TropicalMed, Vol. 7, Pages 400: Inhibiting Human and Leishmania Arginases Using Cannabis sativa as a Potential Therapy for Cutaneous Leishmaniasis: A Molecular Docking Study

1. IntroductionLeishmaniasis is a parasitic infection caused by protozoan parasites that belong to the Leishmania genus and are transmitted to humans through an infected phlebotomine sandfly bite. This infection is associated with the following clinical forms: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL) [1]. In fact, CL, the least severe clinical form of leishmaniasis, causes various types of skin lesions including ulcers, ulcero-crusted lesions, and nodular ones that are located in uncovered areas of the human body and leave disfiguring scars [2,3].About 700,000 to 1 million new cases are reported each year worldwide [1]. CL is considered to be a public health problem especially in North African countries including Morocco and is induced by three species: Leishmania major, L. tropica, and L. infantum [4]. In the absence of a vaccine, the treatment of CL in Morocco is still based on pentavalent antimonials as the primary therapy. Furthermore, other molecules such as amphotericine B, pentamidine, paromomycin, and miltefosine were used as alternative therapies. Nevertheless, the use of these drugs is associated with many side effects such as relapses, high toxicity, and the emergence of drug-resistant parasitic strains [5]. Thus, and for better Leishmaniasis control, the design of an efficient anti-leishmanial drug must focus on the development of natural, less toxic, and cost-effective drugs with greater availability to low-income populations.The parasite life cycle involves two stages: promastigote and amastigote. The promastigote-insect stage is characterized by an extracellular and flagellated form that is found in the vector’s gut. On the other hand, during the amastigote-vertebrate stage, the amastigote resides in mammalian cells, especially inside macrophages [6]. Interestingly, these immune cells are not only the replicative niche of Leishmania, but are also the anti-leishmanial effector cells [7]. To perform these contrasting functions, macrophages are characterized by functional plasticity and adopt different effector states. Through the influence of their microenvironment, naïve macrophages (M0) are activated and polarized toward “classically activated” (M1) or “alternatively activated” (M2) with different disease outcomes [8,9]. In these two types of macrophages, arginine is a key amino acid for two competing metabolic pathways: arginine can be converted to ornithine by arginase or alternatively to the anti-leishmanial agent nitric oxide (NO) by inducible nitric oxide synthase [10]. Classical M1 are activated upon Th1 cytokine exposure and contribute to the increased production of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, hydroxyl radicals, and NO, which are crucial to the elimination of intracellular pathogens such as Leishmania due to their higher microbicidal capacity [9,11,12,13]. In contrast, Th2 cytokines induce the M2 profile, which is characterized by polyamine biosynthesis via activation of the arginase enzyme. This reaction is ensured through the bioconversion of L-arginine into L-ornithine and urea in the presence of manganese atoms. In fact, urea and L-ornithine are beneficial to intramacrophage Leishmania proliferation and disease progression [14,15,16,17].An increased arginase activity associated with a decreased nitric oxide production is involved in leishmaniasis. Interestingly, Leishmania expresses arginase, and its activity has been shown to play an essential role in parasitic vital functions such as polyamine production and infectivity [18,19]. Arginase is also expressed in mammals; two of its isoforms were discovered: the hepatic arginase (ARG-1) and the extra-hepatic arginase (ARG-2), which is highly expressed in the kidney and prostate [20]. Host arginase (ARG-1) is induced by Leishmania, leading in turn to polyamine synthesis and thereby reinforcing Leishmania’s proliferation [16]. Consequently, both parasitic and host arginases are required for parasite growth and survival. Indeed, previous studies showed that patients with CL exhibited higher levels of Arg-1 in plasma and dermal lesions and demonstrated that there was a positive correlation of the CL severity with the host arginase level [9,21,22,23]. Using murine leishmaniasis models, studies have also demonstrated an increased arginase expression in susceptible BALB/c mice, which was associated with Th-2 cell response and a higher parasitic burden, resulting in the disease severity [17,24]. Moreover, the inhibition of host arginase was translated by a remarkable decrease in the parasitic load in infected mice [17,25,26]. Lee et al. also showed that a non-healing strain of L. major was efficiently phagocytized by M2 macrophages in vitro and in vivo. However, a strain that produced self-healing lesions was less phagocytosed by M2 macrophages. The authors stated that the preferential infection of M2 cells played a crucial role in the severity of the cutaneous disease [27]. Collectively, these studies suggested that both parasite and host arginases play a crucial role in the balance of life and death of Leishmania and underline the importance of arginase pathway as a potential and promising therapeutic target.Cannabis sativa (C. sativa) is an annual plant belonging to the Cannabaceae family that is cultivated in different regions around the world and is used for many medicinal purposes [28,29]. C. sativa synthetizes several phytochemical compounds called cannabinoids, terpenoids, flavonoids, and alkaloids [30]. These natural components are the most identified in C. sativa and their production is impacted by specific environmental conditions such humidity, temperature, and others [29]. The phytocannabinoids found in organic extracts of Moroccan C. sativa are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), both of which belong to the most studied cannabinoids [31,32]. In addition to the phytocannabinoids, terpenoids such as α-humulene, β-caryophyllene, β-myrcene, caryophyllene oxide, α-pinene, decane, and limonene were described in different C. sativa essential oils [33,34]. In vitro, C. sativa extracts and active components showed an antimicrobial potential against pathogenic bacteria, fungus, and parasites, including Leishmania [35,36]. In addition, in vivo studies on a CL hamster model showed that this plant extract exhibited remarkable anti-leishmanial and healing potentials. Interestingly, the application of a C. sativa-based cream on CL-associated skin lesions promoted the healing process [37]. Nonetheless, these overall studies did not go further to determine C. sativa’s possible targets and mechanisms of action.

In light of these results and due to the existing interplay between host and parasite arginine, as well as the importance of polyamine metabolism in the outcome of Leishmania infections, our aim was to highlight the molecular interactions of cannabinoids and terpenoids with both arginases through molecular docking. Although this was an in silico work, it will contribute to the discovery of new natural and effective compounds against CL.

4. DiscussionCutaneous leishmaniasis is a neglected tropical infection that requires proper treatment and close monitoring [40]. The drugs used for leishmaniasis treatment are still based on pentavalent antimonials (Glucantime® and Pentostam®), which have severe side effects [5]. Hence, natural compounds derived from plants are characterized by their efficiency, low toxicity, and cost, and are associated with minor side effects. In fact, these natural molecules have been exploited as alternative anti-leishmanial agents [5]. Previously, it was reported that a crude methanolic extract of C. sativa with a high amount of phenolic compounds possessed an in vitro growth-inhibition effect on the L. major parasite [41]. Furthermore, three C. sativa essential oils composed mainly of myrcene, α-pinene, and e-caryophyllene induced an in vivo protective effect in L. tropica-infected mice [42].CL is characterized by localized skin lesions in which arginase is upregulated and involved in Leishmania proliferation [21,43]. Leishmania arginase represents the first enzyme involved in polyamine biosynthesis; its lack blocked L. mexicana as well as L. major growth, but it was restored by adding polyamines [18,44]. Consequently, various studies proved that flavanols such as quercetin as well as verbascoside were effective against extracellular promastigote and intracellular amastigote forms of L. amazonensis by inhibiting parasitic arginase [45,46,47]. Similarly, 2(S)-amino-6-boronohaxanoic acid (ABH) blocked Leishmania and/or human arginase activities [38,43]. Interestingly, our in silico study, which used both parasitic and human arginases, highlighted for the first time the molecular interactions between phytochemical components of C. sativa and the active site of these enzymes. Therefore, our docking interaction results for the L. mexicana arginase active site indicated that delta-9-THC and three other selected ligands interacted with the amino acids HIS 139, HIS 154, ALA 192, and ALA 140. HIS 139 belongs to the amino acids involved in bridging with Mn2+ [48,49]. This latter interaction with nor-NOHA blocked arginase activity, as demonstrated previously [50]. Therefore, this metal bridging is necessary for arginase activity, which led us to suggest that interactions between delta-9-THC and HIS 139 might be responsible for the inhibition of this target enzyme. Furthermore, HIS 154 is directly involved in the L-arginine substrate and binds to L. mexicana as well as L. amazonensis arginases [48,51]. Consequently, interaction between HIS 154 and delta-9-THC might be responsible for arginase inhibition. Another compound, phenylacetamide, as well as caryophyllene oxide, interacted with the ALA 192 amino acid, which resulted in the inhibition of Leishmania arginase activity [49]. In addition, Leishmania arginases sequences, mainly in the active site, possessed a high degree of conservation [47]. Thus, this result could be exploited in the context of different Leishmania species such as L. major, L. tropica, and L. infantum, which are responsible for CL in Morocco.The human arginase amino acid residues involved in our protein ligand links were HIS 126, ASN 139, HIS 141 ASP 181, and PRO 247; some of these amino acids are involved in the active site of human arginase [52,53]. Among the studied cannabinoids, delta-9-THC showed good inhibitory activity, as did CBD. In addition, they shared some properties such as strong antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory activities [54,55,56]. Our molecular analysis demonstrated that caryophyllene oxide was linked to human arginase by different residues that included ASN 139 and HIS 126. Previously, cinnamide derivatives were also reported to inhibit mammalian arginase through interactions with HIS 126, HIS 141, and other target pocket residues [57]. Moreover, lignanamides, which are new compounds that were isolated from C. sativa, were reported as natural inhibitors of bovine arginase [58]. In addition, delta-9-THC and beta-caryophyllene involved different residues while interacting with LmArg and h-Arg. Interestingly, the cannabinoids and terpenoids selected for this study were found to inhibit both human and Leishmania arginases’ active sites via hydrogen bonds as well as via hydrophobic interactions. The interplay of the host, parasite arginine, and polyamine metabolism is decisive in the outcome of Leishmania infections. In addition, the Leishmania parasite has the ability to modulate the host arginase activity and therefore the immune response [10]. Therefore, studies suggested that inhibition of the parasite arginase alone may not be a sufficient therapeutic strategy. Indeed, although the infectivity level was diminished, the arginase-deficient mutants in different Leishmania species were still able to establish infections [10,44,59].

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