molecules 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.