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. 2025 Jul 3;10(27):29087–29097. doi: 10.1021/acsomega.5c01536

Crystallographic Structure of Human Dihydroorotate Dehydrogenase in Complex with the Natural Product Inhibitor Lapachol

Aline D Purificação †,‡, Laila S Benz §,∥, Wemenes J Lima Silva †,⊥, Flavio S Emery †, Carolina Horta Andrade †,⊥,#, Manfred S Weiss ∥, Maria Cristina Nonato †,‡,*
PMCID: PMC12268739  PMID: 40686985

Abstract

Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway, playing a critical role in cellular processes and offering therapeutic potential for antiviral, antineoplastic, and autoimmune treatments. Human DHODH (HsDHODH) utilizes ubiquinone as a second substrate, positioning its quinone-binding site as a promising target for inhibitor development. Lapachol, a natural naphthoquinone, has gained prominence as a valuable natural product for the discovery of novel therapeutic agents, thanks to its wide range of biological activities. In this study, we present the first crystal structure of HsDHODH in complex with lapachol, providing valuable insights into the interactions between this natural product and the enzyme. The structure reveals key binding interactions that mediate lapachol’s affinity for HsDHODH and validates previously proposed computational models. Complementary molecular dynamics simulations further highlight the stability of the complex and the importance of water-mediated interactions in ligand binding. These findings enhance our understanding of how naphthoquinone derivatives, such as lapachol, interact with class 2 DHODHs, offering a foundation for the design of optimized inhibitors for therapeutic applications. By integration of structural and computational data, this study contributes to the rational design of novel HsDHODH inhibitors, paving the way for future exploration of lapachol and its derivatives in drug discovery.

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1. Introduction

Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway, essential for DNA and RNA syntheses in all organisms. Human DHODH (HsDHODH) belongs to class 2 DHODHs, which occur in the mitochondrial membrane and operate through a ping-pong reaction mechanism involving two distinct steps. In the first step, dihydroorotate (DHO) is oxidized to orotate (ORO) while flavin mononucleotide (FMN) is reduced. In the second step, the reduced FMN is reoxidized by a second substrate, varying by organism, ensuring the continuous catalytic cycle of the enzyme. In HsDHODH, the second substrate is ubiquinone, which is reduced to ubiquinol.

The inhibition of DHODH in various organisms is linked to antiparasitic, antineoplastic, and antiviral activities. HsDHODH is a significant therapeutic target for diseases such as cancer, autoimmune disorders, and viral infections. Host-targeted antiviral therapy, a modern approach, utilizes HsDHODH inhibition to reduce the intracellular nucleotide pool, impairing viral replication, as viruses rely on host nucleotides for rapid reproduction.

Teriflunomide, the active metabolite of leflunomide, is a clinically approved HsDHODH inhibitor used to treat rheumatoid arthritis. Several other inhibitors have been described, some of which are undergoing clinical trials for cancer and viral infections (NCT04997993, NCT04575038).

Among natural inhibitors of HsDHODH, lapachol, a 1,4-naphthoquinone derivative extracted from Tabebuia impetiginosa, has been studied since the 19th century for its antineoplastic, antibiotic, and antimalarial properties. , Naphthoquinone derivatives, such as lapachol, demonstrate anticancer activities through various mechanisms, including topoisomerase inhibition, modulation of the tumor suppressor p53, and inhibition of MALT. Recently, we have identified lapachol as a potent inhibitor of both HsDHODH and DHODH of Schistosoma mansoni (SmDHODH). These class 2 DHODHs are monomeric proteins anchored to cellular membranes and require quinones as their physiological oxidizing agent. Given the biological role of quinones in the activity of HsDHODH and SmDHODH, it is anticipated that quinone derivatives like lapachol will interact effectively with these enzymes.

Moreover, previous docking studies suggested the interactions between lapachol and HsDHODH, as well as between lapachol and SmDHODH on the same binding site, that is partially conserved between class 2 DHODHs. However, these studies were limited by the absence of experimental structures of lapachol in complex with class 2 DHODHs, relying solely on computational models. ,

To date, no crystallographic data has been available for lapachol in complex with any class 2 DHODH, despite extensive interest in this compound’s biological activity. The lack of a crystal structure in complex with lapachol, potentially linked to the low solubility of this compound (log P = 2.8), has limited the validation of these computational models and the detailed understanding of the key interactions that underlie the potency and selectivity of lapachol and its derivatives. Our structure fills this gap by providing, for the first time, experimental confirmation of Lapachol’s binding mode on HsDHODH, validating previous docking studies, together with dynamic simulations that highlight the stability of the ligand at the binding site and at the same time expose the stability of a couple of waters at the binding site that may impact ligand affinity.

2. Results and Discussion

The HsDHODH–lapachol complex was solved at 1.31 Å, comprising 365 residues (Thr32 to Arg396), 1 FMN molecule, 1 ORO molecule, 1 acetic acid molecule, 3 glycerol molecules, 3 sulfate ions, 1 lapachol molecule, and 245 water molecules treated as oxygens. The coordinates for this structure have been deposited in the PDB under accession code 9EG9. Data collection and structure refinement statistics are summarized in Tables and . The overall structure reveals the characteristic α-β barrel fold typical of HsDHODH structures (Figure ). The high-resolution structure exhibits excellent geometric quality and a strong fit to the electron density (Figure S2), with the main challenges observed in the N-terminal region, consistent with findings in other HsDHODH structures. ,

2. Details of the Data Collection and Results for Data Processing.

diffraction source beamline BL14.1 (BESSY II)
wavelength (Å) 0.9184
temperature (K) 100
detector PILATUS3 S 6M
crystal-detector distance (mm) 151.61 mm
rotation range per image (°) 0.1°
total rotation range (°) 360°
exposure time per image (s) 0.1 s
space group P3221
a, b, c (Å) 90.46, 90.46, 122.66
α, β, γ (°) 90, 90, 120
resolution range (Å) 48.29–1.31 (1.39–1.31)
total no. of reflections 2780713 (448755)
no. of unique reflections 139283 (22325)
rmeas (%) 11.3 (322.5)
completeness (%) 100.0 (100.0)
redundancy 19.96 (20.1)
⟨I/σ(I)⟩ 18.45 (0.98)
CC half 100.0 (41.2)
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3. Results for Structure Refinement.

resolution range (Å) 48.34–1.31 (1.344–1.310)
completeness (%) 99.94 (99.89)
no. of reflections, working set 132428
no. of reflections, test set 6855
final R working 0.174 (0.330)
final R free 0.180 (0.347)
no. of non-H atoms 3199
protein 2853
ligand 101
water 245
R.m.s. deviations  
bonds (Å) 0.013
angles (°) 1.65
average B factors (Å2)  
protein 21.4
ligand 28.9
water 33.9
ramachandran plot  
most favored (%) 97.8
allowed (%) 2.2
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1.

1

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Structural overview of HsDHODH–lapachol and the binding site. (a) Cartoon representation of the overall fold, showing the C-terminal domain (Met78-Arg396), the linking loop, and the N-terminal domain α1 and α2 helices (Met30-Leu68). Orotate and FMN are orange, and lapachol is blue. The C-terminal domain is colored yellow, the N-terminal domain is colored green, and the linking loop is colored red. (b) Close-up view of the lapachol binding site in the crystal structure with hydrogen bonds depicted as blue dotted lines. The inhibitor and residues involved in hydrogen bonding are colored by atoms: oxygen (red), nitrogen (blue), and carbon (pale green for N-terminal residues, yellow for C-terminal residues, and blue for lapachol). The omit map (contoured at 3 RMSD) is shown around the ligand.

A region of particular interest is the loop comprising residues Asn212 to Leu224, which has been implicated in mediating the orotate release mechanism. While this loop is often disordered in HsDHODH structures complexed with inhibitors, in the HsDHODH–lapachol complex, it is well-defined and can be reliably modeled.

Lapachol binds to the N-terminal region of the enzyme, occupying the proposed ubiquinone-binding site where all currently described HsDHODH inhibitors are bound. This study discloses the first crystal structure of HsDHODH in complex with a quinone, providing strong evidence that this site is indeed the binding region for the second substrate, ubiquinone. The benzoquinone ring of ubiquinone contains redox-active sites, whereas the polyisoprenoid chain is responsible for positioning the molecule within the midplane of the lipid bilayer of various cell membranes. In the case of HsDHODH, the long polyisoprenoid chain of ubiquinol likely interacts with the enzyme’s transmembrane domain, which anchors it to the mitochondrial membrane. Notably, the quinoidal core of ubiquinone closely resembles that of lapachol, while the branched tail of lapachol mirrors the polyisoprene chain of ubiquinone, albeit with a shorter length (10 units in ubiquinone). These structural similarities lend support to a plausible mechanism of the ubiquinone interaction in this binding site.

This pocket is also the binding site for brequinar, one of the most well-characterized HsDHODH inhibitors, extensively studied for potential applications in cancer, , infectious diseases, , and inflammatory disorders. In the crystallographic structure reported here, the hydroxyl group attached to carbon 2 and the carbonyl group attached to carbon 1 of the naphthoquinone moiety of lapachol form interactions with the side chain of Arg136, resembling those mediated by the carboxy group of the five-membered ring of brequinar analogue (PDB ID: 1D3G) (Figure ). Additionally, the hydroxyl group on carbon 2 of lapachol’s naphthoquinone also interacts with the side chain of Gln47, an interaction similar to that of brequinar’s carboxyl group in the same region. This interaction pattern has been classified by Baumgartner et al. as brequinar-like.

2.

2

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Brequinar-like interaction pattern of lapachol is confirmed by the crystal structure. Representation of the binding site that accommodates a brequinar analogue (a) and the lapachol (b), showing the hydrogen bonds as dotted green lines and the residues responsible for hydrophobic interactions. The plot was generated by LigPlus using the crystallographic structures of HsDHODH in complex with a brequinar analogue (PDBID 1D3G) and lapachol. The residues in red highlight the differences in the interaction patterns, as they are residues that interact solely with the brequinar analogue (a) or lapachol (b).

According to Baumgartner’s classification, lapachol interacts with subsites 1 (Met43, Leu46, Ala55), 2 (Arg136, Gln47), 3 (Tyr356), and 4 (Val134). Additionally, lapachol interacts with Pro52, His56, Thr360, and Ala59residues that were described to interact with brequinar but are not included in Baumgartner’s subsite classification. These findings support a “brequinar-like” binding mode for lapachol, as predicted by prior docking studies from our group (Figure ). Notably, the interaction with Val134 is unique to lapachol. Brequinar interacts with residues Pro364, Thr63, Leu67, and Leu68, which may contribute to its higher potency compared to lapachol.

Comparing the crystal structure of HsDHODH–lapachol here presented with the docking model previously obtained by our group, we can see that the docking calculations accurately predicted the lapachol pose and the residues involved in its interactions (Figure ). The primary difference between the predicted pose and the crystallographic structure is observed in the isoprenyl group of lapachol, which likely reflects the conformational flexibility of the isoprenyl chain and does not involve significant rearrangements of the ligand or protein side chains. In the crystal structure, the isoprenyl group interacts with Leu46, whereas in the predicted pose, it interacts with Leu359. Notably, a slight conformational change in the side chain of Arg136 observed in the crystal structure, with a root-mean-square deviation for all atoms (RMSDall) of 1.26 Å, enabled interactions with both the carbonyl group at carbon 1 and the hydroxyl group at carbon 2 of the naphthoquinone core. In contrast, the docking model predicted only the interaction with the hydroxyl group. A minor variation in the side chain of Gln47 (RMSDall = 0.78 Å) also facilitated a hydrogen bond with the quinonoid core of lapachol in the crystal structure, a feature not predicted in the docking model.

3.

3

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Computational prediction of the HsDHODH–lapachol complex closely resembles the experimental structure. (a) Schematic representation comparing the lapachol binding site in the predicted model (blue) and the crystallographic structure (green), highlighting the ligand and the residues involved in interactions, along with their respective RMSDCα and RMSDall values. The residues Leu46 and Leu359 are emphasized, as they interact with the isoprenyl portion of lapachol in different conformations observed in the docking model and the crystal structure. (b) RMSDCα and RMSDall by residue plot showing RMSDCα (blue) and RMSDall (orange) values on the Y-axis for each residue along the X-axis.

Overall, the predicted and experimental structures are highly similar, with the average root-mean-square deviation for Cα carbon (RMSDCα) and RMSDall values of 0.23 and 0.37 Å, respectively, indicating a modest deviation (Figure b). The maximum RMSDCα and RMSDall values are also small, 1.84 and 2.95 Å, respectively, and are observed in the loop region comprising residues Asn212 to Leu224, near the reaction product, orotate. The residue interacting directly with orotate, Asn217, shows the lowest root-mean-square deviation values for this region (RMSDCα = 0.20 Å and RMSDall = 0.22 Å), indicating high consistency between the predicted and experimental structures.

Our group previously reported that lapachol inhibits the enzyme activity of HsDHODH (IC50 100  ±  7 nM) and SmDHODH (IC50 19  ±  2 nM). To further elucidate the similarities and differences in the modes of interaction of lapachol with both enzymes, we conducted molecular docking studies. When comparing our crystallographic structure of lapachol in complex with HsDHODH and our docking model of lapachol with SmDHODH, it is observed that the interaction mode is similar, despite the moderate sequence identity (Figure ). The quinoidal core of lapachol on SmDHODH is flipped in relation to the position found in the crystal structure of HsDHODH in complex with lapachol. This new conformation of the quinoline chain was previously described in the crystallographic structure of the SmDHODH enzyme determined in complex with (2-((4-fluorophenyl)­amino)-3-hydroxynaphthalene-1,4-dione), a simplified analogue of atovaquone (PDB ID 6UY4 ). The isoprenyl chain assumes a different conformation on the binding site of the homologous proteins, allowing hydrophobic interaction with Leu46 on the human enzyme while with Gly351 on the parasitic one. Some of the residues that were found to interact with lapachol in HsDHODH are changed by amino acid groups of dissimilar properties in SmDHODH, but with a similar interaction mode with lapachol, such as Met43 in HsDHODH, which is replaced by Leu36 in SmDHODH, Ala59 by Ser53, Thr360 by Val358, Val134 by Ile128, and Pro52 by Gly46. Key residues such as Arg136, His56, and Ala55 are conserved between the two enzymes. In addition, the interaction of lapachol with Tyr356 is not observed in SmDHODH, although this residue is conserved between the proteins. The similarity in the interaction mode of lapachol with both enzymes makes it difficult to account for the 5-fold difference in potency for the SmDHODH solely based on the structural data, highlighting the need for further studies to investigate key differences in the binding mechanism of these enzymes with lapachol and how these variations influence reaction kinetics.

4.

4

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Interaction mode of lapachol with SmDHODH and HsDHODH. (a) A superposition of the lapachol binding site of the docking model of SmDHODH (green) and the crystallographic structure of HsDHODH (wheat). The protein is represented as a cartoon with transparency, while lapachol and the residues involved in the interaction are represented by sticks. (b) Structural alignment of HsDHODH and SmDHODH, highlighting the residues that interact only with SmDHODH (green), HsDHODH (wheat), or both enzymes (red). (c, d) Representation of the binding site that accommodates lapachol in SmDHODH (c) and HsDHODH (d), showing the hydrogen bonds as dotted green lines and the residues responsible for hydrophobic interactions. The plot was generated by LigPlus using the docking model of lapachol with SmDHODH (c) and the crystallographic structure of HsDHODH in complex with lapachol (d). The residues in red highlight the differences in the interaction patterns, as they are residues that interact solely with the lapachol in the SmDHODH model (c) or in the HsDHODH crystallographic structure (d).

Here, additionally, molecular dynamics (MD) simulations were performed to explore the binding interactions and mechanistic behavior of lapachol within the HsDHODH binding site. The simulations were performed in three independent replicas, each running for 1 μs, with distinct initial geometries and velocities to ensure the variability and robustness in the results. The analyses employed root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and principal component analysis (PCA) to assess the results. The HsDHODH backbone remained stable throughout all simulations, exhibiting RMSD values below 2 Å (Figures a and S3a). Lapachol maintained stable conformations with RMSD values below 2 Å in replicas 1 and 2. In replica 3, stability persisted until 900 ns, after which a conformational shift increased the RMSD to approximately 5 Å, suggesting the emergence of a potential secondary binding mode (Figures b and S1). To evaluate the persistence of this newly observed mode, the MD simulation for replica 3 was extended by an additional 500 ns. The secondary binding mode features an additional hydrogen bond with Thr360, distinguishing it from the usual binding mode observed in both the HsDHODH–lapachol crystal structure and replicas 1 and 2 (Figures and S1d). Furthermore, the conformation of this second binding mode is stable with RMSD values below 2 Å (Figure S3b). RMSF analysis showed minimal fluctuations (<2 Å) in HsDHODH, except in loop regions and the N-terminal region, where variations reached up to 7 Å (Figures c and S3c).

5.

5

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RMSD and RMSF analyses of MD simulations. (a) RMSD analysis of the HsDHODH backbone protein in complex with lapachol. (b) RMSD analysis of the lapachol bound to the HsDHODH binding site. (c) RMSF (Ca) analysis of the HsDHODH protein in complex with lapachol.

6.

6

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Conformational stability of the HsDHODH–lapachol complex system. Free-energy landscape analysis (a, c, e) based on the first two principal components (PC1–PC2) for three replicas of the HsDHODH–lapachol complex. Interaction maps illustrating the binding interactions between lapachol and the HsDHODH binding site (b, d, f) are shown relative to the central frames of the free-energy basins (indicated by yellow stars). Panels (a, b) represent replica 1. Panels (c, d) represent replica 2. Panels (e, f) represent replica 3.

The free-energy landscape provides insights into the energy distribution in biomolecular systems and their conformational behavior. It describes energy barriers, stable states, and transition pathways between different molecular conformations. , The PCA-based free-energy landscapes of the compounds provide an in-depth understanding of the conformational stability of the system (Figure ). In replica 1, the system exhibits a free-energy basin and two regions with metastable states. In contrast, replicas 2 and 3 exhibit distinct free-energy wells (Figure a,c,e). Based on these free-energy landscapes, we extracted the centroid structures of the basins (Figure b,d,f) and used them to analyze the molecular interactions between HsDHODH and lapachol.

The results demonstrated that in all three replicates lapachol forms hydrogen bonds with Arg136 and Gln47, consistent with observations in the HsDHODH–lapachol crystal structure. Additionally, water-mediated hydrogen bonds were observed with residues Leu359, Tyr38, and His56 (Figure b,d,f). A secondary binding mode for lapachol, identified in replicate 3, revealed an additional hydrogen bond with Thr360. Furthermore, the analysis of the final frame from the extended replica 3 MD simulation revealed water-mediated hydrogen bonds among Thr63, Ala55, and Arg136. These findings underscore the critical role of Arg136 and Gln47 in stabilizing lapachol within the HsDHODH binding site while also highlighting the importance of water-bridging interactions in ligand stabilization. Notably, although these water-mediated interactions were absent in the HsDHODH–lapachol crystallographic structure, analogous interactions have been documented in other HsDHODH–ligand complexes. Water-mediated hydrogen bonds with Leu359 and Tyr38 align with observations in the crystal structure of PDB ID 5ZF4. Similarly, water-mediated hydrogen bonding with His56 was observed in the PDB crystal ID 4OQV. As for the extended MD simulation results of replica 3, water-mediated hydrogen bonds with Ala55 were observed in the crystal structures PDB IDs 5HQE, 5HIN, and 5H73 and with Arg136 are consistent with the crystal structures PDB IDs 6ET4, 4ZMG, 3G0X, 3FJL, 3FJ6, and 1D3H. Finally, water-mediated hydrogen bonding performed by Thr63 was not observed in any crystal structure. This observation suggests that solvent dynamics may play a role in modulating inhibitor stability and affinity, offering potential avenues for the rational design of derivatives that exploit these interactions.

Taking all of this structural information on lapachol into account, we propose a series of compounds that could be synthesized in further projects. As illustrated in Figure S4 (Supporting Information), polar and apolar substituents can be introduced to Ring A (electron-withdrawing or electron-donating groups) to understand the impact of stereoelectronic properties and hydrophobicity on enzyme inhibition. According to the structural analysis, it is crucial to retain the hydrogen-bonding acceptor–donor (HBA/HBD) interactions of the carbonyl-hydroxy groups in the quinoidal core (Ring B). For this purpose, we suggest either maintaining this scaffold or replacing it with heterocycles (e.g., imidazole). Additionally, we propose maintaining the hydrophobic interactions at the prenyl group position due to their importance for enzyme binding, and we also suggest substituting the carbonyl moiety adjacent to the prenyl chain with polar groups, such as oximes and oxime-ethers, or heterocycles (e.g., fused-pyrazole ring system) to mimic the water-mediated bridge of lapachol with Leu359 and His56. Future studies will be necessary to experimentally test these analogues and evaluate their potential for enzyme inhibition, antiviral activity, anti-inflammatory effects, and pharmacokinetic properties.

3. Conclusions

The crystallographic and dynamic structural characterization of HsDHODH in complex with lapachol provides a detailed understanding of the molecular interactions underlying the inhibition of class 2 DHODHs by quinoidal compounds, such as lapachol and its derivatives. This work offers experimental validation for previously proposed computational models, enhancing our knowledge of how naphthoquinone derivatives interact with the enzyme’s binding site.

By addressing a key challenge in the study of lapachol derivatives, limited by the low solubility and the absence of structural data, this work supports ongoing efforts to develop therapeutics targeting HsDHODH. Given the enzyme’s role in pyrimidine biosynthesis and its relevance to various therapeutic areas, such as antiviral and antineoplastic strategies, these findings offer practical insights for future research.

Molecular dynamics simulations provided additional insights into the stability and interaction dynamics of the HsDHODH–lapachol complex. The simulations confirmed the stable binding of lapachol with interactions mediated by residues Arg136 and Gln47, consistent with the crystallographic data. The observation of water-mediated hydrogen bonds in the MD simulations, absent in the crystal structure, suggests additional avenues for exploring the influence of solvent dynamics on binding stability.

This study illustrates the importance of integrating structural and computational approaches in drug discovery. Moreover, exploring natural products, particularly lapachol, unveils the chemical and structural innovations developed by nature, providing valuable insights into modulating target protein activity for drug design. The structural data presented here enable a refined perspective on the conserved features of the binding site that facilitate quinone interactions, contributing to the rational design of improved inhibitors.

4. Methods

4.1. Protein Production and Purification

The protein was expressed and purified following an established in-house protocol using Escherichia coli strain BL21 Codon Plus (DE3) cells transformed with the PET28a-Sumo-HsDHODH plasmid. Cells were grown in a rich medium supplemented with kanamycin and chloramphenicol, and to induce protein expression, 100 μM IPTG was added. The cultures were incubated at 18 °C for 24 h, and after expression, the cells were collected by centrifugation and disrupted by sonication.

The initial purification step involved affinity chromatography. The histidine tag was then cleaved using the ULP1 protease. For the final purification, size exclusion chromatography (SEC) was performed at pH 7.4 in a buffer containing 50 mM HEPES (pH 7.4), 400 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA, and 0.05% (v/v) thesit. Fractions corresponding to the homogeneous peak obtained from SEC were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), pooled, and concentrated to 30 mg mL–1. Protein concentration was determined based on a theoretical extinction coefficient (ε280nm) of 15.9 mM–1 cm–1 and a theoretical molecular weight of 39 887 Da.

4.2. Protein Crystallization

Before setting up the crystallization plates, HsDHODH was incubated at a final concentration of 20 mg mL–1 with 2 mM DHO, 20.8 mM N,N-dimethyldecylamine N-oxide (DDAO), and 0.75 mM lapachol for 2 h (adapted from Lewis and collaborators). For crystallization, sitting drop vapor diffusion experiments were performed using 48-well MRC (SWISSCI AG) plates. Each drop, consisting of 1 μL of the protein solution mixed with 1 μL of the reservoir solution, was equilibrated against 250 μL of the reservoir solution at 20 °C. Details of the materials and methods are provided in Table .

1. Details of the Crystallization Protocol.

method sitting drop vapor diffusion
plate type MRC Maxi 48-well crystallization plate (Swissci)
temperature (K) 293.15
protein concentration 20 mg mL–1
buffer composition of protein solution 50 mM HEPES pH 7.4, 400 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA, and 0.05% (v/v) thesit, 2 mM DHO, 20.8 mM DDAO, and 0.75 mM of lapachol
incubation time 2 h
composition of reservoir solution 0.1 M sodium acetate trihydrate pH 4.8, 1.9 M ammonium sulfate, and 30% (v/v) glycerol
volume and ratio of drop 2 μL (1:1)
volume of reservoir 250 μL
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4.3. Data Collection and Processing

The diffraction data was collected at beamline BL14.1 (BESSY II, Berlin, Germany). The data was processed using the automatic processing pipeline XDSAPP. Data collection and processing statistics are summarized in Table .

4.4. Structure Solution and Refinement

Initial phases were obtained by molecular replacement with PHASER using the HsDHODH structure PDB code 5K9C as the search model. Model building and refinement were performed with Coot and Refmac5 through the CCP4 suite. The quality of the final model was validated by MolProbity, and the structure was deposited in the Protein Data Bank (PDB) under accession code 9EG9. Structure refinement statistics are listed in Table .

4.5. Molecular Docking Simulation

Molecular docking simulations were employed to analyze differences and similarities in the interaction mechanisms of the ligands 2-((4-fluorophenyl)­amino)-3-hydroxynaphthalene-1,4-dione and lapachol at the binding sites of SmDHODH (PDB ID 6UY4) and HsDHODH (PDB ID 9EG9), respectively. Ligands were separated from receptors, protonated using the UCSF Chimera program, and assigned AM1-BCC charges and GAFF force field parameters using the AmberTools program antechamber. The FMN cofactor was prepared following the same protocols as those applied to the ligands. Proteins were protonated, and ff14SB charges and parameters were assigned using the AmberTools program tleap. The protonation states of the ionizable residues at pH 7.4 were evaluated by means of the H++ method (http://biophysics.cs.vt.edu/H++). The assembled receptor–ligand complexes were then subjected to a short energy minimization using Amber18 in the Chimera program with strong restraints on all nonhydrogen atoms to relax the system in a controlled manner. The surface of the receptors, without ligand, was determined using DMS with a 1.4 Å probe atom radius, and the binding cavity was filled with docking beads using the DOCK6.12 tool sphgen. Finally, a docking grid was generated for each receptor with the DOCK6.12 accessory program grid within a box that surrounded all spheres with a margin of 8.0 Å in all directions and at a 0.3 Å resolution. Each grid point was assigned Lennard-Jones parameters with attractive and repulsive exponents of 6 and 9, respectively, and included a Coulombic energy term calculated using a distance-dependent dielectric constant of 4π. For redocking and cross-docking experiments, the ligands were treated as flexible based on the FLX protocol described by Mukherjee et al.

4.6. Molecular Dynamics (MD) Simulations

MD simulations were employed to investigate the dynamic behavior of the cocrystal of HsDHODH in complex with lapachol (PDB ID 9EG9). The HsDHODH structure was prepared using the Protein Preparation Wizard available in the Schrödinger Suite (Schrödinger, L. Maestro Schrödinger 2021–4). We added missing atoms, adjusted side chains, and ensured accuracy of the atomic charges. Protonation and tautomeric states of amino acids were modified to match a pH of 7.4. Hydrogen bond sampling and adjustment of water molecule orientations were performed using PROPKA at pH 7.4. Structural water within 5 Å of the protein was preserved, and a minimization process with the OPLS4 force field was executed until an average root-mean-square deviation (RMSD) of 0.3 Å for the nonhydrogen atoms was achieved. In turn, the lapachol structure was assigned a protonation and ionization state at a pH of 7.4 using the LigPrep module (Schrödinger, L. Schrödinger Release 2021–4: LigPrep). Then, MD simulations were performed using the AMBER24 software package. The protein and ligand were treated with the ff14SB force field and general Amber force field version 2.2.20 (GAFF2). The complex was neutralized with a chloride ion, and then the system was solvated in TIP3P in an octahedral box 10.0 Å away from the edge. Subsequently, the system charges were neutralized with 0.15 NaCl. Then, the complex system was first minimized by the steepest descent method and the conjugate gradient method. Seven equilibration steps were applied by gradually decreasing the constraint forces. After the equilibration phase, three independent production simulations of 1 μs each were conducted for the HsDHODH–lapachol complex under NPT ensemble conditions (1 atm and 310 K). To further investigate the stability of an observed conformational state, replica 3 was extended for an additional 500 ns during the production phase (totaling 1.5 μs). The Langevin thermostat was employed for temperature regulation, while the pressure was managed using the Berendsen barostat. The VMD and CPPTRAJ tools were used for trajectory analysis.

Supplementary Material

ao5c01536_si_001.pdf (1.5MB, pdf)

Acknowledgments

The authors sincerely thank the Helmholtz-Zentrum Berlin (HZB) partners for crystallization, data collection, and processing. This work was supported by iNEXT-Discovery [Project Number 871037] funded by the Horizon 2020 program of the European Commission. This work was also supported by the São Paulo Research Foundation (FAPESP), Brazil [Process Number #2021/13237-5 and #2020/06190-0], the Goiás Research Foundation (FAPEG), Brazil [Process Number #202010267000272], the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil [Finance Code 001 number 441038/2020-4], the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil [process numbers #441038/2020-4, #443750/2023-8 and #440373/2022-0], the NIH, USA [process number 5R01AI160379], and Deutsche Forschungsgemeinschaft (DFG), Germany [process number FE2166/1-1].

The coordinates of the model are available in the Protein Data Bank (PDB) under accession code 9EG9. The trajectories of molecular dynamics simulations are available at https://zenodo.org/records/14267492.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c01536.

  • Lapachol secondary binding mode identified by MD showing interactions; RMSD and RMSF analyses of MD simulations for replicate 3; stereoview of the lapachol binding site in the crystal structure; and proposed modifications of the lapachol molecule that could be synthesized in further projects (PDF)

M.C.N. coordinated, designed, and supervised the project. A.D.P. purified the protein, refined the crystallographic structure, and wrote the first draft of the manuscript. M.S.W. supervised, and L.S.B. performed protein crystallization, data collection, and data processing. C.H.A. supervised, and W.J.L.S. performed molecular dynamics simulations and contributed to the first draft of the manuscript. F.S.E. contributed to the second revision and made a new figure. M.C.N., M.S.W., F.S.E., and C.H.A. acquired funding for this project. All authors critically reviewed and contributed to the final version of the paper.

The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brazil (ROR identifier: 00x0ma614).

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c01536_si_001.pdf (1.5MB, pdf)

Data Availability Statement

The coordinates of the model are available in the Protein Data Bank (PDB) under accession code 9EG9. The trajectories of molecular dynamics simulations are available at https://zenodo.org/records/14267492.

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