Leishmaniasis is among the neglected global health problems. Cutaneous leishmaniasis (CL) is caused mainly by the protozoan parasites Leishmania major and L. tropica in Afro-Eurasia. Nearly 90 % of the world cases appear in Iran, Afghanistan, Brazil, Saudi Arabia, Peru, and Syria [1,2]. Although CL caused by L. major is mostly considered self-healing and nonfatal in most instances, its satisfactory cure can last months or even years [3]. Since CL is prone to secondary infection, functional impairment, mutilating facial scars, parasite migration to mucous membranes, or recurrence throughout the recovery period [4,5], the prime aim of therapy is thus to shorten the recovery period and suppress the propagation of the parasite by boosting the host's ability to remove the causative agent while healing of the lesions [6,7].

The current recommended first-line antileishmanial drugs like meglumine antimoniate (MA, Glucantime®) and sodium stibogluconate (Stiboson®) require prolonged courses of parenteral drug administration exerting adverse effects, toxicity, and drug resistance [4]. Disadvantages of other second-line drugs such as amphotericin-B, paromomycin, and miltefosine also include high price, insufficient efficacy, and resistance [7,8]. Consequently, the introduction of safer, less toxic, and more affordable alternative treatments is advantageous. Although copper (Cu) plays a critical role in many biological processes [9], Cu-containing compounds have long been known as potent oxidative stress or cell damage inducers [10]. Metal complexes, in general, have also been shown to interfere with the function of parasite-specific enzymes such as cysteine proteases [11,12], aquaporin, and aquaglyceroporin channels [13], and oxidoreductase enzymes [14]. Besides, the intercalation of copper compounds with nucleic acids has been addressed as an attractive approach to designing bioactive Cu (II) complexes [15]. Organic moieties containing heteroatoms, when utilized as coordinating ligands, may contribute to improved cellular uptake and the biological properties of metal-containing complexes [16]. Literature suggests that metal complexes may be dominantly suited to reach the parasite cell. A homoleptic copper complex containing a Cu (II) ion coordinated with two 4,4′-dimethyl-2,2′-bipyridine (bpy) ligands, abbreviated as CuDMBP here, has already shown antiproliferative activity against human cancer cell lines and exhibited promising interactions with eukaryotic macromolecules in vitro. The effect has partially been attributed hydrophobic interaction of the methyl groups on the bpy rings. [16]. It is quite rational and becoming frequent to choose a compound with proved eukaryotic antiproliferative or cytotoxic activities for antiparasitic studies [9]. Considering the wide range of applicability of metalo-organic complexes [17,18], CuDMBP, makes a good candidate for antileishmanial investigations. Caspases belong to the superfamily of cysteine proteases found in animals and a few viruses. Activated from of the pro-enzymes, which are produced via a proteolytic cleavage reaction, play a central role in apoptotic programmed cell death and therefore are the center of attention [19,20]. In protozoa, plants, and yeasts, the role of caspases is thought to be played by metacaspases (MCs), making these enzymes suitable targets for docking studies [21]. While several metacaspases could be found in bacteria and archaea, protozoan organisms contain only 1 metacaspase [22]. Two prime features can signify metacaspases as key drug investigational targets: MCs are associated with apoptotic programmed death of the parasite cells and they are absent from human cells [23]. ADME evaluations, structure-activity relationship (SAR), and structure-property relationship (SPR) predictions can be achieved using readily available web-based platforms. SwissADME (http://www.swissadme.ch), provided by the Swiss Institute of Bioinformatics [24], is a frequently referred portal for this purpose. It is well-documented that pathogen contamination can induce alterations in cytokine release profiles from macrophages [25]. For instance, intracellular amastigotes of Leishmania have been able to trigger the release of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α), an effect for which the parasite's zinc metalloprotease GP63 has been assumed to be responsible [26]. Interleukin-12 (IL-12) is a Th1-type immunity activation marker against Leishmania infections, whereas IL-10 is the primary index of type 2 T-helper cells (Th2) represents a key regulatory cytokine in leishmaniasis to facilitate parasite persistence and dissemination [27,28]. Moreover, reports project a dual role for nitric oxide (NO) in regulating the immune response [15]. Indeed, the proinflammatory NO is produced in macrophages and other innate immune cells by the inducible nitric oxide synthase (iNOS) and performs an essential role in the host immune response against bacterial, viral, and parasitic contamination [29].

This study aimed to explore the in silico binding affinity of CuDMBP towards a parasitic metacaspase and in vitro assessments of its inhibitory effects against L. major promastigotes using the MTT colorimetric technique. We also predicted CuDMBP's ADME properties and explored its apoptotic profile by flow cytometric analyses. The potency of the complex to modulate the cellular expression of iNOS, IL-10, and IL-12 genes in the treated amastigote-infected model murine macrophages was also evaluated.