In silico Elucidation of Dihydroquinine Mechanism of Action against Toxoplasma gondii

Dihydroquinine (DHQ), is a quinine-based compound with anti-malarial properties. However, little is known about its mechanism of action against T. gondii inhibition, which shares similar biology with Plasmodium spp. In order to explore DHQ activity as an inhibitor of T. gondii using in vitro assays, we first used an in silico approach to decipher its mechanisms of action based on previous knowledge about its disruption of nucleic acid and protein synthesis. An in silico study was performed on T. gondii parasite replication, transcriptional and translational machinery to decipher the binding potentials of DHQ to some top selected enzymes. We report for the first time, using an in silico analysis that showed that DHQ binds strongly to DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase and thus could affect DNA replication, transcriptional and translational activities in T. gondii. Also, we found DHQ to effectively bind to mitochondria detoxifying enzymes (i.e., superoxide dismutase (SOD), peroxidoxin, and Catalase (CAT)). In conclusion, DHQ could be a lead compound for the treatment of toxoplasmosis when successfully evaluated using in vitro and in vivo models to confirm its effectiveness and safety.


Introduction
Zoonotic parasitic infections continue to cause serious public health, veterinary and socioeconomic predicament globally (Ben-Harari and Connolly, 2019; Pappas et al., 2009;Flegr et al., 2014;Dubey, 2010). For example, toxoplasmosis, the disease caused by T. gondii affects nearly 1/3 of the human population worldwide (Pappas et al., 2009;Flegr et al., 2014). More worrisome, a recent study, revealed that over 35 to 76% of wild and domestic felids are infected with T. gondii (Montazeri et al., 2020). This suggests that its infection in man and animals are on the increase globally. To overcome these parasitic diseases, different strategies are required which include, management, prevention, and treatment with safe and effective chemical inhibitors. Although few drugs are available for the treatment of individuals (humans and animals) infected with T.gondii, they are faced with limitations such as toxicity, high cost, and more so, most drugs are ineffective in treating the latent stage (bradyzoite) that continuously persist in the brain (Shammaa DHQ is a pharmaceutical impurity associated with quine production (Nontprasert et al., 1996). It has been reported to have anti-malarial properties ( Gorka et Brossi et al., 1971;Polet and Barr, 1968). Furthermore, its mechanism of action has been predicted in Plasmodium species to target nucleic acid and protein synthesis (Brossi et al., 1973;Brossi et al., 1971). However, its mechanism of action against T. gondii, which shares a similar phylum with Plasmodium is yet to be investigated.
Here, using computational analysis, we seek to assess the molecular mechanism of action of DHQ in T. gondii using selected replicative, transcriptional, translational, and mitochondria machinery such as DNA gyrase, CDPKs, MIF, RNA synthase, SOD, peroxidoxin, and Catalase.

Author Summary
Early deciphering of compounds' mechanism of action is crucial for drug discovery and development. This approach saves time, resources and provides an insight into the possible mechanism of action of compounds of interest before wet experimental work could be carried out. In this paper, we used in silico approach as a first point of deciphering the mechanism of DHQ, a quinine derivative, that has been reported to have anti-Plasmodium activity in vitro and in vivo. The prediction showed that DHQ binds strongly to very important replicative, transcriptional, translational, and mitochondrial-associated proteins. Thus, this process was carried out as the first approach to predict DHQ's possible mechanism of action against T. gondii before performing wet labs to gain insight into the compound's inhibitory activity in T. gondii growth in vitro.

In silico Docking of DHQ with T. gondii Replicative and Translational Machinery
Based on our cellular and biochemical assay results, we used in silico docking analysis to decipher whether DHQ could be targeting the following enzymes/receptors: DNA Gyrase, TgCDPK1, tRNAs, MMIF, ROP5B, and ROP5C. These proteins play crucial roles in the parasite's invasion, survival, and replication. Herein, we analyzed for the theoretical binding affinities between DHQ and the enzymes/receptors as well as their ligand-receptor interactions at the docking pocket. The docking analysis was performed using extracted crystalized structures of the receptors from the RCSB website or modeled using the online modeling software SWISS-MODEL (https://swissmodel.expasy.org/). DHQ, 3-Amino-1H-pyrazole-4-carboxamide, and Moxifloxacin were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).

DHQ interaction with T. gondii DNA Gyrase interactions
To stop the progression of multidrug-resistant tuberculosis, fluoroquinolone has been shown to have high potency, and the mechanism of action of fluoroquinolone is known to target DNA gyrase (Blower et al., 2016). It has also been proven in parasites (e.g., Trypanosomes) that some quinolone drugs bind to DNA gyrase to form a complex that blocks transcription by RNA polymerase (Willmott et al., 1994). DNA gyrase of T. gondii has also been indicated as a potential target for drugs against T. gondii (Lin et al., 2015). While the crystal structure of the DNA gyrase of multidrug-resistant tuberculosis has been discovered, the DNA gyrase of T. gondii is currently unavailable. In this work, we used the crystal structure of multidrug-resistant tuberculosis as a model study receptor to dock DHQ as a ligand and to analyze for their binding affinities (ΔG). To also show if there exist any contrasting binding affinities between DHQ and fluoroquinolone for the M. tuberculosis DNA gyrase as well as T. gondii DNA gyrase, their binding affinities were compared and the specific amino acids interacting with these compounds were analyzed for the type of interactions occurring at the binding pocket. While fluoroquinolone (also known as moxifloxacin) showed a slightly lower binding affinity of -7.2 kcal/mol (  Figure 2A, 2B and 2E). As shown in Figure 1, the 3D rendition of DHQ in red color interacting with gyrase in green color, also in Figure 1, a 2D depiction of DHQ interacting with gyrase at the binding pocket indicates that DHQ forms a strong hydrogen bond with ARG234 producing a bond distance of 1.90Å, for which DHQ acts as the hydrogen acceptor and ARG234 the donor. The ALA255 of TgDNA gyrase binds to DHQ via hydrophobic interactions, forming a pi-alkyl bond with DHQ with a bond distance of 4.69 Å in the binding pocket. Forming a hydrogen bond at 2.97Å with DHQ, TgDNA gyrase via ARG234 acts as a hydrogen donor for which DHQ is the acceptor. DHQ was able to form a stable hydrogen bond with TgDNA gyrase via the gyrase's ASP200 amino acid residue which acts as a hydrogen acceptor. A bond distance of 1.99 Å is established between them. ARG195 interacts with DHQ via hydrogen bond also, for which ARG195 is the hydrogen acceptor. LEU257 of TgDNA gyrase establishes pi-sigma interaction with DHQ to establish a hydrophobic bond of distance 3.38 Å.

DHQ and T. gondii prolyl tRNA synthetase interactions
Prolyl-tRNA synthetase belongs to the aminoacyl-tRNA synthetase family and plays a crucial role in protein translation in living cells. The tRNA synthetase of T. gondii has been a target for several compounds used as therapeutics against T. gondii and other Apicomplexans, e.g., the use of febrifugine and halofuginone (Mishra et al., 2019). This current work revealed that DHQ targets the prolyl tRNA synthetase of T. gondii with a high relative free binding energy of -7.7 kcal/mol ( Table 1). Figure 3C is the 3D depiction of the DHQ interacting with the tRNA synthetase of T.
gondii. Also illustrated in Figure 3C and Figure 3D), a 2D illustration of the contacts of DHQ at the binding site to tRNA synthetase of T. gondii indicates that DHQ shares two hydrogen bonds at amino acids ARG594, and GLN555. There exists also a van der Waals interaction between the compound and the amino acid HIS560, a pi-alkyl interaction at amino acid PHE415 and pi-cation interaction with ARG470.
This analysis shows that there is a stable DHQ-prolyl tRNA synthetase interactions, pointing to the capacity of DHQ as a T. gondii inhibitor by interrupting the T. gondii protein translational activity by binding to the parasite's prolyl tRNA synthetase.   Figure 4C to interact with ROP5C, and in Figure 4D, the 2D diagram shows a stable interaction at the binding pocket in which LYS263 of ROP5C makes hydrogen bond contact with the compound, VAL248 makes a pi-sigma contact, whereas ARG241 makes an alkyl contact with DHQ. MET337 however, makes a pi-sulfur bond with DHQ.

DHQ and Macrophage Migration Inhibitory Factor of T. gondii (MIF) Interactions
Collectively, these interactions create stable and strong bonds between DHQ and ROP5C, pointing to the capability of DHQ as a T. gondii inhibitor by the mechanism of binding to and disrupting the T. gondii pathogenesis process.  Figure 5C, and 5D).
LYS338 however, shows a strong unfavorable donor-donor interaction with the hydrogen moiety of 3-Amino-1H-pyrazole-4-carboxamide. This interaction therefore might have caused a considerable loss of affinity of 3-Amino-1H-pyrazole-4-carboxamide to TgCDPK1 compared to DHQ. Hence, the strong unfavorable donor-donor interaction recorded seems to slightly dampen this stability of 3-Amino-1H-pyrazole-4-carboxamide to TgCDPK1 interaction, hence the recorded -5.5 kcal/mol binding affinity, DHQ on the other hand which produced a very high affinity of -8 kcal/mol recorded no unfavorable bonds in its binding pocket. Hence, we predict a higher inhibitory potential for DHQ on TgCDPK1 than 3-Amino-1H-pyrazole-4-carboxamide. To validate whether DHQ had any way of counteracting the mitochondrial enzymes that aid T.
gondii to detoxify drugs and other induces of ROS, we screen DHQ against the above-mentioned enzymes. Interestingly, DHQ interaction with T. gondii peroxidoxin (PRX), SOD3, and CAT had binding affinities of -7.0, -6.9, -6.8 kcal/mol, respectively. Figure 6A and 6B depicts DHQ (red) bound to the SOD3 molecule in the binding pocket. As shown in Figure 6B, DHQ establishes a stable hydrogen bond with GLU210. It also interacts with SOD3 via alkyl and pi-alkyl hydrophobic interactions through amino acids HIS80, TYR84, PHE168, and ARG221. Other weaker van der Waals interactions established between SOD3 and DHQ were recorded on amino acids LYS166, GLY169, ASN87, THR165, HIS167, TRP 209, HIS211, ASN 219, ASP220, and GLY222. Together these interactions enhanced the stability of the binding of DHQ to the SOD3 molecule. Figures 6C and 6D depict the interaction of DHQ (red) with the SOD2 molecule. As shown in Figure 6C, DHQ formed a stable hydrogen bond with ALA118 and HIS115. It also interacts with SOD2 via alkyl and pi-alkyl hydrophobic interactions through amino acids LYS122, TYR119, ARG260, and PRO261. The weak van der Waals interactions established between SOD2 and DHQ were recorded on amino acids ASN151, ALA203, PHE206, GLY207, TRP248, GLU249, ASN258, and ASP259. Accumulatively these interactions boosted the stability of the biding of DHQ to SOD2 molecule. Figure 7A and 7B depicts DHQ (red) bound to Tg Catalase chain A. As depicted in Figure 7B, DHQ forms a stable hydrogen bond with HIS183 and shares a pi-sigma bond with VAL291 and VAL443 respectively. VAL291 and VAL443 can also interact with DHQ via alkyl hydrophobic interactions. Tg Catalase also can bind to DHQ via its PRO293 by another hydrophobic alkyl interaction with the hydroxyl group of the DHQ. Figures 7C and 7D depict the interaction of DHQ (red) with the Tg peroxidoxin molecule. As shown in Figure 7D, Tg peroxiredoxin binds tightly to DHQ via two hydrogen bonds established between DHQ at amino acids AS204 and TYR164.
It is also able to stabilize DHQ in the binding pocket via hydrophobic interactions such as pi-pi-T shaped interactions, alkyl, and pi-alkyl interactions through the amino acids TYR164, IL3106, VAL206, LEU128, PHE132, LEU228, and PRO 127. Also, a weaker van der Waal force exists between ALA163, SER188, LEU190, THR131, and ALA269 with DHQ. These altogether stabilized DHQ in Tg peroxiredoxin binding pocket.

Conclusion
DHQ was highly effective in binding to replicative, transcriptional, and translational machinery of T. gondii in silico. Thus, could cause inhibition of T. gondii growth, invasion, and egress. Also, based on the in-silico data obtained regarding mitochondria machinery association with DHQ, we believed that the compound might cause ROS generation in T. gondii tachyzoites and eventually mitochondrial membrane disruption. All these predictions will need further investigation using in vitro experiments.

Molecular Docking of DHQ
The protein-ligand docking approach was employed to analyze and identify the specific amino acid interactions between DHQ and the respective receptors found on T. gondii. The binding affinities afford a good prediction of the ability of DHQ to inhibit T. gondii via various mechanisms modulated by proteins/enzymes (DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase) understudy.
The following platforms were used for the computational studies of the effect of DHQ on various replicative, and translational machinery; Vina Autodock, Pymol (2) (Schrodinger, LLC), PyRx, and Discovery Studio 2021.

TgDNA gyrase structure modeling
Currently, the TgDNA gyrase crystal structure is not available. Therefore, an in silico model was made using the TgDNA sequence. In summary, the TgDNA gyrase subunit B sequence (ID KFG61327.1) was downloaded from the NCBI website. The sequence was converted into FASTA format using the sequence format converter (http://avermitilis.ls.kitasato-u.ac.jp/readseq.cgi), afterward, the sequence was modeled into its predicted 3D structure using the online SWISS-MODEL tool (https://swissmodel.expasy.org/). The model with the highest score in Global Model Quality Estimation (GQME) was utilized for the binding study.

Ligand preparation
The DHQ was prepared as a ligand for docking onto the following receptors, 4DH4, ROP5C, ROP5B, and enzymes (TgDNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase). These receptors/enzymes were selected for the molecular docking analysis to confirm the in vitro mechanism of action(s) based on their association with replication, invasion, and survival of T. gondii. Additionally, previous works on Plasmodium spp implicated dihydroquinine to inhibit DNA, RNA, and protein synthesis (Brossi et al., 1973;Brossi et al., 1971;Polet and Barr, 1968). The chemical structure of DHQ was extracted from the PubChem database.
The ligand was uploaded into PyRx software via the Open babel plugin, and the 3D and geometry optimizations with energy minimization of DHQ structure were carried out. DHQ structure was converted to autodock DHQ.pdbqt ligand file via the same Open babel plugin in PyRx. The Vina wizard was used to load the DHQ.pdbqt file as a ligand to the respective receptors/enzymes used in the docking process. Huang et al. (2015), demonstrated that 5-aminopyrazole-4-carboxamide derivatives were selectively potent inhibitors of 4YJN (Calcium-Dependent Protein Kinase 1 (TgCDPK1) from T. gondii. They also showed the effectiveness of the structure-activity relationship of this compound in a mouse model against T. gondii in the brain, spleen, and peritoneal fluids. In this computational investigation, we analyzed the binding affinity of 5aminopyrazole-4-carboxamide also to 4YJN to serve as an experimental control for all the binding studies of DHQ on the various molecules. The 2D structure (PDB format) of 5-aminopyrazole-4carboxamide was downloaded from the PubChem database (CID_79254) and processed for docking. 5-aminopyrazole-4-carboxamide was prepared for docking following the same process as started earlier.

Preparation of protein structures and grid generation
The Crystal structures of virulent alleles ROP5B (3Q5Z) (