Structural basis for the design of selective inhibitors for Schistosoma mansoni dihydroorotate dehydrogenase

Biochimie 158 (2019) 180e190

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Research paper

Structural basis for the design of selective inhibitors for Schistosoma mansoni dihydroorotate dehydrogenase
dua a, Juliana S. David a, Renata A.G. Reis a, M. Cristina Nonato a, *, Ricardo A.P. de Pa Giovani P. Tomaleri a, Humberto D'Muniz Pereira b, Felipe A. Calil a a ~o Preto, Universidade de Sa ~o Paulo, Ribeira ~o Preto, SP, 14040
rio de Cristalografia de Proteínas, Faculdade de Ci^ Laborato encias Farmac^ euticas de Ribeira 903, Brazil b ~o Carlos, Universidade de Sa ~o Paulo, Sa ~o Carlos, SP, 13560-970, Brazil Centro de Biotecnologia Molecular Estrutural, Instituto de Física de Sa

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 September 2018 Accepted 10 January 2019 Available online 19 January 2019

Trematode worms of the genus Schistosoma are the causing agents of schistosomiasis, a parasitic disease responsible for a considerable economic and healthy burden worldwide. In the present work, the characterization of the enzyme dihydroorotate dehydrogenase from Schistosoma mansoni (SmDHODH) is presented. Our studies demonstrated that SmDHODH is a member of class 2 DHODHs and catalyzes the oxidation of dihydroorotate into orotate using quinone as an electron acceptor by employing a ping-pong mechanism of catalysis. SmDHODH homology model showed the presence of all structural features reported for class 2 DHODH enzymes and reveal the presence of an additional protuberant domain predicted to fold as a flexible loop and absent in the other known class 2 DHODHs. Molecular dynamics simulations showed that the ligand-free forms of SmDHODH and HsDHODH undergo different rearrangements in solution. Well-known class 2 DHODH inhibitors were tested against SmDHODH and HsDHODH and the results suggest that the variable nature of the quinone-binding tunnel between human and parasite enzymes, as well as the differences in structural plasticity involving rearrangements of the N-terminal a-helical domain can be exploited for the design of SmDHODH selective inhibitors, as a strategy to validate DHODH as a drug target against schistosomiasis. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Keywords: Dihydroorotate dehydrogenase Homology modeling Enzyme kinetics Schistosoma mansoni Schistosomiasis Inhibitor

1. Introduction Dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway [1,2]. DHODHs follow a ping-pong mechanism of catalysis, where in the first half reaction, DHO is oxidized, accompanied by the reduction of the prosthetic group flavin mononucleotide FMN to FMNH2. In the next halfreaction, a second substrate (electron acceptor) reoxidizes the flavin cofactor for new cycles of catalysis [3]. DHODHs are divided into two major classes, class 1 and class 2, according to their cell location and preferences for electron


rio de Cristalografia de Proteínas, Departamento * Corresponding author. Laborato ^ncias Farmace ^uticas de Ribeir~ de Física e Química, Faculdade de Cie ao Preto, USP. Av.
S/N, Monte Alegre, Ribeira ~o Preto, SP, 14040-903, Brazil. Cafe E-mail address: [email protected] (M.C. Nonato).

acceptors. Class 1 DHODHs are cytosolic enzymes and utilize soluble substances such as fumarate as their final electron acceptor [3]. On the other hand, class 2 DHODHs are found anchored to membranes and use respiratory quinones to reoxidize the flavin group [4e7]. The depletion of nucleotide pools by the selective inhibition of DHODH has been exploited for the development of different therapeutic strategies [8]. Moreover, the orotic acid produced by DHODH was proposed to control transcription, suggesting that inhibition of DHODH might interfere with cell development by alternative mechanisms rather than only nucleotide shortage [9,10]. The drugs teriflunomide and brequinar, which target the class 2 human enzyme, were reported to be effective in the treatment of cancer, autoimmune and viral-mediated diseases [11e14]. Leflunomide (Arava), the prodrug of teriflunomide, was approved by the FDA for treatment of rheumatoid arthritis [15] and other DHODH inhibitors were also investigated as antibiotics [16e18], antifungal [19] and antiviral drugs [20].

https://doi.org/10.1016/j.biochi.2019.01.006 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

M.C. Nonato et al. / Biochimie 158 (2019) 180e190

In addition, there is a large interest in investigating DHODH inhibition as a strategy for the development of anti-parasitic drugs. The evaluation of DHODH as therapeutic target against trypanosomiasis and leishmaniasis is currently in progress [21e24]. Recently, studies demonstrated that Plasmodium falciparum, Plasmodium berghei and Plasmodium vivax, the parasites responsible for human malaria, are susceptible to class 2 DHODH inhibition [25e29]. In fact, Malarone, used nowadays for the treatment and prevention of malaria, is a combined preparation of praguanil hydrochloride and atovaquone, the latter being an ubiquinone analogue which inhibits class 2 DHODHs. Given the biological relevance of DHODH enzymes and their structural and functional similarities, we found reasonable to hypothesize that existing DHODH inhibitors, including approved drugs, could offer alternative routes for the control of a wide range of diseases. In particular, the search for new treatments against neglected parasitic diseases could largely benefit from the existing knowledge in drug development based on the selective inhibition of DHODH. Within this context, we are interested in evaluating DHODH as a potential target for the development of anti-schistosomiasis drugs. Schistosomiasis is a chronic parasitic disease caused by different species of blood fluke parasites of the genus Schistosoma. This disease is the deadliest among the neglected tropical disease (NTDs) and according to World Health Organization (WHO) is responsible for over 230 million of human infections spread through 77 countries worldwide. Despite being a global public health issue, the treatment for schistosomiasis depends almost exclusively on a single drug, praziquantel, a 30 years old medicine, which despite rare side effects, has induced several cases of drug resistance [30e32]. Surprisingly, not much attention has been devoted to the study of nucleotide metabolism in Schistosoma parasites. It is known that Schistosoma mansoni, responsible for intestinal schistosomiasis, cannot synthesize purine bases de novo and depends exclusively on the salvage pathway to supply their purines requirements [33]. On the other hand, all pathways of the pyrimidine metabolism are functional: de novo, salvage [34e36] and thymidylate cycle [37]. In fact, the genome sequencing project for S. mansoni reveals that this parasite possesses all six enzymes involved in de novo uridine monophosphate (UMP) biosynthesis [34,35], including DHODH (SmDHODH), suggesting the relevance of this metabolic pathway for parasite survival. Moreover, maintaining the pyrimidine pool is not the only important role played by DHODHs. In Toxoplasma gondii, for instance, DHODH plays a second essential function [38] possibly coupled to the mitochondrial respiratory activity, where it replenishes ubiquinol levels and prevents reactive oxygen species formation [39]. As a first step towards evaluating the potential of DHODH as a drug target against schistosomiasis, we cloned gene fragments encoding SmDHODH and its human homologue, HsDHODH, using identical constructs. Both enzymes were overexpressed in E. coli and purified using metal ion affinity chromatography. Tag free SmDHODH and HsDHODH were kinetically characterized and an inhibition assay was optimized to allow for the screening of ligands and cross-validation studies. Well known class 2 DHODH inhibitors, atovaquone, A77126, brequinar [5] and DSM265 [29,40,41] were tested against both enzymes and a multimethod approach combining homology modeling and comparative analysis of molecular dynamics trajectories were used to provide the molecular basis of inhibitor selectivity. Our results indicate that selective inhibition of SmDHODH is feasible and can be used towards the development of novel chemical entities to be tested for the treatment of schistosomiasis.

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2. Experimental procedures 2.1. Cloning The gene sequence coding for SmDHODH (GenBank ID: XM_002578964) was amplified by PCR using the forward primer 50 AGT GGA TCC TTG TAC TCT GGA AAT GAG CAC TTT TAT AAA G 30 and the reverse primer 50 CTG CTC GAG TTA AGA TGT CAT TTT TAA TTT ATT AAT TTC T 3’ (BamHI and XhoI restriction sites are underlined, respectively). The primers were designed to obtain a truncated version of the enzyme (Leu23- Ser379) (GenBank ID: CCD78646), named DpepSmDHODH which lacks the signal peptide and the mitochondrial membrane spanning domain. The PCR mixture consisted of 100 pmol of each primer, 0.2 mM dNTP mix, 3 mM MgCl2, 5 U Taq DNA Polymerase (Invitrogen), 1x Taq Reaction Buffer (Invitrogen) and S. mansoni cDNA plasmid library as a template in a 100 mL volume. The PCR was carried out using initial denaturation at 96  C for 180 s, followed by 35 cycles of 30 s denaturation at 96  C, annealing at 58  C for 60 s, extension at 72  C for 90 s and a final extension step of 30 min. The amplified band corresponding to the expected DpepSmDHODH size was excised from a 1% agarose ethidium bromide containing gel and purified using Wizard® SV Gel and PCR Clean-Up System (Promega). The gene was cloned into a linearized pTZ19R vector (Fermentas) and then propagated using E. coli DH5a accordingly to the manufacturer's instructions. Blue/ white screen was used to select positive colonies and after overnight culture in liquid media, pure plasmids were obtained using Wizard® SV Miniprep DNA Purification System (Promega). The DpepSmDHODH-pTZ vector and a modified version of the pET-28 vector (Novagen), containing the SUMO protein sequence [42], were digested with BamHI and XhoI restriction enzymes (New England Biolabs). The digested DpepSmDHODH gene and the pET28-SUMO vector were gel purified as earlier described. T4 DNA Ligase (New England Biolabs) was used to clone DpepSmDHODH into pET-28-SUMO generating the pET-28-SUMO-DpepSmDHODH construct. Cloning of the human DHODH (Met29-Arg395), GenBank ID: AAA50163, named DpepHsDHODH, was performed into pET-28SUMO as previously reported [14]. 2.2. Expression A single colony of E. coli Rosetta (DE3) transformed with pET-28SUMO-DpepSmDHODH or pET-28-SUMO-DpepHsDHODH plasmid was grown overnight in 10 mL of medium (20 g/L bacto-tryptone, 15 g/L yeast extract, 2 g/L Na2HPO4, 1 g/L KH2PO4 and 8 g/L NaCl) containing 30 mg/mL kanamycin and 34 mg/mL chloramphenicol at 37  C and 180 rpm and used to inoculate 1 L of fresh medium. When the O.D600nm reached 0.5, isopropylthio-b-galactoside (IPTG, Fermentas) was added to 100 mM final concentration and the temperature was lowered to 18  C. After 24 h the cells were isolated by centrifugation at 10,000 g for 8 min and kept in a freezer at 20  C until used. 2.3. Purification The same purification protocol was used for both DpepDHODHs. Cell pellets corresponding to 333 mL of culture were resuspended in 20 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 600 mM NaCl, 0.33% Thesit (Sigma), 1 mM phenylmethanesulfonylfluoride (PMSF), 10% glycerol and EDTA free SigmaFAST™ (Sigma) protease inhibitor cocktail). The cells were lysed using 15 cycles of 30 s sonication with 30 s intervals on ice with an output power of 10 W. The lysate was then maintained on ice for 30 min on a rocking shaker and centrifuged at 16,100 g for 30 min at 4  C.

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The soluble fraction was poured into 2 mL of Ni-NTA resin (Qiagen) previously equilibrated with buffer A (50 mM Tris-HCl pH 7.5, 600 mM NaCl, 10% glycerol, 0.05% Thesit) and packed in a PolyPrep chromatography column (BioRad). The wash steps consisted of passing through the column 20 mL of buffer A containing 10 mM imidazole followed by 20 mL of buffer A containing 25 mM imidazole. 6xHis-SUMO-DpepDHODH was eluted with 20 mL of buffer A containing 500 mM imidazole. The eluted sample was concentrated to 500 mL using a 10 kDa cutoff Amicon Centrifugal filter unit and loaded in a 5 mL HiTrap Desalting € column connected to an AKTA-Purifier system (GE Life Sciences). An isocratic chromatographic run was carried out with buffer A at 1 mL/min. Homemade 6xHis-ubiquitin-like protein 1 (ULP1) protease [42] was incubated with the desalted protein, for 16 h at 4  C. The sample was loaded into the Ni-NTA resin pre-equilibrated in buffer A. Tag free DpepDHODH was collected in the flow through. The 6xHis-SUMO tag and 6xHis-ULP1 were eluted from the resin with buffer A containing 500 mM imidazole. Protein purity was assessed by SDS-PAGE and quantification was carried out by absorbance using the extinction coefficient of the protein cofactor FMN, ε456nm ¼ 13080 M1 cm1 for DpepSmDHODH; and ε454nm ¼ 14260 M1 cm1 for DpepHsDHODH, calculated as described elsewhere [43]. 2.4. SmDHODH steady-state kinetics The production of orotate by DpepSmDHODH was monitored spectrophotometrically at 300 nm (3 ¼ 2650 M1cm1) in buffer 50 mM Tris pH 8.15, 150 mM KCl, 0.05% Thesit and varied concentrations of quinone Q0 (1000, 750, 500, 250, 125 and 62.5 mM) and DHO (1500, 1000, 500, 250, 125 and 62.5 mM). In a 96 well UVtransparent microplate, 198 mL of reaction mixture were added to 2 mL of the enzyme (90 nM final concentration) and the rate of orotate production was determined over time. The experiments were performed at 25  C in a SpectraMax Plus 384 plate reader (Molecular Devices). The kinetic parameters, kcat and Km were estimated from the non-linear regression fit of the data to equation (1):

kobs ¼

kcat ½DHO½Q  K m DHO ½Q  þ K m Q ½DHO þ ½DHO½Q 

(1)

Where kobs is the initial rates, kcat is the turnover number, [DHO] and [Q] are the concentrations of dihydroorotate and coenzyme Q0, respectively and KmDHO and KmQ are the Km values for the substrates dihydroorotate and quinone, respectively. 2.5. Enzyme inhibition assays Enzyme activity of both DpepSmDHODH and DpepHsDHODH was measured in the presence of the inhibitors atovaquone, brequinar, and teriflunomide (purchased at Sigma Aldrich); and DSM265 (synthesized and provided by the Broad Institute). Because these compounds can also absorb light near 300 nm, an indirect assay was employed and consisted of monitoring 2,6dichloroindophenol (DCIP) reduction at 610 nm in 96 well microplates. To start the reaction 200 mL of a solution containing 50 mM Tris pH 8.15, 150 mM KCl, 0.1% Triton X-100, 500 mM DHO, 100 mM Q0, 60 mM DCIP and different concentrations of inhibitors were gently mixed to 5 mL of enzyme. 20 nM and 40 nM were the final optimized concentrations of human and parasite enzymes, respectively. The percentage of inhibition, considering initial rates of the reaction in absence of inhibitor as 100% active, was plotted against the logarithm of inhibitor concentration and the curve

fitted using non-linear regression to equation (2) implemented on Origin Lab software:

V i ¼ V min þ

ðV max  V min Þ 1 þ 10½ðlog½IlogIC 50 Þh

(2)

where Vi is the initial velocity (s1) in presence of [I], where [I] is the inhibitor concentration. Vmin and Vmax represent the minimum and maximum plateaus, respectively, in velocity units. IC50 is the inhibitor concentration relative to the halfway velocity between Vmin and Vmax values, and h is the hill coefficient. IC50 data were determined over a range of inhibitor concentrations using triplicates in a single experiment. 2.6. Modeling Homology modeling strategy was used to predict the tertiary structure of SmDHODH. The homology model of SmDHODH was built using the coordinates from X-ray structures of rat (Rattus norvegicus), human (Homo sapiens), Plasmodium falciparum and E. coli DHODHs (PDB IDs: 1UUM [44], 3F1Q [45], 3SFK [40], 1F76 [46], respectively). Briefly, the program Modeller (version 9.11) [47] was used to generate optimized models based on the variable target function method with conjugate gradients and the refinement was performed using molecular dynamics with simulated annealing. The model was further refined with REFMAC structure idealization tool [48] and validated using MolProbity [49]. Electrostatic potential surfaces were calculated by solving the Poisson-Boltzmann equation implemented by APBS plugin [50] in the PyMOL program [51]. 2.7. Molecular dynamics simulation All simulations were performed with GROMACS 4.5.5 [52] using the TIP3P water model [53] and the OPLS-AA forcefield [54] in an orthorhombic simulation box with periodic boundary conditions. The production MD simulations were pursued for independent 100 ns at 309 K. The NVT ensemble was maintained by the v-rescale
-Hoover thermostat [55] during thermalization and by the Nose thermostat [56] during production runs. All interactions were cut off at 1 nm and the long range electrostatic interactions were treated by the PME method [57]. The crystallographic structure of HsDHODH, PDB ID 2FPV, and the homology model of SmDHODH were considered as the starting state of two independent simulations. The different conformations generated during MD simulation was clustered using the g_cluster program's default RMSD cut-off 0.1 nm from the center structure. All the conformations were sorted into two clusters. First cluster contains structures with all conformations from 30 to 60 ns, SmDHODH, and from 20 to 40 ns, HsDHODH. Second cluster contains structures with all conformations from 75 to 90 ns, SmDHODH, and 60e80 ns, HsDHODH. 3. Results and discussion 3.1. DpepDHODHs properties and enzyme production Signal P 4.1 server [58] predicted the first 22 residues of SmDHODH to constitute a signaling peptide sequence and the mitochondrial membrane spanning domain [59]. Multiple sequence alignment with representative members of class 2 DHODHs (Fig. 1) indicated the presence of the hydrophobic region, responsible for anchoring the enzyme at the intermembrane side of the mitochondria inner membrane [60e62], the quinone binding domain [2,6] and the conserved catalytic base serine at position

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203, unique features of the class 2 DHODHs [2]. Previous studies on class 2 DHODHs showed that truncation of the signal peptide and the mitochondrial membrane spanning domain did not impact on their in vitro activity [63,64]. Thus, anticipating solubility problems, we expressed an N-terminally truncated SmDHODH construct, DpepSmDHODH, where this region was intentionally deleted (Fig. 1). Tag free DpepSmDHODH was purified with the excellent yield of 40 mg/L of cell culture. SDS-PAGE analysis showed that the obtained protein migrated between 30 and 45 kDa (Supplementary Fig. S1A). This result is in agreement with the analysis made in the ProtParam server, which revealed that DpepSmDHODH, comprising residues 23 to 379, has a predicted molecular mass of 38908.4 Da [19]. This band was further confirmed to be DpepSmDHODH by N-terminal sequencing (data not shown). A similar construct for the truncated version of the human homologue enzyme, DpepHsDHODH, where the predicted mitochondrial target signal and transmembrane domain (residues 1 to 28) have been deleted (Fig. 1), was also expressed in E. coli. Tag free DpepHsDHODH was purified using the same protocol developed for DpepSmDHODH with a yield of 10 mg/L of culture and shows a similar purification profile on the SDS-PAGE (Supplementary Fig. S1B). DpepHsDHODH comprises residues 29 to 378 and has a predicted molecular mass of 39496.1 Da. 3.2. SmDHODH employs a ping-pong reaction mechanism

DpepSmDHODH showed to catalyze the oxidation of DHO to orotate using Q0 as oxidizing substrate (Fig. 2). The results obtained for the steady-state kinetic analysis demonstrated that DpepSmDHODH employs a two-site Ping-Pong steady-state mechanism of catalysis, also used by other DHODHs [67e70]. In this mechanism, in the first and reductive half-reaction, the enzyme oxidizes DHO to orotate with the concomitant reduction of FMN to FMNH2. In the second and oxidative half-reaction, FMNH2 is reoxidized by reducing the quinone (Q) to quinol. The estimated values for the kinetic parameters were: KmDHO ¼ 230 ± 30 mM; KmQ0 ¼ 170 ± 20 mM and kcat ¼ 31 ± 2 s1 (Fig. 2A). Under the same experimental conditions and using the same electron (Q0) DpepHsDHODH kinetic parameters were estimated as: KmDHO ¼ 290 ± 30 mM; KmQ0 ¼ 350 ± 40 mM and kcat ¼ 78 ± 4 s1 (Fig. 2B). 3.3. SmDHODH overall structure and dynamics As expected from the significant sequence similarity with template structures (Fig. 1), the homology model built for SmDHODH possesses all structural features reported for class 2 DHODH enzymes [5,44,46,71]. SmDHODH folds as a two-domain protein connected by an extended loop: one a/b barrel where DHO is oxidized and a hydrophobic N-terminal domain composed by two helices, which forms the binding site for class 2 inhibitors such as teriflunomide, brequinar, atovaquone, and their derivatives, and is the predicted to be the binding site for quinone (Fig. 3). A worth noting difference between SmDHODH and other class 2 DHODHs structures was observed at the top of the catalytic a/b barrel, consequence of a 10-residue insertion (Figs. 1 and 3). This sequence, mainly composed of hydrophobic residues and predicted to fold as a flexible loop, assembled as a protuberant domain and it is absent in the others known class 2 enzymes (Figs. 1 and 3). To evaluate the quality of the model, analysis by MolProbity was performed. The Ramachandran plot analysis for the refined model revealed that 92.6% of residues are located in the most favored regions and 99.4% are located in allowed regions. The only two outliers, Val43 and Lys375, are found solvent exposed and in flexible

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regions: Val43 is present at the hinge loop connecting the two Nterminal helices and, Lys375 is located at the C-terminal end. Overall SmDHODH and HsDHODH shares approximately 47% of sequence identity (Fig. 1) and the large majority of residue substitutions between SmDHODH and HsDHODH are randomly distributed over the 3D structure model of SmDHODH. The DpepSmDHODH homology model allowed us to predict that the residues Ala89, Ala90, Gly91, Gly113, Thr114, Asn169, Lys241, Ser271, Ser303, Gly304, Leu307, Val331, Gly332, Gly333, Leu353, Tyr354 and Thr355 are involved in the binding of the co-factor FMN while residues Arg140, Cys141, Gly142, Phe143, Ser203 and Asn205 are important for binding of the substrate dihydroorotate. The fully conserved residues Asn139, Asn200, Asn272 and Thr273 bind both the co-factor and substrate (Fig. 1). All residues identified are conserved with the exception of Thr114, Cys141 and Ser271, that in HsDHODH corresponds to Ser119, Tyr146 and Thr282, respectively (Fig. 1). Worth mentioning that the same pattern of substitutions described for SmDHODH is observed for Plasmodium falciparum DHODH (PfDHODH) (Fig. 1), a validated target against malaria [72]. Although a substitution of a cysteine by a tyrosine seems drastic, a closer inspection revealed that Tyr146 interacts with FMN through hydrogen bonds involving main-chain atoms and the substitution to Cys141 in SmDHODH does not alter this framework of FMN interactions. Nevertheless, the side chain of both Tyr146 and Cys141 are pointing towards the hydrophobic channel described to be the binding pocket for class 2 DHODH inhibitors and also hypothesized to be the quinone binding site [5], suggesting that besides not interfering with FMN coordination, a substitution at this position can be exploited to enhance inhibitor selectivity. Molecular dynamics simulations for both SmDHODH and HsDHODH were used to map conformational dynamics and revealed the distinct conformations that each enzyme tend to acquire in a solvated environment. Three distinct representative conformations for both SmDHODH and HsDHODH structures were identified over 100 ns MD simulation (Fig. 4). First, superposition of all three representative structures was used to estimate RMSD values for Ca atoms during trajectory (Supplementary Fig. S2 and Table S1). While HsDHODH structure remained stable during the entire simulation with averaged RMSD values for Ca atoms of approximately 1 Å, SmDHODH suffered major conformational rearrangements concerning both the helical domain (Gly26 to Gly60) and the protuberant flexible loop (Gly285 to Lys294). Concomitantly, a local displacement within SmDHODH catalytic domain enabled the formation of a cavity that provided clear access to N5 of the isoalloxazine ring of FMN (Fig. 4). The helical domain is well described as part of the quinone/inhibitor binding site [5] and the catalytic domain responsible for the oxidation of dihydroorotate, however the function of the additional hydrophobic loop regarding protein stability and dynamics and/or enzymatic activity remains to be elucidated. Work towards this goal is currently in progress. SmDHODH was also compared to HsDHODH representative structures (Supplementary Fig. S3 and Table S1) and a remarkable difference was noted between both enzymes regarding the conformations adopted by the N-terminal helical domain with respect to the catalytic domain (Fig. 3). In agreement with our results, the mobility of the N-terminal domain in HsDHODH was previously described for class 2 DHODHs [44]. The superposition of representative conformations for SmDHODH also revealed a movement between the two a-helices and, using the position of the N-terminal Gly26 Ca atom as reference, the N-terminal a-helix showed to exhibit a relative displacement of 12.8 Å (Fig. 5). This V shaped a-helical domain adopted a parallel conformation in SmDHODH but remained angled in HsDHODH (Supplementary Fig. S4). Likewise, this parallel

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Fig. 2. 3D plot for the reaction rate as a function of Qo and DHO concentrations. (A) DpepSmDHODH KmDHO ¼ 230 ± 30 mM; KmQ0 ¼ 170 ± 20 mM and kcat ¼ 31 ± 2 s1 (B) DpepHsDHODHs KmDHO ¼ 290 ± 30 mM; KmQ0 ¼ 350 ± 40 mM and kcat ¼ 78 ± 4 s1. Surface shows data fit to equation (1) that describes the ping-pong mechanism of catalysis. Error represents standard error of the fit. Single experiment in triplicate.

domain converged to this parallel conformation even in absence of inhibitors suggests that this state may be adopted by different DHODHs and could be stabilized by ligand binding. In addition, two features identified in SmDHODH may favor the mobility of the Nterminal domain which includes the presence of Gly46 (Pro51 in HsDHODH and RnDHODH) and the insertion of Arg44 which make the loop longer and proned to adopt different conformations. Since the two N-terminal a-helices form an entrance to the FMN and defines the site for inhibitor binding, this difference in can also grant some selectivity for the compounds when comparing SmDHODH and HsDHODH. 3.4. Differences in the inhibitor binding site among class 2 DHODHs Four chemically distinct class 2 inhibitors were tested against

DpepDHODHs: brequinar, a quinoline carboxylic acid [44], which is

Fig. 3. Cartoon representation of SmDHODH homology model. The hydrophobic Nterminal domain composed by two helices is illustrated in yellow. The catalytic central barrel composed of eight parallel b strands and surrounded by eight a-helices is illustrated in pink. The FMN group is located at the top of the barrel and is illustrated as a ball-and-stick model. The catalytic residue Ser203 is shown in green. The main structural difference between SmDHODH and class 2 DHODHs is the presence of a tenresidue peptide that folds as a protuberant subdomain and is illustrated in slate blue.

conformation was previously observed for Rattus norvegicus DHODH (RnDHODH) in complex with atovaquone [44]. The fact that in our molecular dynamics simulations SmDHODH N-terminal

an immunosuppressive drug and cytostatic agent; teriflunomide, the active metabolite of the prodrug leflunomide [5]; atovaquone, a structural analogue of ubiquinone [44], used as a broad-spectrum antiparasitic drug in the treatment of malaria, toxoplasmosis and pneumonia; and DSM265, a triazolopyrimidine-based inhibitor of Plasmodium falciparum DHODH and the first DHODH inhibitor to reach clinical development for treatment of malaria [29,41]. The molecules showed to selectively inhibit either the parasite or human enzyme (Fig. 6 and Table 1). For instance, brequinar displayed a selectivity index of almost 540 times towards the human enzyme. Teriflunomide showed similar properties inhibiting HsDHODH 156 times more than SmDHODH. On the other hand, atovaquone showed a higher potency against SmDHODH and displayed a

Fig. 1. Sequence alignment of selected Class 2 DHODHs: HsDHODH (Human DHODH), RnDHODH (Rattus norvergicus DHODH), SmDHODH (Schistosoma mansoni DHODH), PfDHODH (Plasmodium falcipurum DHODH) and EcDHODH (Escherichia coli DHODH). Similar residues are coloured based on their physical-chemistry properties: polar neutral amino acids (S,T,Q,N) are brown, polar basic residues (K,R,H) are cyan, polar acidic (D,E) are red, non-polar aromatic (F,Y) are blue, and non-polar aliphatic (A,V,L,I,M) amino acids are pink. G and P are coloured in orange. Signal peptide and transmenbrane domain predicted for HsDHODH are highlighted in green. Signal peptide predicted for SmDHODH is highlighted in yellow. Starting point for truncated SmDHODH (DpepSmDHODH) and HsDHODH (DpepHsDHODH) constructs is indicated by a blue arrow. The N-terminal microdomain responsible for the protein anchoring to the membrane and, harboring the respiratory quinones for FMN reoxidation is highlighted in pale yellow. The serine catalytic residue of class 2 DHODH is highlighted in black. The residues involved in FMN binding, orotate binding, or both FMN and orotate binding are indicated by red, green and pink stars, respectively. Residues thought to be involved in inhibitor binding are indicated by grey arrows. Residue numbering for each sequence is shown at the left. The alignment was performed using MULTALIN [65] and graphically displayed using ESPript 3.0 [66].

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Fig. 4. Molecular surface for the representative structures of SmDHODH and HsDHODH. SmDHODH_1 and HsDHODH_1 refer to t ¼ 0 ns simulation; SmDHODH_2 and HsDHODH_2 refer to the averaged structures between 30 and 60 ns, and 20e40 ns, respectively; and SmDHODH_3 and HsDHODH_3 refer to the averaged structures between 75 and 90 ns, and 60e80 ns, respectively. The hydrophobic N-terminal domain is illustrated in yellow. The catalytic central barrel is illustrated in pink. The main structural difference between SmDHODH and class 2 DHODHs is the presence of a ten-residue peptide that folds as a protuberant subdomain and is illustrated in slate blue.

Fig. 5. Cartoon representation for the representative structures SmDHODH_1 and SmDHODH_2. The hydrophobic N-terminal domain composed by two helices is highlighted. The difference between the two representative structures of SmDHODH is observed in the flexibility for the N-terminal domain. The position of the N-terminal Gly26 Ca atom exhibits a relative movement of 12.8 Å between the structures. SmDHODH_1 and SmDHODH_2 is illustrated in deepteal and greencyan, respectively.

selectivity index of 6 towards the parasite enzyme. Special attention was given to the inhibitor binding site of SmDHODH, hypothesized to also be the coenzyme Q0 binding pocket [5]. Structural comparison and molecular dynamics simulation were used to probe structural differences that could account for the observed species-related preferential inhibition for brequinar, teriflunomide, atovaquone and DSM265. First, multiple structural alignment of SmDHODH homology model and crystallographic structures of RnDHODH (PDB ID 1UUM and 1UUO [44]), PfDHODH (PDB ID 4RX0 [41]), and HsDHODH (PDB ID 1D3H [5]) bound to the inhibitors atovaquone, brequinar, DSM265 and teriflunomide/A771726, respectively; were used to map static variations in both steric and chemical distributions (Supplementary Tables S2eS5). Both teriflunomide and brequinar were described to display similar interactions with DHODH conserved residues. They were found to bind DHODH enzymes by exploring a set of chemical interactions with residues belonging to the helices of the N-terminal domain, the connecting loop and the a/b barrel catalytic domain [44]. The carboxyl group of brequinar form a hydrogen-bonded ring system to the sidechain of Gln47 (replaced by Arg40 in SmDHODH) and forms a salt bridge with Arg136 (Arg130 in SmDHODH) [5]. For teriflunomide, the carbonyl is hydrogen bonded to a water, which in turn is bound to the conserved Arg136, while the enolic OH is hydrogen bonded to Tyr356 (fully conserved among class 2 DHODHs). The stacking between the fluoroquinoline ring with the imidazole ring of His56 (His50 in SmDHODH) was previously shown to be crucial for brequinar potency [73]. Both biphenyl group of

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Fig. 6. Inhibition profile for atovaquone, brequinar, DSM265 and teriflunomide against DpepDHODHs. Lines show data fit to equation (2). Errors represent standard deviation of each triplicate. Single experiment.

Table 1 Inhibitory potential (IC50) in nM for the compounds tested against HsDHODH and SmDHODH and their selectivity index. Error represents standard error of the fit. Single experiment in triplicate. IC50 (nM) Enzyme HsDHODH SmDHODH Selectivity index

Atovaquone 2600 ± 200 430 ± 20 6 (SmDHODH)

Brequinar 37 ± 2 20000 ± 1000 540 (HsDHODH)

brequinar and the trifluoromethyl-containing aromatic ring in teriflunomide display hydrophobic contacts with numerous sidechains of this hydrophobic tunnel (Tables S3 and S5). Val134 in HsDHODH, is found only 3.2 Å away from the fluorine of brequinar and 3.9 Å from the methyl of teriflunomide. The corresponding residue in SmDHODH is Ile128, which shows to promote a steric clash and could contribute for the ligand selectivity towards the human enzyme observed for both brequinar and teriflunomide. Inhibition efficiency displayed by atovaquone against HsDHODH

DSM265 7000 ± 1000 21000 ± 1000 3 (HsDHODH)

Teriflunomide 320 ± 40 50000 ± 2000 156 (HsDHODH)

is found less pronounced than brequinar and teriflunomide. However, atovaquone shares many previously described interactions to both of them. The only interactions present for brequinar/teriflunomide but not for atovaquone are Tyr38, Leu46 and Thr63 (Table S2). When comparing all the residues that interacts with atovaquone, one could hypothesize that differences between HsDHODH and SmDHODH could confer selectivity towards the parasite enzyme [74] (Table S2). However, this argument cannot explain the fact that atovaquone was found to display high potency

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and selectivity against RnDHODH [44,74], which shares high sequence identity with its human homologue enzyme. Considering that the binding of atovaquone required a rearrangement of the Nterminal helical domain [44], we speculate whether rather than variation in static interactions, mobility, as observed for RnDHODH and SmDHODH, plays major role in atovaquone binding. DSM265 is a potent and selective inhibitor of PfDHODH with IC50 values in the low nanomolar range [40]. The amino acid composition of the binding site is highly variable between PfDHODH and HsDHODH, and this is thought to grant strong selectivity for the parasite enzyme. DSM265 interacts with PfDHODH by exploiting both hydrophobic and hydrogen bond interactions (Table S4). Surprisingly, even though SmDHODH and PfDHODH share high sequence similarity at the inhibitor binding site (Fig. 1), DSM265 showed to be a poor inhibitor for SmDHODH. This selectivity towards PfDHODH enzyme reinforce the fact that the few but important substitutions, Leu176 (replaced by Arg40 in SmDHODH), Cys184 (Ala48), Ile272 (Val137) and Leu531 (Phe357) are sufficient to compromise potency and selectivity. Overall, our results suggest the feasibility of identifying selective and potent inhibitors for SmDHODH and suggest that the atovaquone scaffold should be a starting point in the search for compounds to treat schistosomiasis, based on the inhibition of DHODH. In particular, we propose a rational approach that involves to exploit conformational dynamics and variations at the inhibitorbinding site, including important substitutions such as Leu42 and Leu359 (HsDHODH) that are replaced by the bulkier and rotationally constrained rigid ring structures of Phe357 and Phe35 (SmDHODH), respectively; Ala59 (HsDHODH) replaced by Ser53 (SmDHODH); Met111 (HsDHODH) replaced by Ala105 (SmDHODH) and Pro52 replaced by Gly46 (SmDHODH). 4. Conclusions The discovery and development of novel antischistosomal drugs is of utmost importance. It is our interested to evaluate DHODH as a drug target against schistosomiasis. As a first step towards this goal, DHODH from Schistosoma mansoni was expressed in E. coli and characterized by combining biochemical, biophysical and theoretical methods. SmDHODH showed to catalyze the conversion of dihydroorotate into orotate using quinone as electron acceptor following a pingpong mechanism. Despite the overall resemblance to the human homologue enzyme, substitutions and differences in the conformational states adopted by HsDHODH and SmDHODH structures may allow the identification of selective inhibitors against the parasite enzyme. In addition, comparison between SmDHODH and PfDHODH structures suggest that inhibitors of PfDHODH could be exploited as potential inhibitors of SmDHODH. In fact, we believe that the search for SmDHODH inhibitors as well as the evaluation of SmDHODH as a drug target against schistosomiasis can be strongly benefited by the extraordinary work already performed in the development of class 2 DHODH inhibitors. Based on the structural and biochemical similarities shared among class 2 DHODHs, it is reasonable to consider that the strategy of testing chemical scaffolds already developed to inhibit class 2 DHODHs, including chemotherapeutic agents already in the market or undergoing clinical trials, can shortcut the identification of pharmacophore groups as well as potent SmDHODH inhibitors. Thus, considering the biological relevance of nucleotide biosynthesis, and the limited investment on the development of new treatments against neglected diseases, it is reasonable to consider that the possibility of repurposing existing drugs based on the selective inhibition of class 2 DHODHs can be a useful strategy to accelerate the drug development for schistosomiasis due to lower costs, reduced risk

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