that the mechanism of action of 5376 is indeed the inhibition of the S. aureus TrxR as well as optimize the 5376 lead molecule.

Bacterial Species
For the routine MIC screening, a panel of four American Type Culture Collection (ATCC) strains was tested. They included S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922, and P. aeruginosa ATCC 27853. The following S. aureus strains were then used for additional MIC analysis to assess anti-S. aureus activity: N315 (USA100), MW2 (USA400), and Newman (MSSA)(provided by Jean Lee (Brigham and Young Hospital, Boston, MA, USA); JE2 (USA300 provided by the Network on Antimicrobial Resistance in Staphylococcus aureus strain repository), and multidrug-resistant Interestingly, other research groups have also shown that the Trx system is a novel drug target. A class of benzoisoselenazol compounds were shown to be potent inhibitors of the thioredoxin reductase of Bacillus anthracis, the causative agent for anthrax [11]. Biochemical studies found that ebselen and its analogs inhibited the activity of TrxR at a subnanomolar concentration. This study also found that these compounds exhibited a high-barrier against resistance development in Bacillus subtilis, S. aureus, Bacillus cereus, and M. tuberculosis. Another study proposed that the mechanism by which ebselen inhibits is by covalent modification of the enzyme's cysteine residues [12].
In addition, auranofin-an orally bioavailable FDA-approved anti-rheumatic drug-was shown to have potent bactericidal activities against both replicating and non-replicating M. tuberculosis based on a cell-based screen for bactericidal compounds [15]. Auranofin was also active against other Gram-positive bacteria, including B. subtilis, E. faecalis, E. faecium, and S. aureus. Their biochemical study also showed that auranofin inhibited the bacterial TrxR. They hypothesized that auranofin decreases the reducing capacity of target bacteria, thereby sensitizing them to oxidative stress from the host's immune system.
The efficacy of auranofin and five of its analogs were further evaluated in vitro against H. pylori. Auranofin completely inhibited bacterial growth at 1.2 µM and inhibited the activity purified H. pylori TrxR in a cell-free assay (IC50~88 nM). However, the compounds were not as toxic against HEK-293T human embryonic kidney cells. Interestingly, they discovered a synergy between auranofin and amoxicillin, suggesting that H. pylori infections can be treated by targeting both the reductive enzyme TrxR and cell wall synthesis.
We have identified a new class of antibiotics built on a pyrazolone scaffold that inhibits the growth of drug resistant S. aureus strains, potentially by inhibiting the S. aureus TrxR protein. Future work will try to confirm that the mechanism of action of 5376 is indeed the inhibition of the S. aureus TrxR as well as optimize the 5376 lead molecule.

Bacterial Species
For the routine MIC screening, a panel of four American Type Culture Collection (ATCC) strains was tested. They included S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922, and P. aeruginosa ATCC 27853. The following S. aureus strains were then used for additional MIC analysis to assess anti-S. aureus activity: N315 (USA100), MW2 (USA400), and Newman (MSSA) (provided by Jean Lee (Brigham and Young Hospital, Boston, MA, USA)); JE2 (USA300 provided by the Network on Antimicrobial Resistance in Staphylococcus aureus strain repository), and multidrug-resistant MRSA clinical isolates MC7606 (USA300), MC7769 (USA100), MC7827 (USA100), and MC7846 (a vancomycin-intermediate resistance USA100) were supplied by Marshfield Clinic. Strains of M. smegmatis and M. marinum were obtained from the University of Wisconsin-La Crosse culture collection.

Minimum Inhibitory Concentration (MIC)/Minimum Bactericial Concentration (MBC)
In vitro MIC and MBC determinations were performed on the compounds according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [20] for ATCC strains of S. aureus, E. faecalis, E. coli, and P. aeruginosa that were screened. Tetracycline, oxacillin, and vancomycin (Sigma-Aldrich, St. Louis, MO, USA) were included as control antibiotics and correlated with established MIC values for the ATCC strains. All anti-Mycobacterium MIC evaluations were performed using MIC assays in Middlebrook 7H9 broth with 10% oleic acid albumin dextrose complex (OADC) as previously described [21]. Isoniazid was used as the positive control for the mycobacterial MICs. All MIC numbers are a compilation of modal values plus range from at least three separate runs.

Thioredoxin Reductase-DTNB Assay
The activity of M. tuberculosis TrxR was examined using a DTNB (5,5 -dithiobis(2-nitrobenzoic acid)) reduction assay [22]. TrxR and TrxC were expressed and purified as previously described [23]. In the assay, TrxR used NADPH to oxidize TrxC, which spontaneously reduced DTNB. The production of TNB (1,3,5-trinitrobenzene) was monitored by measuring its absorbance at 412 nm in a SpectraMax M5 multi-mode plate reader (Molecular Devices, Sunnyvale, CA, USA). The assay was used to examine the inhibition kinetics of 5376. Each 200 µL assay contained 200 mM KPO 4 pH 7.2 mM EDTA, 2 µM bovine serum albumin, 1 mM DTNB, 500 µM NADPH, 10 nM TrxR, and 1% dimethyl sulfoxide. Concentrations of CSD 3 5376 tested were 30, 10, 3, and 0 µM, and concentrations of TrxC tested were a seven-point 1:2 dilution series ranging between 16 µM and 0.25 µM, in duplicate Assays were initiated by the addition of TrxR. Assays were done in clear 96-well non-treated microplates (Greiner BioOne 655101, Monroe, LA, USA). Since 5376 has color, compound background absorbance control wells were included that lacked TrxR and TrxC, but included all other assay components. Reaction progress was monitored for 30 min. The compound background absorbance for each concentration was subtracted from experimental wells for each time point before calculating the rate of reaction. The absorbance values were converted to concentration of TNB produced using the extinction coefficient 14,150 M −1 ·cm −1 . The rate of reaction was calculated after 8.25 min to assure a linear rate (see Appendix A Figure A1). The data were fitted to a competitive inhibition model using Graphpad Prism 6.04.

Docking
The structures of the TrxR proteins in this study were obtained from the protein data bank (www.pdb.org) [24]. The accession code for the M. tuberculosis thioredoxin reductase is 2A87, and the accession code for the S. aureus subsp. aureus Mu50 strain is 4GCM. Compounds were docked into PDB files 2A87 and 4GCM that were stripped of their ligands, water molecules, and counterions. We have decided to dock with FAD included in the structures, since both crystal structures have FAD co-crystallized. Using UCSF Chimera s Dock Prep module, histidine protonation states were calculated, and incomplete side chains were automatically filled [25]. Three-dimensional conformations of the compounds were generated using Open Babel, and were positioned using a rigid receptor and flexible ligand orienting code in AutoDock 4.2.1 (AD4, Scripps Research Institute, La Jolla, CA, USA) [26]. Both receptor and ligand were seeded with Gasteiger charges. AD4 was used for all dockings in this study. In general, the docking parameters were kept to their default values. However, the number of independent genetic algorithm runs was increased from 10 to 50, and was processed using the built-in clustering analysis with a 2.0 Å cutoff [27].

Abbreviations
The following abbreviations are used in this manuscript: DTNB Ellman′s Reagent (5,5-dithio-bis-(2-nitrobenzoic acid)) DTT 1,4-Dithiothreitol Figure A1. Reaction progress curves of wells containing varying concentrations of TrxC and 30 µM 5376. The rate of the reaction was calculated after 8.8 minutes of data collection, which is where progress curves become linear.