Molecular Docking and Evaluation of Antileishmania Activity of a Ruthenium Complex with Epiisopiloturine and Nitric Oxide

Leishmaniasis is an infectious disease that affects both animals and humans, caused by flagellated parasites belonging to the genus Leishmania. The disease is estimated to reach about 700,000 to 1 million people, causing the deaths of 20 to 30,000 individuals annually. Thus, the present study aims to perform molecular docking tests and evaluation of antileishmania activity in vitro of a ruthenium complex with epiisopiloturine and nitric oxide. AutoDockTools-1.5.6 software was used to perform molecular docking tests. Molecular targets were considered rigid, and Epiruno 2 considered flexible. The genetic algorithm Lamarckian (AGL) with global search and pseudo-Solis and Wets with local search were the methods adopted in the docking. The most promising results of molecular interaction were achieved in the targets Pteridine reductase and UDP-glucose Pyrophosphorylase with rates of −10.68 Kcal∙mol −1 and −10.51 Kcal∙mol −1 , respectively. This demonstrates that Epiruno 2 has molecular affinity with the targets of L. major. In vitro assays prove the antileishmania activity of there is a clear need to search for new compounds with pharmacological potential and low toxicity by alternative methods that bring reliability in their results, speed and cost benefit. Thus, computational quantum chemistry presents itself as a promising alternative, using several computational tools that predict molecular properties related to a pharmacological potential. Using the laws of quantum chemistry and various programming techniques that are capable of predicting energy state, molecular structures, vibrational frequencies of atomic and molecular systems and molecular interaction between two molecules de-vel-oping virtual models saving time and materials that would be wasted on ex-periments in the field laboratory [9] [10]. the complex in the face of promastigote forms with inhibition of growth, concluding through this study that the Epiruno 2 complex has antileishmania activity.


Introduction
Leishmaniasis is a disease that affects more than 98 countries worldwide, with about 700,000 to 1 million new cases reported annually, and an annual rate of 20 to 30,000 deaths [1]. There are several ways for the disease to manifest clinically, and may present as cutaneous, mucocutaneous and visceral. Infection with Leishmania major (L. major) species has a chronic evolution that affects the structures of the nasopharyngeal epidermis and cartilage, either localized or diffuse [2]. The parasitic cycle results from the abundance of carbohydrates on the surface of Leishmania, which includes lipophosphoglycans, glycosylphosphatidylinositol lipid-anchored proteins and proteophosphoglycans [3]. These glycoproteins are part of the promastigote infectious glycocalyx, which is the most important process in host infectivity [4] and phlebotomine interaction [5].
After diagnosis, the patient undergoes treatment that depending on the infecting strain will be treated with some of the drugs available on the pharmaceutical market, they are: pentavalent antimonial; AmBisomew; liposomal; amphotericin B; miltefosine and diamidines, among others. All of these drugs are potentially toxic and have reduced efficacy in addition to adverse side effects. That in many cases, the patient chooses not to undergo treatment so that he does not suffer from side effects caused by drugs [6] [7].
Besides this problem, the pharmaceutical industries neglect investments in the search for new pharmacological agents that present high inhibition rates with new mechanisms of action and low toxicity. This lack of interest is related to market demand, as it is a neglected disease, that is, it affects only underdeveloped and developing countries, the sector has high risks of not making profits on their investments, because the population would not be able to afford it. With the costs of treatment, even the state would not be able to finance the services offered by the industries [8].
Thus, there is a clear need to search for new compounds with pharmacological potential and low toxicity by alternative methods that bring reliability in their results, speed and cost benefit. Thus, computational quantum chemistry presents itself as a promising alternative, using several computational tools that predict molecular properties related to a pharmacological potential. Using the laws of quantum chemistry and various programming techniques that are capable of predicting energy state, molecular structures, vibrational frequencies of atomic and molecular systems and molecular interaction between two molecules devel-oping virtual models saving time and materials that would be wasted on experiments in the field laboratory [9] [10].
In this sense, ruthenium (Ru) complexes have become attractive in pharmacological studies because they have low toxicity and are an excellent conductor of energy when dealing with a transition metal, where it plays an important role in the bioactive process of a compound in reaction with a target disease, having little energy loss in its path [11] [12]. The Ru complex with epiisopiloturin and nitric oxide (Epiruno 2 ) was synthesized by  in anti-schistosoma mansoni studies, where the Epiruno 2 complex showed schistosomicidal activity in sílico and ex vivo studies. There was a 10-fold increase in the biological activity of Epiisopiloturin (EPI) when coupled with the Ru complex against Schistosoma parasites, eliminating 60% of male worms at a concentration of 50 µM within 72 hours, showing antiparasitic activity [13].
Associated with the antiparasitic schistosomicidal effect presented by the Epiruno 2 complex in studies by , we assume that the complex has antileishmania activity. Thus, the present study aims to perform molecular docking tests and evaluation of antileishmania activity in vitro of a ruthenium complex with epiisopiloturine and nitric oxide.
The three-dimensional molecular structure of the Epiruno 2 complex was designed using GaussView 5.0 software [17] and optimized by DFT (Density Functional Theory) calculation using the B3lyp functional and the 6-311 ++ G (d, p) available in Gaussian 09W software [18] [19].
The molecular docking process followed the protocol developed by Rocha and collaborators, with some modifications [20]. All molecular docking procedures were performed by AutoDockTools-1.5.6 software [21]. L. major targets and the Epiruno 2 complex were prepared for docking simulations, where targets were considered rigid and Epiruno 2 was considered flexible. Partial charges were calculated after the addition of all hydrogens. The nonpolar hydrogen atoms of the protein and binder were subsequently fused. A 60 × 60 × 60 point cubic box with a spacing of 0.375 Å between grid points was generated for the simulations. The molecular affinity grid centers were defined from the coordi-nates of the atoms of their respective active sites Asn376, Lys380, Gly91, Asn109 and Asp15, respectively.
The Lamarckian global search (LGA) genetic algorithm [22] and the pseu-

In Vitro Trials on Promastigote forms MHOM/IL/80/Friedlin of L. major
For in vitro assays, the method adopted by Carneiro and collaborators was used, with some modifications [25].

Molecular Docking
The evaluation criteria were defined by the results that showed lower cluster conformation with lower a bind G energy, besides the hydrogen bridge interactions and inhibition constant presented by the Epiruno 2 complex against the L. major molecular targets. The most intense interactions between the target protein and the Epiruno 2 complex occur between the residues Asp232, Lys198, Ser111 and Ser227, places where the highest intermolecular forces act ( Figure 1).
The Epiruno 2 complex also showed excellent molecular affinity results with the target protein 5nzg of L. major, obtaining a a bind G energy of −10.51 Kcal•mol −1 ( Table 1)    both residues with C8 ( Figure 2). These interactions at the edges of the active site make the Epiruno 2 complex have a very promising high inhibitory action, and the tertiary structure is part of recognition elements that facilitate the molecular interactions between protein and ligand, in this case the Epiruno 2 complex [33] [34].
Molecular docking between the 5c7p protein and the Epiruno 2 complex formed three hydrogen bridges at amino acids Arg104, Asn114 and Ser98  (Table 1). These results are promising against this indispensable protein for the maintenance of intracellular nucleoside triphosphate (NTP) levels [35]. They carry the γ-phosphoryl group from an NTP to a nucleoside di-   Table 1). The most intense interactions between the complex and the protein occur at residues Pro11 and Gln40, the two hydrogen bonds formed (Figure 2).

In Vitro Trials on Promastigote Forms MHOM/IL/80/Friedlin of L. major
In these trials we evaluated the leishmanicidal effects of the Epiruno 2 complex against L. major promastigote MHOM/IL/80/Friedlin parasites. The Epiruno 2 complex showed 50.53% inhibition of promastigote growth at a concentration of 800 µg/mL (Figure 3), a significant reduction by analyzing the half maximal inhibitory concentration (CI-50) ( Table 2) showing antileishmania activity, confirming the results presented in sílico tests by molecular docking. However, these J. L. Araújo et al.
values are not considered clinically relevant, according to Santos et al. [40], which defines in their studies that only IC-50 lower than 500 µg/mL can be considered therapeutically relevant.
It is observed that the results presented in molecular docking analyzes were more promising than the results presented in vitro assays. This may be related to the topological polar surface area (TPSA), which uses functional groups obtained from a structural database, avoiding calculations of the ligand's three-dimensional (3D) structures, in this case the Epiruno 2 complex or the confirmation of which conformation. Since this biological method is relevant, this method is used in 2D structures for 14 sets of diverse pharmacological activity data. This methodology is promising for classic 2D descriptors such as calculated LogP (ClogP) and calculated molar refractivity (CMR) in the 2D-QSAR literature [41].
The discovery of new antileishmania chemical compounds has long been rea-

Conclusions
The Epiruno 2 complex presented antileishamania activity both in sílico studies by molecular docking and in vitro study. Its best molecular affinity parameter presented in docking studies was for target proteins 1e7w and 5nzg with a bind G energies −10.68 Kcal•mol −1 and −10.51 Kcal•mol −1 , respectively. In addition to these two targets, it was found that the complex has molecular affinity for the other molecular targets of L. major analyzed in this study. In vitro assays proved the antileishmania activity of the complex against L. major promastigotes MHOM/IL/80/Friedlin with significant growth inhibitions. However, the values are not considered clinically relevant, concluding from in sílico and in vitro studies that the Epiruno 2 complex has antiparasitic activity that can be tested on other Leishmania targets such as L. amazonensis and L. Chagasi and also in other pathogens.