Structural, functional and docking analysis against Schistosoma mansoni dihydroorotate dehydrogenase for potential chemotherapeutic drugs

Sequence retrieval, extraction of DHODH proteins and potential drugs

SchDHODH protein sequences were obtained from the NCBI protein database. The sequences were confirmed in SchistoDB for S. haematobium (two protein sequences) and GeneDB for S. mansoni (two protein sequences) and S. japonicum (one protein sequence). All sequences were obtained in FASTA format. Each of these protein sequences were used for a BLASTp search of the non-redundant protein database on NCBI, employing the protein-protein BLAST algorithm. Results with similarities above 50% and with a query coverage and expectation value of >70% and 0.0, respectively, were obtained and combined for phylogenetic tree construction. Hypothetical and unknown proteins were excluded, even when there was high identity. After combining these sequences, duplicate protein sequences were also excluded. Host DHODH protein sequences were also obtained from NCBI using the search terms as targets “Homo sapiens + DHODH”, “Mus musculus + DHODH” and “Rattus rattus + DHODH” separately. The protein sequences that had 98% similarity to the targets were retrieved for further analyses. One HsDHODH, one MmDHODH and two RrDHODH sequences were retrieved. Each SchDHODH sequence was used to search DrugBank (version 5.1.2, released 2018-12-20)²⁶ for potential compounds that may bind. All settings were at default.

Functional domain analysis

Functional domains for each SchDHODH, MnDHODH and HsDHODH were computed using the normal and genomic mode of SMART^27,28. The functional domains predicted were validated using other webtools; NCBI Conserved Domains^29–32, PROSITE³³, InterPro³⁴ and Pfam version 32.0³⁵. Default parameters were used for these analyses.

Prediction of intrinsic disorder in SmDHODH

Since, for many disordered proteins, binding affinity with their receptors is regulated by post-translational modification, SmDHODH protein was analyzed for intrinsically disorder. SmDHODH was the only protein used in this analysis because it was used for modeling and protein-drug interaction. PONDR, which can predict natural disordered regions in a protein sequence, was used. This was validated using other similar tools such as SLIDER webserver³⁶ and DisEMBL Intrinsic Protein Disorder Prediction 1.5³⁷.

Physical and chemical properties of SchDHODHs, HsDHODHs and MmDHODHs

Each protein’s physiochemical properties, which includes the number of residues, molecular weight and extinction coefficient were predicted using the ProtParam web tool³⁸. These properties were validated using the PepCalc peptide property calculator and Protein Physicochemical Properties Prediction Tool (PPPPT) web tools.

SchDHODH, HsDHODH and MmDHODH sequence alignment

All SchDHODH, HsDHODH and MmDHODH sequences were compared by Multiple Sequence Alignment (MSA) and the conserved and deleted regions were analyzed. The MSA analysis was carried out using Clustal Omega tools in Jalview³⁹. The MSA was analyzed for conserved properties and regions of similarity.

Phylogenetic studies and evolutionary conservation analysis

Phylogenetic trees were constructed using MEGA version 7 software⁴⁰. The constructed phylogenetic trees were validated using Phylogeny.fr⁴¹. The tree file, in Newick format, was exported and visualized in FigTree software version 1.4.2⁴² for proper annotation. The pairwise distances were also estimated, using the Poisson correction model in MEGA⁴⁰.

Protein-protein interactions

Protein-protein interactions were determined using the STRING database for functional protein association networks. Each of the SchDHODH, HsDHODH and MmDHODH protein sequences were searched on the STRING and the interactions were downloaded in jpeg format.

3D modelling

SmDHODH sequences were modelled using SWISS-MODEL^43–47. The template 3u2o.1.A, with a sequence identity of 47.95%, GMQE of 0.74 and QMEAN of -0.98 was selected as the model of choice for SmDHODH sequences.

Molecular docking and analyses

Molecular docking was carried out using DockingServer⁴⁸ and Gasteiger partial charges were added to the ligand atoms⁴⁹, as described by Kumar, 2011⁵⁰. Proteins were uploaded, protein charges were calculated, and solvation parameters were calculated and cleaned. All advance docking parameters were left at default.

Sequence retrieval of DHODHs and drugs

In total, five SchDHODH sequences were retrieved for further analyses. One for S. japonicum and two proteoforms each for S. haematobium and S. mansoni. One HsDHODH sequence, two MmDHODH sequences and two RrDHODH sequences (Figure 1 and Project 1, Extended data)⁵¹ were included in the study for proper comparison as mammals, including humans, are the hosts, while rats and mice are commonly used in the laboratory for schistosomiasis studies. Of 12,110 drugs in the database, 26 compounds that can bind to SchDHODH proteins were identified and retrieved from DrugBank. These drugs were retrieved with E value: 7.46761e-71, Bit score: 224.172, query length: 379 and alignment length: 314. These results were the same for each of the SchDHODH sequences searched. All of the drugs were at the experimental phase, except flavin mononucleotide, capecitabine and leflunomide, which are approved for the treatment of other diseases, and manitimus, which is still at the investigational phase. The mode of action of most of these retrieved drugs are not known, except for atovaquone, leflunomide and teriflunomide, which act as inhibitors to known proteins other than DHODH.

Figure 1. Alignment analysis of Schistosoma dihydroorotate dehydrogenases alongside the corresponding dihydroorotate dehydrogenases of host species.

Conserved amino acids across SchDHODHs are marked and indicated in purple boxes, while across SmDODHs and ShDHODHs are marked and indicated in red boxes. The red arrows show where the mutation in SjDHODH compare to SmDODHs and ShDHODHs.

Domains of DHODHs and intrinsic disorder predictions

The protein domain analysis (see Project 3, Extended data)⁵² shows that all analyzed proteins have the dihydroorotate dehydrogenase domain (DHO_dh), while ShDHODH proteins have dynein light domains and transmembrane helix regions, which are not seen in the other proteins. The disorder predictions show that a small fraction of the SmDHODH protein is disordered, as shown in Project 2, Extended data⁵³.

DHODHs of S. haematobium and S. mansoni evolved from a single ancestral species

Physiochemical parameters (see Project 1, Extended data)⁵¹ and alignment (Figure 1), as well as phylogenetic analyses of all the SchDHODH sequences (Figure 2), show that the DHODH proteins of S. haematobium and S. mansoni could be evolutionary closer than that of S. japonicum. However, S. japonicum evolved 0.8928 million years ago (mya), compared to S. haematobium which evolved 0.8207 mya and S. mansoni which evolved 0.8012 mya. The last common ancestor for DHODH of both S. haematobium and S. mansoni was 0.8379 mya. The host DHODHs evolved more recently, as shown in Figure 2. These results could also explain the similarities between DHODHs that are shown in the sequence alignment (Figure 1). Mutations that occurred in ShDHODHs were also observed in the SmDHODHs, though there are some points where all three Schistosoma species shared mutation points. However, the similarities between ShDHODH and SmDHODH sequences do not correlate with the physiochemical parameters; ShDHODH theoretical pI, extinction coefficients, instability index, aliphatic index and grand average of hydropathicity scores are more similar to SjDHODH than SmDHODH (see Project 1, Extended data)⁵¹.

Figure 2. Phylogenetic tree of the dihydroorotate dehydrogenase proteins from Schistosoma pathogens, mice and human.

All the SchDHODHs shows clearly that the SchDHODHs of S. haematobium and S. mansoni could be evolutionary closer compared to S. japonicum.

SchDHODHs, HsDHODHs and MmDHODHs protein-protein interaction analysis

The prediction of possible in vivo interactions of these DHODHs showed that SchDHODH binds to NADPH (gene Smp_166580), putative glutamate synthase (Smp_128380.2) and NADH-cytochrome B5 reductase (Smp_053230) (Figure 3). These four proteins bind to each other; however, it is unclear whether they form a complex. SchDHODHs have an inhibitory effect on orotate phosphoribosyltransferase (Smp_050540), which has an inhibitory transcriptional regulation on aspartate carbamoyltransferase (Smp_186670) and vice versa. HsDHODH and MmDHODHs non-specifically bind to collapsin response mediator protein 1 (Crmp1) and different proteoforms of dihydropyrimidinase (Dpys). However, HsDHODH and MmDHODHs have an inhibitory role on carbamoyl-phosphate synthetase (Cps1) and uridine monophosphate synthetase (Umps).

Figure 3.

Interaction of proteins with (a) SmDHODH (b) HsDHODH and (c) MmDHODH.

Docking of selected ligands against modelled SchDHODH protein structure

SmDHODH was selected for modeling and protein-drug interaction studies as a representative for the three SchDHODHs, since sequence alignment and phylogenetic analysis showed that they are all closely related. The modelled SmDHODH used for docking against the 26 potential compounds is shown in Figure 4. After the molecular docking, 13 drugs had a binding affinity of less than -6kcal/mol, suggesting a strong bond²¹, with SchDHODH (Table 1 and Figure 5) and details of these interactions are shown in Figure 6. The results show that 3-{[(3-fluoro-3'-methoxybiphenyl-4-yl)amino]carbonyl}thiophene-2-carboxylic acid (accession number DB07976) and 3-{[(3-fluoro-3'-methoxybiphenyl-4-yl)amino]carbonyl}thiophene-2-carboxylic acid (accession number DB07978) have the highest binding affinity, with an estimated free energy of binding (kcal/mol) of -10.18 and -10.66, respectively. Leflunomide (accession number DB01097) and N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (accession number DB08006) both have an estimated free energy of binding of -8.12. The SchDHODH protein residues that interact with the different drugs in Table 1, are shown stick while drugs cartoon. It was observed that SmDHODH interacts with the various compounds using hydrogen bonds, polar, hydrophobic, pi-pi, halogen and other forms of bonding.

Figure 4. Modelled SmDHODH protein.

Figure 5. The structure of compounds retrieved after searching the drug database with the SmDHODH sequence.

a) 2-({[2,3,5,6-TETRAFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENTA-1,3-DIENE-1-CARBOXYLIC ACID (Accession Number: DB07978); b) 3-{[(3-FLUORO-3'-METHOXYBIPHENYL-4-YL)AMINO]CARBONYL}THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07976); c) 3-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07977); d) Flavin mononucleotide (Accession Number: DB03247); e) 2-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENT-1-ENE-1-CARBOXYLIC ACID (Accession Number: DB07975); f) 5-Carbamoyl-1,1':4',1''-terphenyl-3-carboxylic acid (Accession Number: DB04583); g) 5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08008); h) Manitimus (Accession Number: DB06481); i) Capecitabine (Accession Number: DB01101); j) Brequinar Analog (Accession Number: DB03480); k) (2Z)-N-biphenyl-4-yl-2-cyano-3-cyclopropyl-3-hydroxyprop-2-enamide (Accession Number: DB08169); l) Leflunomide (Accession Number: DB01097); m) N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08006).

Figure 6. The binding site of the interaction between the SmDHODH protein and the retrieved compounds.

a) 2-({[2,3,5,6-TETRAFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENTA-1,3-DIENE-1-CARBOXYLIC ACID (Accession Number: DB07978); b) 3-{[(3-FLUORO-3'-METHOXYBIPHENYL-4-YL)AMINO]CARBONYL}THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07976); c) 3-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)THIOPHENE-2-CARBOXYLIC ACID (Accession Number: DB07977); d) Flavin mononucleotide (Accession Number: DB03247); e) 2-({[3,5-DIFLUORO-3'-(TRIFLUOROMETHOXY)BIPHENYL-4-YL]AMINO}CARBONYL)CYCLOPENT-1-ENE-1-CARBOXYLIC ACID (Accession Number: DB07975); f) 5-Carbamoyl-1,1':4',1''-terphenyl-3-carboxylic acid (Accession Number: DB04583); g) 5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08008); h) Manitimus (Accession Number: DB06481); i) Capecitabine (Accession Number: DB01101); j) Brequinar Analog (Accession Number: DB03480); k) (2Z)-N-biphenyl-4-yl-2-cyano-3-cyclopropyl-3-hydroxyprop-2-enamide (Accession Number: DB08169); l) Leflunomide (Accession Number: DB01097); m) N-anthracen-2-yl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (Accession Number: DB08006).

Underlying data

SJCHGC02326 protein [Schistosoma japonicum], Accession number AAW26221: https://identifiers.org/ncbiprotein/AAW26221

Dihydroorotate dehydrogenase (quinone), mitochondrial [Schistosoma haematobium], Accession number KGB36135: http://identifiers.org/ncbiprotein/KGB36135

Dihydroorotate dehydrogenase (quinone), mitochondrial [Schistosoma haematobium], Accession number XP_012795900: https://identifiers.org/ncbiprotein/XP_012795900

dihydroorotate dehydrogenase [Schistosoma mansoni], Accession number CCD78646: http://identifiers.org/ncbiprotein/CCD78646

dihydroorotate dehydrogenase [Schistosoma mansoni], Accession number XP_018651255: https://identifiers.org/ncbiprotein/XP_018651255

dihydroorotate dehydrogenase (quinone), mitochondrial precursor [Mus musculus], Accession number NP_064430 : http://identifiers.org/ncbiprotein/NP_064430

Chain A, DIHYDROOROTATE DEHYDROGENASE [Rattus rattus], Accession number 1UUM_A: http://identifiers.org/ncbiprotein/47169292

Chain B, DIHYDROOROTATE DEHYDROGENASE [Rattus rattus], Accession number 1UUM_B: http://identifiers.org/ncbiprotein/47169293

Crystal structure of human dihydroorotate dehydrogenase at 1.7 A resolution [Homo sapiens], Accession number 5K9D: https://identifiers.org/pdb/5K9D

Extended data

Figshare: Extended data 1_Physiochemical Properties.docx. https://doi.org/10.6084/m9.figshare.8019683.v1⁵¹

Figshare: Prediction of intrinsic disorder of SmDHODH.docx https://doi.org/10.6084/m9.figshare.8050541.v1⁵³

Figshare: Extended data 3_Protein domain analysis of DHODHs of the Schistosoma sp and Host.docx https://doi.org/10.6084/m9.figshare.8051777.v1⁵²

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).