Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans

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Article Inhibition of Urease by Disulﬁram, an FDA-Approved Thiol Reagent Used in Humans

Ángel Gabriel Díaz-Sánchez *,†, Emilio Alvarez-Parrilla, Alejandro Martínez-Martínez, Luis Aguirre-Reyes †, Jesica Aline Orozpe-Olvera, Miguel Armando Ramos-Soto, José Alberto Núñez-Gastélum, Bonifacio Alvarado-Tenorio and Laura Alejandra de la Rosa

Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua 32310, Mexico; [email protected] (E.A.-P.); [email protected] (A.M.-M.); [email protected] (L.A.-R.); [email protected] (J.A.O.-O.); [email protected] (M.A.R.-S.); [email protected] (J.A.N.-G.); [email protected] (B.A.-T.); [email protected] (L.A.R.) * Correspondence: [email protected]; Tel.: +52-656-688-1800 † These two authors contribute equally to this work.

Academic Editor: James W. Gauld Received: 8 October 2016; Accepted: 21 November 2016; Published: 26 November 2016

Abstract: Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (L-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,20-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a “hinge” located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

Keywords: urease; inhibition; disulﬁram

1. Introduction Urease activity (E.C. 3.5.1.5) constitutes one of the biological steps in the global nitrogen cycle [1,2]. It is present in bacteria, fungi and plants and is one of the enzymes selected as a target for controlling medical [3], agricultural [4] and environmental issues [5]. Structural and mechanistic features of urease are largely addressed in the literature [6–19]. It is accepted that the active site architecture and mechanism of action are similar for all ureases, independent of the extraction source [7,13,16,18,20]. Urease is an amidohydrolase that rapidly produces the decomposition of urea into ammonia and carbamate, followed by the spontaneous decomposition of carbamate into bicarbonate and a second ammonia molecule. The reaction involves the participation of two catalytic Ni2+ ions bound to a carbamylated Lys that fulﬁlls the structural function of the active site [6,7,10,11,16,21] but does not participate directly in the catalysis and an important His residue functioning as the general acid catalyst [7]. The Ni centers and the carbamylated Lys residue are located in the active site and are relatively immobile, but His593 (Jack bean urease, JBU numeration, here after used) is located in a

Molecules 2016, 21, 1628; doi:10.3390/molecules21121628 www.mdpi.com/journal/molecules Molecules 2016,, 21,, 1628 1628 22 of of 14 15 a flexible flap that opens and closes [10] during the catalytic cycle. Therefore this acid-acting residue isflexible not always flap that in the opens proton and transfer closes [subsite,10] during and the seems catalytic to be required cycle. Therefore only inside this the acid-acting active site residue of the productiveis not always complex in the to proton assist transfer in catalysis subsite, once and the seemsflap is toin the be requiredclosed conformation only inside the[18,22,23]. active siteIt has of beenthe productive shown that complex blocking to the assist closure in catalysis of active once sitethe flap flap produces is in the a significant closed conformation inhibition [of18 urease,22,23]. activityIt has been [10,12,24,25], shown that most blocking likely by the obstructing closure of the active correct site accommodation flap produces a of significant catalytic His593. inhibition Thus, of oneurease of the activity strategies [10,12 to,24 control,25], most urease likely activity by obstructing is the use the of correctcompounds accommodation that block the of catalytic freedom His593. of the flexibleThus, one flap. of Inhibition the strategies studies to control together urease withactivity site-directed is the mutagenesis use of compounds showed that the block role of the Cys592 freedom as aof flap the hinge flexible in flap.the observed Inhibition flex studiesibility together[12,25,26]. with Mutation site-directed of the mutagenesisCys592 to Ala showed showed the that role the of HelicobacterCys592 as a pylori flap hingemutant in enzyme the observed was less flexibility susceptible [12, 25to ,inhi26].bition Mutation by epigallocatechin of the Cys592 to and Ala quercetin showed [25],that thesupportingHelicobacter the pyloriproposedmutant role enzyme for this was residu lesse. susceptible The description to inhibition of compounds by epigallocatechin interacting and blockingquercetin Cys [25], residues supporting in urease the proposed enzymes, role especial for thisly residue. residue The 592, description is extensiv ofe compounds[9,24,25,27–33]. interacting These compoundsand blocking include Cys residues those that in urease contain enzymes, groups that especially are reactive residue to 592, thiols. is extensiveIt is suggested [9,24,25 that,27– the33]. mechanismThese compounds of action include of such those compounds that contain is their groups interaction that are with reactive Cys592. to thiols. Here we It is used suggested the urease that fromthe mechanism seeds of Citrullus of action vulgaris of such (CVU), compounds a plant is enzyme, their interaction to demonstrate with Cys592. that disulfiram Here we (DSF)—a used the reactiveurease from sulphur-containing seeds of Citrullus compound vulgaris (CVU), that is a approved plant enzyme, by the to demonstrateFDA for clinical that use disulfiram in humans—is (DSF)—a a potentialreactive sulphur-containing effective urease inhibitor. compound We that also is approvedused four bycompounds the FDA for that clinical contain use groups in humans—is that are a reactivepotential to effective thiols to urease describe inhibitor. the potential We also usedinteraction four compounds of DSF with that Cys592 contain by groups means that of kinetic are reactive and molecularto thiols to docking describe experiments. the potential Molecular interaction docking of DSF play withs Cys592an important by means role ofin kineticthe rational and moleculardesign of drugs,docking being experiments. a useful tool Molecular which dockingreasonably plays predicts an important the best role orientation in the rational of one design molecule of drugs, within being the putativea useful tooltarget, which allowing reasonably the performance predicts the of best reliable orientation virtual ofscreening one molecule processes; within for the instance, putative see target, [34– 36].allowing Here the the docking performance approach of reliable seems virtualto be adequate screening to predict processes; if compounds for instance, of seethis [kind34–36 can]. Here interact the withdocking relevant approach Cys residues. seems to be adequate to predict if compounds of this kind can interact with relevant Cys residues. 2. Results 2. Results 2.1. Kinetic Characterization of Urease from C. vulgaris Seeds 2.1. Kinetic Characterization of Urease from C. vulgaris Seeds CVU kinetic parameters on the reaction of urea hydrolysis were determined. We observed a Vmax CVU kinetic parameters on the reaction of urea hydrolysis were determined. We observed a Vmax of 3841.00 ± 67.15 U/mg of protein and a Km of 2.08 ± 0.18 mM at pH of 6.80 (Figure 1). These values of 3841.00 ± 67.15 U/mg of protein and a K of 2.08 ± 0.18 mM at pH of 6.80 (Figure1). These values were compared with those reported elsewherem in the literature [37] and found to be in agreement were compared with those reported elsewhere in the literature [37] and found to be in agreement with previous work done at pH of 8.00, which are: Vmax of 3700 U/mg of protein and a Km value of 8 with previous work done at pH of 8.00, which are: V of 3700 U/mg of protein and a K value of mM. Given the observed results we assumed that ourmax CVU preparation was suitable to performm the 8 mM. Given the observed results we assumed that our CVU preparation was suitable to perform the inhibition studies. inhibition studies.

4000

3200

2400

0 v 1600 (U/mg Prot) (U/mg 800

0 0 1224364860 [Urea] mM

Figure 1. SaturationSaturation kinetics kinetics of of CitrullusCitrullus vulgaris vulgaris ureaseurease (CVU) by urea. The The observed observed initial velocities of reaction are plotted against dife diferentrent concentrations of urea. U is defined defined as the amount of enzyme + −1 −1 that produces one micromol of NH+ min−1 −·1mL . The grey line shows the best curve fitting to that produces one micromol of NH3 min3 ·mL . The grey line shows the best curve fitting to Equation ® (1).Equation Non-linear (1). Non-linear curve fitting curve and fitting plot were and plotprepared were using prepared Graph using Pad Graph Prism Pad 5® (GraphPad Prism 5 (GraphPad Software, Inc.,Software, La Jolla, Inc., CA, La Jolla,USA) CA, and USA) one of and three one typical of three experimental typical experimental results is results used ishere used as hereindicated as indicated in the Methodsin the Methods section. section.

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2.2. Kinetic Characterization of DSF Inhibition over Citrullus vulgaris Urease 2.2. Kinetic Characterization of DSF Inhibition over Citrullus vulgaris Urease It is known that DSF reacts with solvent-exposed Cys residues in some enzymes [38,39], forming a covalentIt is knownadduct thatand DSFleaving reacts a diethylthiocarbamate with solvent-exposed Cysmoiety residues (DTC) in in some the enzymesprotein (Figure [38,39], 2A). forming If this modificationa covalent adduct occurs and in leavingresidues a diethylthiocarbamatethat account for activity, moiety an (DTC) inhibition in the is protein expected. (Figure To2 A).explore If this the susceptibilitymodification of occurs CVUin to residues inhibition that by account DSF, the for urease activity, activity an inhibition was monitored is expected. in Tothe explore presence the of differentsusceptibility concentrations of CVU toof inhibitioncompound, by over DSF, differe the ureasent times activity of incubation was monitored at 37 in°C. the DSF presence produces of a ◦ time-dependentdifferent concentrations progressive of compound, loss of urease over activity different that times follows of incubation a pseudo-first at 37 C. order DSF kinetics, produces that a reachtime-dependent a plateau and progressive depend on loss inhibitor of urease concentration activity that follows (Figure a 2B). pseudo-first The observed order kinetics, remaining that enzyme reach activitya plateau at andplateau depend apparently on inhibitor corresponds concentration to the (Figure uninhibite2B). Thed enzyme observed fraction remaining at equilibrium. enzyme activity Each dataat plateau set wasapparently fitted to Equation corresponds (2). The to theobserved uninhibited inactivation enzyme kinetic fraction constant at equilibrium. increases Eachin a non-linear data set trendwas fittedas a function to Equation of (2).DSF The concentration observed inactivation used in the kinetic experiment, constant increases consistent in awith non-linear an inactivation trend as a function of DSF concentration used in the experiment, consistent with an inactivation mechanism mechanism consisting of two or more steps. One of these steps may be the binding of DSF and at least consisting of two or more steps. One of these steps may be the binding of DSF and at least the second the second could be accounting to the reaction of one of the enzyme Cys residue with the inhibitor could be accounting to the reaction of one of the enzyme Cys residue with the inhibitor and subsequent and subsequent formation of the DTC derivative. formation of the DTC derivative.

FigureFigure 2.2. KineticKinetic characterization characterization of of the the inhibition inhibition of of CVU CVU by by disulfiram. disulfiram. (A (A) )DSF DSF general general reaction reaction of solvent-accessibleof solvent-accessible Cys Cys residues residues in inenzymes. enzymes. Susce Susceptibleptible Cys Cys residues residues are carbamylatedcarbamylated with with a a diethylthiocarbamate moiety (DTC) followed by the release of a proton together with the free DTC diethylthiocarbamate moiety (DTC) followed by the release of a proton together with the free DTC moiety. (B) Time courses of inactivation of CVU pre-incubed with different DSF concentrations at moiety. (B) Time courses of inactivation of CVU pre-incubed with different DSF concentrations at 37 37 ◦C. Solid grey lines show the best curve fitting to a single exponential decay equation. Each curve is °C. Solid grey lines show the best curve fitting to a single exponential decay equation. Each curve is labelled with the DSF concentration used. (C) Inhibition kinetics of urease observed by the incubation labelled with the DSF concentration used. (C) Inhibition kinetics of urease observed by the incubation with DSF. The solid grey line shows the best curve fitting to Equation (3). The dashed grey line shows with DSF. The solid grey line shows the best curve fitting to Equation (3). The dashed grey line shows the concentration of DSF at 50% of total inhibition. (D) Inhibition kinetic pattern obtained by measuring thethe concentration saturation kinetics of DSF of CVUat 50% by ureaof total at variable/fixed inhibition. (D concentrations) Inhibition kinetic of DSF. pattern The solid obtained grey line by measuringshows the the best saturation curve fitting kinetics to Equation of CVU (5).by urea The at insert variable/fixed in D shows concentrations the Lineweaver–Burk of DSF. plotsThe solid of greythe line kinetic shows pattern the wherebest curve intersection fitting to of Equation linear curves (5). toThe abscise insert axis in D is observed,shows theconsistent Lineweaver–Burk with a plotsnon-competitive of the kinetic inhibition. pattern where Here U intersection is defined as of the line amountar curves of enzyme to abscise that producesaxis is observed, the change consistent of one withunit a of non-competitive the absorbance at inhibition. 558 nm per Here milligram U is ofdefi protein.ned as Non-linear the amount and of linear enzyme curve that fitting produces and plots the changewere preparedof one unit using of the Graph absorbance Pad Prism at 5558®. Thenm presentedper milligram experiments of protein. are Non-linear one of three and typical linear results. curve fittingChemical and plots structures were wereprepared drawn using using Graph ACD/ChemSketch Pad Prism 5®.® The(Advanced presented Chemistry experiments Development, are one of Inc., three typicalToronto, results. ON,Canada). Chemical structures were drawn using ACD/ChemSketch® (Advanced Chemistry Development, Inc., Toronto, ON, Canada). In these conditions it was observed that 50 minutes of incubation with DSF is enough time to assureIn these that apparentconditions equilibrium it was observed is reached that at50any minu usedtes concentration,of incubation with i.e., inDSF incubation is enough of time CVU to −1 assurewith that 50 µ apparentM an apparent equilibrium half-life is reached time of 13.80at any min, used a concentration,kobs of 0.050 ± i.e.,0.007 in incubation min and of aCVUplateau with 50at µM 63.60% an apparent± 1.40% half-life of the initial time velocityof 13.80 weremin, a observed kobs of 0.050 and ± after 0.007 50 min min−1 theandplateau a plateauwas at reached. 63.60% ± 1.40% of the initial velocity were observed and after 50 min the plateau was reached. For 80 µM, an apparent half-life time of 7.50 min, a kobs of 0.092 ± 0.024 min−1 and a plateau at 53.48% ± 3.05% of the initial velocity were observed around 25 min after addition of DSF.

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−1 For 80 µM, an apparent half-life time of 7.50 min, a kobs of 0.092 ± 0.024 min and a plateau at 53.48% ± 3.05% of the initial velocity were observed around 25 min after addition of DSF. In order to estimate the apparent inhibition constant (IC50), CVU was incubated for an hour at 37 ◦C with different concentrations of DSF and the remaining initial velocities of enzyme were then measured (Figure2C). The inhibition followed a hyperbolic trend with an IC50 value of −4 80.02 ± 1.30 × 10 µM, obtained by fitting data to Equation (3). This apparent Ki value was used to design an inhibition pattern experiment (Figure2D) to better explain the mechanism of DSF inhibition. Lineweaver–Burk plots of the inhibition pattern indicated a non-competitive inhibition mechanism (Figure2D insert); thus, initial velocity data in Figure2D were globally fitted to Equation (5). The obtained inhibition constant (Ki) was 67.60 ± 7.00 µM, which is comparable to IC50 value. The observed non-competitive inhibition could be explained by the depletion of active CVU forms, i.e., DSF reacts with Cys592 forming an enzyme-inhibitor complex, we hypothesized that the active site flap in enzyme-DSF complex is unable to completely close, a position that is essential for the correct accommodation of the catalytic His593.

2.3. Inhibition of C. vulgaris Urease by Other Thiol Reactive Compounds In order to investigate the susceptibility of CVU to inhibition by other known thiol reagents, and associate DSF inhibition of CVU to its possible interaction with a Cys residue, the effect of the thiol reagent DPS and other drugs that possess groups that react with thiols were tested: captopril, bithionol and quercetin-these compounds inhibit urease activity in a time dependent trend in a similar manner than for DSF. The IC50 values between these compounds were of the same order, except for DPS, which was near the submicromolar range (Figure3 and Table1). It was demonstrated that the reactivity and titration of essential thiol of urease from Klebsiella aerogenes (KAU) is affected by the presence of substrate and competitive inhibitors [40]. It was also demonstrated that pre-incubation of KAU with the active site competitive inhibitor phenyl-phosphorodiamidate (PPD), protects one Cys residue per catalytic unit from DPS modification during thiol group titration experiments, showing a possible interaction between PPD, or DPS and the active site Cys, most likely with Cys592. It has also been previously reported that quercetin exerts inhibition of KBU by its interaction with Cys592 [25]. Given these findings, it is assumed that, DPS reacts in specific manner with Cys residues, so the complete inhibition observed in CVU is produced by its interaction with essential Cys residues. It was also surmised that enzyme activity protection against DPS elicit by the pre-incubation of CVU with less reactive inhibitors could be used as a tool to detect compounds that interact with relevant Cys residues. Whether captopril, bithionol, and quercetin (here called I2) compete for at least one same site as DPS was investigated by measuring their protective effect over DPS full inhibition (Figure3E). After the incubation of CVU with I 2 for one hour, DPS was then added, and the residual activity was measured. It was found that pre-incubation of enzyme with thiol reactive compounds protected urease from the full inhibition elicited by DPS. Our results suggest that the inhibition produced by DSF is most likely due to the blocking of Cys592. The competition experiments between PPD and DPS [40] and with quercetin [25] performed in KBU also support this suggestion.

Table 1. IC50 values of CVU inhibition by Sulphur-reactive-compounds and quercetin. Span represents 1 the percentage of the inhibition, and 2 of the span was calculated from the difference between the plateau obtained by the ﬁtting and 100% of the initial activity.

1 Compound IC50 (µM) Plateau (% of Initial) 2 of Span (% of Initial) DSF 80.02 ± 1.30 × 10−4 0.00 50.00 Aldrithiol (DPS) 3.35 ± 0.81 0.00 50.00 Captopril 523.90 ± 39.62 0.00 50.00 Bithionol 376.50 ± 120.40 34.53 ± 18.23 67.26 Quercetin 154.70 ± 10.15 58.86 ± 1.52 79.43 MoleculesMolecules 20162016,, 2121,, 1628 1628 55 of of 14 15

A 120 D 120

100 100

80 80

60 60 OH (%) (%) S OH 40 NS 40 HO N residual activity residual residual activity 20 20 OH OH O 0 0 0 100 200 300 400 0 100 200 300 400 500 600 700 [DPS] μM (Aldrithiol) [Quercetin] μM B 120 E 100 HS a I2 + DPS 80 O ab quercetin N I2 60 OH c

(%) DPS 40

residual activity residual I2 + DPS a 20 M μ bithi onol I2 b 0 0 100 200 300 400 500 600 700 DPS c [Captopril] μM 120 I2 + DPS a C captopril 100 I2 ab

80 I50 = [I] Inhibidor DPS c

60 Cl Cl

(%) I + DPS 2 a 40 HO I DSF S 2 b residual activity residual OH 20 DPS c Cl Cl 0 0 100 200 300 400 500 600 700 0 25 50 75 100 125 [Bithionol] μM inhibition (%)

Figure 3. Inhibition of CVU with a specific thiol-reagent and other reactive drugs. (A) Inhibition Figure 3. Inhibition of CVU with a specific thiol-reagent and other reactive drugs. (A) Inhibition kinetics of CVU by DPS; (B) Captopril; (C) Bithionol and (D) Quercetin. The solid lines in A and kinetics of CVU by DPS; (B) Captopril; (C) Bithionol and (D) Quercetin. The solid lines in A and B B show the best curve fitting to Equation (3), and the solid lines in C and D show the best curve show the best curve fitting to Equation (3), and the solid lines in C and D show the best curve fitting fitting to Equation (4); the latter corresponds to a partial inhibition equation, where the initial and to Equation (4); the latter corresponds to a partial inhibition equation, where the initial and equilibrium residual activity (span) is considered for the IC50 calculation. The chemical structures equilibrium residual activity (span) is considered for the IC50 calculation. The chemical structures of of inhibitors are depicted in the corresponding graph and were drawn using ACD/ChemSketch®. inhibitors are depicted in the corresponding graph and were drawn using ACD/ChemSketch®. The The inhibition curve produced by DSF is depicted in Figure2C. The dashed grey line shows the inhibitionconcentration curve of inhibitorproduced at by 50% DSF of totalis depicted inhibition in (FigureE) Percentage 2C. The of dash inhibitioned grey produced line shows by drugs the concentration of inhibitor at 50% of total inhibition (E) Percentage of inhibition produced by drugs (I2: captopril, bithionol and quercetin) and DPS at the concentration equal to the IC50 value. In I2 + DPS, (ICVU2: captopril, was incubated bithionol with and a quercetin) given drug an ford DPS 1 h andat the then concentration DPS was added equal and to the immediately IC50 value. theIn I2 residual + DPS, CVU was incubated with a given drug for 1 h and then DPS was added and immediately the residual activity was measured, showing that I2 protects CVU from full DPS inhibition. The experiment was activityreproduced was threemeasured, times, showing and mean that and I2standard protects errorCVU arefrom plotted. full DPS A one-way inhibition. ANOVA The experiment was performed was reproducedto find significant three differencestimes, and betweenmean and treatments. standard The error different are plotted. characters A depictedone-way onANOVA bars indicate was performedsignificant differencesto find significant at p < 0.05. differences Plots were between prepared treatments. using Graph The Paddifferent Prism characters 5®. depicted on bars indicate significant differences at p < 0.05. Plots were prepared using Graph Pad Prism 5®. 2.4. Binding of DSF to Citrullus vulgaris Urease Revealed by Fluorescence Auenching Experiments 2.4. Binding of DSF to Citrullus vulgaris Urease Revealed by Fluorescence Auenching Experiments The apparent dissociation constant of DSF from CVU was determined by measuring protein fluorescenceThe apparent quenching. dissociation We found constant that of incubation DSF from of CVU enzyme was withdetermined DSF produces by measuring a quenching protein in fluorescenceCVU autofluorescence quenching. (Figure We found4). The that change incubation in fluorescence of enzyme quenching with DSF of produces CVU exercised a quenching upon the in CVUaddition autofluorescence of DSF to CVU (Figure was in 4). a hyperbolicThe change trend,in fluorescence thus fluorescence quenching data of wasCVU fitted exercised to Equation upon the (6). addition of DSF to CVU was in a hyperbolic trend, thus fluorescence data was fitted to Equation (6). The resulting DSF dissociation constant (Kd = 54.90 ± 3.90 µM) is comparable to that of IC50 and Ki Theconstants. resulting The DSF change dissociation in autofluorescence constant (K producedd = 54.90 ± by 3.90 DSF µM) was is also comparable time-dependent, to that and,of IC therefore,50 and Ki constants.all measurements The change were in made autofluorescence an hour after produced addition ofby the DSF drug. was Thisalso timetime-dependent, was sufficient and, to assuretherefore, that allthe measurements maximum changes were weremade reached. an hour after addition of the drug. This time was sufficient to assure that the maximum changes were reached.

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150

120 ) 3 340nm 90 -em 60 IF(UA x10 IF(UA 290nm Δ

ex 30

0 0 60 120 180 240 300 [Disulfiram] μM

FigureFigure 4. 4.Binding Binding of of DSF DSF to toC. C. vulgaris vulgarisurease urease at at equilibrium. equilibrium. The The change change in in the the intrinsic intrinsic fluorescence fluorescence (∆(ΔIF)IF) was was measured measured an an hour hour after after the the addition addition of of the the indicated indicated concentrations concentrations ofof DSF.DSF. A A non-linear non-linear curvecurve fit fit to to Equation Equation (5) (5) was was made made and and is indicatedis indicated by theby the solid solid grey grey line. line. Curve Curve data data is obtained is obtained in one in ofone three of identicalthree identical binding binding saturation saturation experiments. experi Non-linearments. Non-linear curve fitting curve and fitting plots and were plots prepared were ® usingprepared Graph using Pad Graph Prism Pad 5 . Prism 5®.

2.5.2.5. Binding Binding of of DSF DSF to to Citrullus Citrullus vulgaris vulgaris Urease Urease Revealed Revealed by by Docking Docking Experiments Experiments SinceSince thethe genetic genetic information information ofof CVUCVU isis not not yet yet available, available, the the possibility possibility of of DSF DSF of of interacting interacting withwith active active site site flap flap Cys592 Cys592 was was investigated investigated by docking by docking approach approach and by and a comparison by a comparison of the residues of the presentresidues in present the flap in of the all flap ureases of all homologs ureases homologs found in found the NCBI-non-redundant in the NCBI-non-redundant protein library. protein For library. the dockingFor the modelling,docking modelling, the crystallographic the crystallographic structure of st JBUructure (PDB of code JBU 4AL3)(PDB code and the 4AL3) three-dimensional and the three- modeldimensional of DSF model (ChemSpider of DSF (ChemSpider ID: 3005) were ID: used. 3005) Thewere mechanistic used. The mechanistic and structural and aspectsstructural of ureaseaspects enzymesof urease are enzymes well documented, are well documented, and it is accepted and it thatis accepted ureases that work ureases within work a general within mechanism a general regardlessmechanism of regardless whether they of whether come from they a plant,come from fungi a or plant, bacteria. fungi Here or bacteria. it is anticipated Here it thatis anticipated docking resultsthat docking of DSF results in JBU of could DSF in give JBU a could good give idea a ofgood what idea is of happening what is happening in CVU and in CVU also and what also would what happenwould happen in other in ureases. other ureases. Our docking Our docking results results show thatshow DSF that binds DSF binds to the to active the active site of site JBU of inJBU the in openthe open flap flap conformation conformation where where a direct a direct interaction interactio betweenn between one one of of the the disulfur disulfur of of the the tetrathiuram tetrathiuram andand thethe sulfursulfur atomatom ofof Cys592Cys592 isis observed observed (Figure(Figure5 A).5A). Also, Also, docking docking binding binding DPS DPS experiments experiments (ChemSpider(ChemSpider ID:ID: 58603), 58603), Captopril Captopril(ChemSpider (ChemSpider ID:ID:40130), 40130),Bithionol Bithionol(ChemSpider (ChemSpider ID:ID: 2313)2313) and and QuercetinQuercetin (ChemSpider(ChemSpider ID:ID: 4444051)4444051) werewere performedperformed (Figure(Figure5 C–F).5C–F). All All compounds compounds interact interact with with activeactive sitesite flap,flap, anan observationobservation thatthat supportssupports thethe suggestionssuggestions mademade onon thethe basisbasis ofof thethe inhibitioninhibition experiments.experiments. The The presence presence of of two two neighboring neighboring His His residues residues around around Cys592 Cys592 is is suggestive suggestive of of the the origin origin ofof the the observed observed high high thiol thiol reactivity. reactivity. It isIt wellis well known know thatn that this this type type of residue of residue causes causes a decrease a decrease in the in intrinsicthe intrinsic pKa ofpK thisa of group.this group. For theFor possibilitythe possibility of DSF of DSF to inhibit to inhibit any knownany known urease, urease, an analysis an analysis of the of evolutionarythe evolutionary conservation conservation of residues of residues located located in the active in the site active flap ofsite urease flap enzymesof urease was enzymes performed. was Basedperformed. on the Based premise on thatthe premise the motive that Cys592-His593-His594 the motive Cys592-His593-His594 accounts for accounts the inhibition for the exerted inhibition by compoundsexerted by compounds that contain that sulfur-reactive contain sulfur-reactive groups, it isgroups, proposed it is proposed that any ureasethat any containing urease containing at least theseat least three these residues three wouldresidues be inhibitedwould be byinhibited this type by of this compounds. type of compounds. A multiple alignmentA multiple with alignment a 1000 non-redundantwith a 1000 non-redundant bacterial sequences bacterial as sequences well as another as well multiple as another alignment multiple with alignment 1035 non-redundant with 1035 non- plantredundant and fungi plant sequences and fungi were sequences performed. were The performed. region corresponding The region corresponding to the flexible flapto the in allflexible bacteria, flap plantsin all bacteria, and fungi plants were and plotted fungi in were a webLogo plotted graph in a webLogo (Figure5B). graph A conserved (Figure 5B). domain A conserved for all ureases domain VCHHLfor all ureases was observed VCHHL thatwas observed corresponds thatto corresponds position 591 to position to 595 in 591 plant to 595 ureases in plant and ureases 318 to and 322 318 in bacterialto 322 in ureases. bacterial The ureases. fact that The VCHHL fact that is VCHHL a conserved is a clusterconserved from cluster bacteria from trough bacteria fungi trough and plants fungi suggestsand plants that suggests these residues that these play residues an important play an andimportant universal and role universal in the functionrole in the of function urease enzymes. of urease Meanwhile,enzymes. Meanwhile, residues of residues the C-terminal of the C-terminal region are region variable. are Sincevariable. it appears Since it that appears in all that urease in all flexible urease flaps,flexible the flaps, CHH the is present,CHH is seemspresent, that seems any that compound any compound containing containing reactive reactive sulphur sulphur groups cangroups inhibit can anyinhibit urease. any Considering urease. Considering the biochemical the biochemical characterization, characterization, the docking the and docking the sequence and conservationthe sequence results,conservation it is suggested results, it that is suggested DSF is a universal that DSF urease is a universal inhibitor. urease inhibitor.

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FigureFigure 5. 5. BindingBinding model model of of DSF DSF and and other other reactive reactive compounds compounds to to urease. urease. (A (A) )Model Model of of the the binding binding of of 2+ DSFDSF to to JBU JBU enzyme enzyme showing showing the the interaction interaction wi withth Cys592 Cys592 (dashed (dashed line, line, distance distance in in Å). Å). Ni 2+ ionsions are are shownshown in in ball ball representations, representations, acti activeve site site flexible flexible flap flap is is shown shown in in cartoon cartoon representations representations with with Cys592 Cys592 andand His593 His593 depicted depicted in in stick stick models models and and the the rest rest of of JBU JBU is is in in surface surface representation. representation. (B (B) )Frequency Frequency of of residuesresidues present present in in the the active active site site flexible flexible flap. flap. The The multiple multiple alignm alignmentent of urease of urease enzymes enzymes available available in Datain Data Bank Bank was wasused used to plot to plotfrequencies frequencies in the in lo thego. logo.The amino The amino acid residues acid residues are depicted are depicted as color as codecolor on code the on basis the basisof their of their physicochemical physicochemical proper propertiesties as asstated stated default default it itthe the weblogo weblogo server server (http://weblogo.berkeley.edu/logo.cgi:(http://weblogo.berkeley.edu/logo.cgi polar: polar amino amino acids acids (G,S,T,Y,C,Q,N) (G,S,T,Y,C,Q,N) are are green, green, basic basic (K,R,H) (K,R,H) blue,blue, acidic acidic (D,E) (D,E) red red and and hydrophobic hydrophobic (A,V,L,I,P,W,F,M) (A,V,L,I,P,W,F,M) are black). ( (CC)) Model Model of of the the binding binding of of DPS DPS ((DD)) Captopril; Captopril; (E (E) )Bithionol Bithionol and and (F (F) )Quercetin Quercetin to to JBU. JBU. All All structural structural images images were were generated generated using using USCF-ChimeraUSCF-Chimera and and are are represented represented in in a a similar similar way way to to A. A.

3.3. Discussion Discussion

3.1.3.1. Potential Potential Use Use of of Disulfiram Disulfiram to to Control Control Urease Urease Activity Activity AlthoughAlthough inhibition inhibition of of urease urease enzyme enzyme has has been been widely widely described, described, new new urease urease inhibitors inhibitors are still are beingstill being sought. sought. Inhibition Inhibition by natura byl naturalproducts products isolated isolatedfrom plants from asplants well as as other well synthetic as other designed synthetic compoundsdesigned compounds [24,30,31,41–49] [24,30,31 showed,41–49] showedin most in mostcases casescompetitive competitive or ornon-competitive non-competitive inhibition inhibition mechanismsmechanisms with with ICIC5500 rangingranging from from 22 to to 300 300 µM.µM. In In many many instances, instances, inhibition inhibition has has been been attributed attributed to to thethe interaction interaction of of compounds compounds with with a a Cys Cys residue residue in in the the hinge hinge flap flap [8,9,29–32]. [8,9,29–32]. In In KleibsiellaKleibsiella aerogenes aerogenes urease,urease, substitution substitution of of Cys Cys for for Ala Ala in in the the flexible flexible loop loop produced produced an an enzyme enzyme that that shifted shifted from from being being inhibitedinhibited by by epigallocatechin epigallocatechin and and quercetin, quercetin, to to a a barely barely inhibited inhibited form form [25], [25], suggesting suggesting that that inhibition inhibition waswas produced produced by by the the interaction interaction of of this this compound compound with with the the Cys Cys in in the the flexible flexible loop. loop. Our Our results results are are consistentconsistent with with reports reports describing describing the the interaction interaction of of inhibitors inhibitors with with Cys592 Cys592 [25,26,40]. [25,26,40]. The The observed observed valuevalue of of DSF DSF KKi iisis in in the the range range of of other other compounds compounds propos proposeded as as potential potential inhibitors inhibitors to to control control the the activityactivity of of urease urease enzymes. enzymes.

Molecules 2016, 21, 1628 8 of 15

Finding new applications for a known drug constitutes one of the strategies for discovering new antimicrobial agents. Here the inhibition produced by DSF over CVU is presented. It was shown that DSF inhibits the mitochondrial aldehyde dehydrogenase 2, through the modification of the essential Cys302 [50,51]. This is how the drug exerts its effect over ethanol metabolism in patients with chronic alcoholism. Although it is known that the thiocarbamate group of DSF interacts with multiple targets, and doses of 500 mg have been accepted in humans even with their side effects [52]. In this context, it was recently shown that DSF inhibits the carbamate kinase of Giardia lamblia and kills their trophozoites. The basis of these inhibitions was recently described in terms of structural crystallographic data [39]. This is produced by modification of a Cys residue located at the edge of the active site, preventing during the reaction an important conformational transition of a loop adjacent to the ADP/ATP binding site. DSF also inhibits betaine aldehyde dehydrogenase from Amaranthus hypochondriacus and Pseudomonas aeruginosa [51,53], and it was also suggested that DSF may be used to inhibit the growth of P. aeruginosa during infections [38,51]. Structural studies showed that the inhibition of these dehydrogenases is produced by modifying the catalytic active site Cys residue [38,51]. It has also been demonstrated that DSF and copper ions can kill Mycobacterium tuberculosis synergistically [54]. It was also shown that DSF could inhibit HCV replication to a similar extent as the clinically used antiviral agent [55]. In this study it is proposed that observed DSF urease inhibition involves the formation of a disulphide bond between DSF and the hinge Cys of the flexible flap and blocks its closure. Briefly, the proposed and accepted catalytic mechanism of urease [15,18,19] consists in the following (Figure5): (1) CVU may be in equilibrium in the closed and open conformations; (2) urea substrate binds to the enzyme in the open conformation; (3) this is followed by a change to the closed conformation, in which His593 approaches the active site and functions as the general acid needed for catalysis [7]; (4) urea + − decomposition to NH4 and carbamate ion (NH2COO ) takes place in the active site; (5) finally, the products are released of urease in the open conformation. Taking this general mechanism into account it is suggested that DSF reacts with Cys592 producing N,N-diethyldithiocarbamate (DTC) that impedes the closure of the flap. This yields a less active urease because the proton transfer steps are compromised.

3.2. Proposed Mechanism of Urease Inhibition by Disulfiram The observed inhibition of CVU by DSF is due to a reaction that produces a thiol–disulfide exchange. In Giardia lamblia carbamate kinase DSF reacts with Cys242 and forms a covalent product detected by crystallography and mass spectrometry [39]. The products consist in a DTC moiety as the one showed in Figure2A. In the case of betaine aldehyde dehydrogenase, a similar thiol–disulfide exchange with the catalytic Cys was also proposed [38,51,53]. Although in urease enzymes Cys591 seems to be highly reactive, the underlying molecular mechanism needs to be addressed. But given the large amount of information about the mechanism of function of urease, we anticipated that Cys592 in the open flap conformation is susceptible to attacking the thiuram group of DSF (Figures6 and7). (1) It is suggested that His593 is able to take the proton from thiol group of Cys592 producing the thiolate form, which is more reactive. (2) After this Cys activation, thiolate produces an attack to the thiuram moiety of DSF and generates the Urease-DTC complex. (3) Finally, Cys modification causes interference not only with the closure of the active site flap but blocks general acid His593 to efficiently exchange a proton during catalysis. MoleculesMolecules 20162016,, 2121,, 1628 1628 99 of of 14 15 Molecules 2016, 21, 1628 9 of 14

Afaster pathway faster pathway urea A (1) (2) (3) (4) urea kcat (1)Urease (2) (3) (4) + - Ureaseclosed open Ureaseopen-urea Ureaseclosed-urea kcat Ureaseclosed-NH4 -NH2-COO + - Ureaseclosed Ureaseopen Ureaseopen-urea Ureaseclosed-urea Ureaseclosed-NH4 -NH2-COO DSF DSF (5) B slower pathway (5) slower pathway DSF B + - Ureaseopen non-competitive inhibition Ureaseopen-NH4 -NH2-COO DSF + - Ureaseopen non-competitive inhibition Ureaseopen-NH4 -NH2-COO DTC DTC Urease*open Kinc DTC DTCurea Urease*open Kinc urea kcat’ DTC k ’ + - + - Urease*DTC Urease* -urea cat Urease*DTC-NH -NH2-COO NH4 + NH2-COO openDTC openDTC openDTC 4 + - + - Urease*open Urease*open-urea Urease* -NH -NH2-COO NH4 + NH2-COO open 4 FigureFigureFigure 6. 6.Scheme SchemeScheme of ofof the thethe proposed proposedproposed kinetic kinetickinetic mechanism mechanism forfor thethe inhibitioninhibition inhibition of of ureaseurease urease enzymesenzymes enzymes byby by disulfiram.disulfiram.disulfiram. (A ( A()A )In) In In the the the absence absenceabsence of of inhibitor inhibitor inhibitor ur ur ureaeaea binds binds binds to to tourease urease urease inin inthethe the open open conformation; conformation; followedfollowed followed by by thebythe thechange change change to to the to the the closed closed closed conformation conformation conformation and and and allowiallowi allowingngng generalgeneral general basebase base to to acco accommodatemmodate inin in thethe the correctcorrect position;position; subsequently subsequently subsequently the the the hydrolysis hydrolysishydrolysis is isis produced producedproduced and and the the flapflap flap changeschanges to to the the openopen open conformation;conformation; conformation; allowingallowingallowing the the the release release release of of of products productsproducts and andand water/-OH water/-OH exchange. exchange. exchange. ((BB) () B InIn) Inthethe the presencepresence presence of DSF,DSF, of DSF, formationformation formation of of adductofadduct adduct with with with urease urease urease in in inthe the the open open open conformation conformation conformation imim impedespedespedes thethe the fullfull full closureclosure closure of of the the flap flap andand and thusthus thus DSFDSF derivativederivativederivative obstructing obstructing obstructing the the the accommodation accommodation accommodation of ofof the thethe gene generalgeneralral acid acid producingproducing a a less less active active formform form ofof of urease.urease. urease. Urease*DTC-openUrease*DTC-openUrease*DTC-open is is isthe the the proposed proposed proposed form formform of ofof the thethe tra trappedtrappedpped enzyme enzyme asas opposed opposed to to a a not not fully-closedfully-closed oror or fully-openfully-openfully-open inhibited inhibited inhibited conformation. conformation. conformation.

open open H O 2 closedclosed H 2O flexibleflexible flap flap openopen* flexibleflexible flapflap openopen** OO O OO NHNH flexibleflexible flap flap O NHNH NH O Cys592Cys592 NHNH OO Cys592 NH NHNH OO Cys592 NHNH Cys592Cys592 His593 O His593 His593 His593His593 flexibleflexible flap flap SHSH O SS OO NH O O OO NH NH S NH S + NH SS N N S NH S - SS + NHNH NH NH O O S S NN NN - NH NN Cys592 SS DTC DTCDTC Cys592 S NN DTC S N SS SSHH SS S S N S NN NN SH O O His593His593 S SH 1 1 22 3 SS DSFDSF DSF NHNH N N ureaurea ureaurea urea urea protonproton transfer transfer urea urea bindingbinding site site bindingbinding site site binding site binding site positionposition binding site binding site wat wat wat wat wat wat wat Asp watwat watwat AspAsp wat wat Asp wat wat wat Asp wat wat Asp wat wat watwat Asp wat Asp His 2+ 2+ His 2+ 2+ His 2+ 2+ His 2+ 2+ His 2+ 2+Ni His His 2+Ni Ni2+ His His 2+ His 2+ Ni 2+ His Ni Ni His Ni Ni His Ni2+ Ni His Ni Ni His Ni His Ni Ni His Ni His O O His His O O His His O O His His O O His His O O His O O His His O O His His O O His metal center metal center metal center metal center N metal center N metal center N metal center N metal center Lys490 N Lys490 N Lys490 Lys490 N Lys490 Lys490 Lys490 N Lys490 Figure 7. Scheme of the proposed chemical mechanism for the inhibition of urease enzymes by FigureFigure 7. SchemeScheme of of the the proposed proposed chemical chemical mechanis mechanismm for for the the inhibition inhibition of of urease urease enzymes enzymes by by disulfiram: Empty urease is able to be in the closed or open conformation; (1) DSF binds to CVU in disulfiram:disulfiram: Empty urease isis ableable toto be be in in the the closed closed or or open open conformation; conformation; (1 )(1) DSF DSF binds binds to CVUto CVU in thein the open conformation and (2) Cys592 thiolate produces the attack to the thiuram group; (3) the theopen open conformation conformation and (and2) Cys592 (2) Cys592 thiolate thiolate produces prod theuces attack the toattack the thiuram to the group;thiuram (3 )group; the enzyme (3) the is enzyme is then modified by DTC moiety and block the flap to achieve the closed conformation; finally, enzymethen modified is then bymodified DTC moiety by DTC and moiety block and the block flap to th achievee flap to the achieve closed the conformation; closed conformation; finally, the finally, other the other DTC moiety is released from the enzyme. Chemical structures were drawn using theDTC other moiety DTC is released moiety from is released the enzyme. from Chemical the enzyme. structures Chemical were drawn structures using ACD/ChemSketchwere drawn using®. ACD/ChemSketch®. ACD/ChemSketch®. 4.4. Materials Materials and and Methods Methods 4. Materials and Methods 4.1. Urease Preparation 4.1. Urease Preparation 4.1. Urease Preparation CVU was purified to apparent homogeneity using DEAE-Sepharose (GE, Healthcare Life Sciences, CVU was purified to apparent homogeneity using DEAE-Sepharose (GE, Healthcare Life Pittsburgh, PA, USA) and Q-Sepharose Hi-trap cromatography (GE, Healthcare Life Sciences). Briefly, Sciences,CVU wasPittsburgh, purified PA, to apparentUSA) and homogeneity Q-Sepharose usHi-traping DEAE-Sepharose cromatography (G(GE,E, HealthcareHealthcare LifeLife Sciences,100Sciences). g of cleanPittsburgh, Briefly, and dry100 PA, seedsg of USA) clean were andand pulverized dryQ-Sepharose seeds in we are blenderHi-trap pulverized atcromatography room in a temperature, blender at(G roomE, andHealthcare temperature, the resulting Life Sciences).powderand the was resultingBriefly, mixed 100 powder for g four of cleanwas hours mixed and (here dry for after fourseeds sample hours were (here waspulverized handledafter sample in in a cold) blenderwas handled with at 100 room in mL cold) temperature, of extractionwith 100 andbuffermL the of (50resulting extraction mM Hepes, powder buffer 50 was(50 mM mixedmM NaCl, Hepes, for at four pH50 hoursmM of 6.8). NaCl, (here Then, afterat pH the sample of homogenate 6.8). was Then, handled the was homogenate in centrifugated cold) with was 100 at mL12,000centrifugated of rpmextraction for anat buffer hour12,000 and rpm(50 supernatant mMfor an Hepes, hour was and50 recoveredmMsupernatant NaCl, and at was pH passed recovered of 6.8). through Then, and 50 passed the mL homogenate DEAE-Sepharose through 50 mLwas centrifugatedcolumn,DEAE-Sepharose then at loaded 12,000 column, columnrpm forthen wasan loaded hour washed andcolumn supernatant with was 300 washed mL was of withrecovered extraction 300 mL and buffer of passed extraction and through eluted buffer using 50 and mL a DEAE-Sepharoselineareluted gradient using a oflinear column, NaCl gradient from then 50of loaded NaCl to 500 from column mM 50 in to was extraction 500 washedmM in buffer extraction with using300 buffer mL FPLC of using extraction equipment FPLC equipmentbuffer (Bio-Rad, and ® elutedBioLogic(Bio-Rad, using, Hercules,BioLogic a linear ®gradient CA,, Hercules, USA). of FractionsNaClCA, USA). from with 50Fractions to urease 500 mMwith activity in urease extraction were activity pooled buffer were and using dialyzedpooled FPLC and exhaustively equipment dialyzed (Bio-Rad,inexhaustively a membrane BioLogic in tube a® , membraneHercules, of 3 kDa (Sigma,CA,tube USA). of Toluca,3 kDaFractions Estado(Sigma, with deToluca, México,urease Estado activity México) de were México, against pooled extractionMéxico) and dialyzedagainst buffer. exhaustively in a membrane tube of 3 kDa (Sigma, Toluca, Estado de México, México) against

Molecules 2016, 21, 1628 10 of 15

The desalted urease, was passed through 5 mL Hi-Trap Q-Sepharose HT column, washed with 300 mL of extraction buffer and eluted with a NaCl linear gradient from 50 to 500 mM in extraction buffer. Finally, the fractions containing urease activity were pooled and used for further analysis.

4.2. Enzyme Activity Assay and Kinetic Characterization The speciﬁc ureolytic activity was assayed spectrophotometrically at 37 ◦C in a Fluostar Omega (BMG LABTECH Inc, Cary, NC, USA) spectrometer. The reaction was monitoring by the increase of ammonia concentration following the absorbance at 558 nm by The Phenol Red Assay, taking similar considerations as the one described elsewhere [15,56]. The standard urease test consisted in 0.3 mL of reaction buffer: 0.5 mM MES, 0.016 mM phenol red, and 50 mM urea, at pH 6.8. All assays were initiated by the addition of the enzyme (≈0.6 ng/mL). Initial velocity rates were determined from the initial linear portion of the reaction course-times. Saturation kinetics experiments were performed under the same conditions, except that different concentrations of urea were used (0.5 to 50 mM), and data were ﬁtted to Michaelis-Menten model (Equation (1)). All experiments presented were reproduced at least three times. Equation (1): Michaelis-Menten, V [S] v = max (1) Km + [S] where: v, is the initial velocity; Vmax, is the Maximum Velocity; [S], is the substrate concentration and Km, is the Michaelis-Menten Constant.

4.3. Inhibition Kinetic Characterization To evaluate the time and concentration-dependence of the inhibition of urease activity, the initial rates of urea hydrolysis were measured in the standard reaction mixture in the presence of DSF (tetraethylthiuram disulphide), (C2H5)2N(C=S)S–S(S=C)N(C2H5)2, during different incubation periods. DSF was purchased from Sigma-Aldrich (Toluca, Mexico). In these experiments urease reactions were started by the addition of urea to a ﬁnal concentration of 2 mM. The residual activity in percentage of the initial was plotted against the time of incubation and ﬁtted to an exponential decay equation with a plateau (Equation (2)). Equation (2): Enzyme inactivation,

−kt residual activity = (A0 − plateau) e + plateau, (2) where the residual activity is the observed activity upon the inhibition; A0, is the activity observed in the absence of the inhibitor; the plateau, is the value of Y minimum; k, is the observed one phase kinetic constant, and t is the time of incubation. Activity was transformed to percentage, where activity in the absence of DSF is 100%. To evaluate the saturation of CVU with inhibitors, enzyme was incubated for one hour at 37 ◦C with different concentrations of inhibitors in the standard reaction mixture, the reactions were started by the addition of urea to a ﬁnal concentration of 2 mM, followed by the measurement of the residual activity. Percentages of residual activity were plotted against inhibitor concentration. Since two types of behavior were observed, two different equations were used to ﬁt the data. In one case, we observed a trend that produced a full loss of activity (Equation (3)), and in the other case a partial inhibition is observed, meaning that at enzyme saturation activity value is non-zero (Equation (4)). Equation (3): Full-Inhibition Saturation Kinetics,

A ICh residual activity (%) = 0 50 (3) h h IC50 + [I] Molecules 2016, 21, 1628 11 of 15

where h, is the Hill coefﬁcient; IC50 is the apparent inhibition constant; [I], is the inhibitor concentration. Residual activity and A0 is the same as in Equation (2). Equation (4): Partially-Inhibition Saturation Kinetics,

A ICh residual activity (%) = 0 50 + plateau (4) h h IC50 + [I] where parameters and variables are same as in Equation (3), with the difference that a plateau is included. For the inhibition pattern urea saturation kinetics experiments were performed at fixed/variable concentrations of DSF and data were globally fitted to Equation (5). Equation (5): Non-Competitive Inhibition Mechanism, ! Vmax [I] [S] 1+ K v = i (5) Km + [S] where Ki, is the Non-Competitive Inhibition Constant, and all other parameters and variables are previously defined. Inhibition of CVU by Aldrithiol (2,20-dipyridyl disulphide, here in after called DPS for dipyridine sulfide), captopril ((S)-1-(3-Mercapto-2-methyl-1-oxopropyl)-L-proline), bithionol (2,20-Sulfanediylbis(4,6-dichlorophenol)) and quercetin were performed in a similar manner as for inhibition experiments with DSF. These inhibitors were purchased from Sigma-Aldrich (Toluca, Mexico). Inhibition kinetics data were fitted to the indicated equation. The protection of the activity by a given inhibitor (called I2), at a concentration equal to the IC50 value, over the inhibition produced by DPS was monitored measuring changes in initial velocity and plotting the percentage of inhibition for each treatment and expressed as mean ± SEM. Student t test was used to estimate statistical significance. Measurements of variance were performed using a one-way ANOVA. Differences were considered significant when p was less than 0.05. All experiments were performed at least three times, and in all cases the SEM justifies the accuracy of the values, except in the case of inhibition kinetics of bithionol and quercetin in which SEM is higher than 10% of the estimated value.

4.4. Binding Equilibrium Experiments The binding of DSF was monitored after apparent equilibrium by measuring the intrinsic tryptophan fluorescence intensity changes at 37 ◦C using a spectrometer (Fluostar Omega, BMG). CVU was excited at 290 nm, and fluorescence intensity was recorded at 340 nm after an hour of incubation at 37 ◦C in the absence or presence of different concentrations of DSF. In order to discard solvent fluorescent changes, a control experiment was performed in which the different volumes of solvent (same as in the titration volumes used in ligand additions) were added to urease. Fluorescence intensity changes were plotted against the DSF concentrations and fitted to Equation (5). Equation (6): Fluorescence binding saturation,

B [DSF] ∆IF = max (6) Kd + [DSF] where ∆IF, is the change in ﬂuorescence intensity at 340 nm; Bmax, is the maximum ∆IF and corresponds to the maximum binding; [DSF], is the disulﬁram concentration and Kd, is the dissociation constant.

4.5. DSF Docking In order to test the possibility of DSF interacting with Cys592 of urease, docking experiments in JBU (PDB code: 3LA4) were performed by using AutoDock Vina and analyzing the 9 best scored Molecules 2016, 21, 1628 12 of 15

solutions [35] with DSF and N,N-diethyldithiocarbamate (DTC). JBU three-dimensional structure was downloaded from PDB, and DSF was downloaded from Zinc Database (http://zinc.docking. org/substance/1529266). Three-dimensional structures where dock prepared using USCF-Chimera (https://www.cgl.ucsf.edu/chimera/), followed by the function Autodock Vina which was used to perform docking using a grid that corresponds to the whole tetrameric urease structure. The most abundant auto-validated complex model was analyzed, and the best model is presented.

5. Conclusions Our kinetic experiments, docking models and comparative study of available urease sequences support the potential use of disulfiram to control urease activity. On the other hand, disulfiram and other compounds that contain thiol reactive groups can be used to redesign better and specific urease inhibitors. Here the docking approach seems to be adequate to predict whether compounds of this kind can interact with relevant Cys residues.

Acknowledgments: We are grateful to Consejo Nacional de Ciencia y Tecnología (CONACyT) of México and Universidad Autónoma de Ciudad Juárez (UACJ) for the retention funding to AGD-S. We also acknowledge to “Programa para el Desarrollo Profesional Docente (PRODEP)” for funding to the AGD-S project: “Apoyo a la Incorporación de Nuevos PTC (No. UACJ-PTC-322)”. Author Contributions: A.G.D.-S., E.A.-P., and A.M.-M. conceived and designed the experiments; A.G.D.-S., L.A.-R., J.A.O.-O. and M.A.R.-S. performed the experiments; A.G.D.-S. and M.A.R.-S. performed the docking experiments; A.G.D.-S., E.A.-P., and A.M.-M. analyzed the data; A.M.-M., J.A.N.-G., B.A.-T., L.A.R. contributed with reagents and analysis tools; A.G.D.-S., A.M.-M. and E.A.-P. wrote the paper. All authors contributed substantially to the work reported. Conﬂicts of Interest: All authors declare no conﬂicts of interest.

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Sample Availability: Samples of all the compounds and enzyme preparation are available from the authors.

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