Inhibiting Human and Leishmania Arginases Using Cannabis sativa as a Potential Therapy for Cutaneous Leishmaniasis: A Molecular Docking Study

Cutaneous leishmaniasis (CL), a vector-borne parasitic disease caused by the Leishmania protozoan, is a serious public health problem in Morocco. The treatment of this disease is still based on pentavalent antimonials as the primary therapy, but these have associated side effects. Thus, the development of effective, risk-free alternative therapeutics based on natural compounds against leishmaniasis is urgent. Arginase, the key enzyme in the polyamine biosynthetic pathway, plays a critical role in leishmaniasis outcome and has emerged as a potential therapeutic target. The objective of this study was to test Cannabis sativa’s phytochemical components (cannabinoids and terpenoids) through molecular docking against Leishmania and human arginase enzymes. Our results showed that delta-9-tetrahydrocannabinol (THC) possessed the best binding energies of −6.02 and −6.35 kcal/mol with active sites of Leishmania and human arginases, respectively. Delta-9-THC interacted with Leishmania arginase through various amino acids including His139 and His 154 and linked to human arginase via His 126. In addition to delta-9-THC, caryophyllene oxide and cannabidiol (CBD) also showed a good inhibition of Leishmania and human arginases, respectively. Overall, the studied components were found to inhibit both arginases active sites via hydrogen bonds and hydrophobic interactions. These components may serve as therapeutic agents or in co-administrated therapy for leishmaniasis.


Introduction
Leishmaniasis 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. studies on a CL hamster model showed that this plant extract exhibited remarkable antileishmanial 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.

Collection and Preparation of C. sativa Ligands
The C. sativa main components used in this study were: delta-9-tetrahydrocannabinol (delta-9-THC), cannabidiol (CBD), caryophyllene oxide, beta-caryophyllene, α-pinene, αhumulene, myrcene, and limonene. Glucantime ® was used as our reference drug because it is used as the first-line cutaneous leishmaniasis treatment in Morocco. These ligands were obtained from published research papers and downloaded from the PubChem database (Table 1) in an SDF file and converted to a 3D PBD file using Open Babel GUI (version 2.4.1). 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 antimi crobial potential against pathogenic bacteria, fungus, and parasites, including Leishmani [35,36]. In addition, in vivo studies on a CL hamster model showed that this plant extrac exhibited remarkable anti-leishmanial and healing potentials. Interestingly, the applica tion of a C. sativa-based cream on CL-associated skin lesions promoted the healing proces [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 Leishmani infections, our aim was to highlight the molecular interactions of cannabinoids and terpe noids 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 agains CL.

Collection and Preparation of C. sativa Ligands
The C. sativa main components used in this study were: delta-9-tetrahydrocannabino (delta-9-THC), cannabidiol (CBD), caryophyllene oxide, beta-caryophyllene, α-pinene, α humulene, myrcene, and limonene. Glucantime ® was used as our reference drug because it is used as the first-line cutaneous leishmaniasis treatment in Morocco. These ligand were obtained from published research papers and downloaded from the PubChem da tabase (Table 1) in an SDF file and converted to a 3D PBD file using Open Babel GUI (ver sion 2.4.1). 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 antimi crobial 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 extrac exhibited remarkable anti-leishmanial and healing potentials. Interestingly, the applica tion 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 terpe noids 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.

Collection and Preparation of C. sativa Ligands
The C. sativa main components used in this study were: delta-9-tetrahydrocannabino (delta-9-THC), cannabidiol (CBD), caryophyllene oxide, beta-caryophyllene, α-pinene, α humulene, myrcene, and limonene. Glucantime ® was used as our reference drug because it is used as the first-line cutaneous leishmaniasis treatment in Morocco. These ligands were obtained from published research papers and downloaded from the PubChem da tabase (Table 1) in an SDF file and converted to a 3D PBD file using Open Babel GUI (ver sion 2.4.1). caryophyllene oxide, α-pinene, decane, and limonene were described in different C. sativ essential oils [33,34]. In vitro, C. sativa extracts and active components showed an antimi crobial potential against pathogenic bacteria, fungus, and parasites, including Leishmani [35,36]. In addition, in vivo studies on a CL hamster model showed that this plant extrac exhibited remarkable anti-leishmanial and healing potentials. Interestingly, the applica tion of a C. sativa-based cream on CL-associated skin lesions promoted the healing proces [37]. Nonetheless, these overall studies did not go further to determine C. sativa's possibl targets and mechanisms of action. In light of these results and due to the existing interplay between host and parasit arginine, as well as the importance of polyamine metabolism in the outcome of Leishmani infections, our aim was to highlight the molecular interactions of cannabinoids and terpe noids 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 agains CL.

Collection and Preparation of C. sativa Ligands
The C. sativa main components used in this study were: delta-9-tetrahydrocannabino (delta-9-THC), cannabidiol (CBD), caryophyllene oxide, beta-caryophyllene, α-pinene, α humulene, myrcene, and limonene. Glucantime ® was used as our reference drug becaus it is used as the first-line cutaneous leishmaniasis treatment in Morocco. These ligand were obtained from published research papers and downloaded from the PubChem da tabase (Table 1) in an SDF file and converted to a 3D PBD file using Open Babel GUI (ver sion 2.4.1).

Retrieval of Arginases' 3D Structures
The crystal structure of Leishmania mexicana arginase (LmArg) (accession ID: 4ITY) and human arginase (h-Arg) (accession ID: 3kv2) were obtained from the RCSB Protein Data Bank in PDB format (https://www.rcsb.org, accessed on 13 August 2022) [38,39]. It was demonstrated that Leishmania species' arginases exhibited highly conserved DNA and amino acid sequences [40]. For further corroboration, we blasted the protein sequences of L. mexicana and the CL-induced species in Morocco (L. major and L. tropica), which showed 95% similarities. In addition, the 5% variation did not impact the active site's amino acids.

Molecular Docking
A molecular docking study was performed using Auto Dock tools (ADT) (version 1.5.7) to analyze the conformations of the major phytochemical compounds.
Then, the proteins were prepared independently for docking by deleting all nonstandard residues and water molecules and adding polar hydrogen atoms and Kollman charges to the macromolecule. For each compound, 100 docking runs were performed using the Lamarckian genetic algorithm (LGA) to search for the best binding pose. Then, the optimal conformations were analyzed using Biovia Discovery Studio 2021 software.

Results
In this study, we performed a molecular docking of the main C. sativa compounds with Leishmania and human arginase proteins in order to visualize the mechanism by which the binding was carried out. The referent anti-leishmanial drug Glucantime ® was used as a control.

C. sativa Components and Leishmania Arginase: Molecular-Interaction Analysis
The docking results are presented in Table 2. Among the tested ligands, while interacting with LmArg, the delta-9-THC, caryophyllene oxide, beta-caryophyllene, and α-humulene showed the best binding scores of −6.02, −5.88, −5.79, and −5.55 kcal/mol, respectively. Although limonene had a score of −4.49 kcal/mol, the lowest ligand-protein affinity was in fact assigned to Glucantime ® at −4.30 kcal/mol. These results were correlated with inhibition constants ranging between 38.63 µM for delta-9-THC and 702.95 µM for Glucantime ® . Delta-9-THC has established conventional hydrogen bonds with HIS 154 and hydrophobic interactions with HIS 139 and ALA 140. The pi-donor hydrogen-bond interaction was also observed for TRH 257. There were also 14

C. sativa Components and Human Arginase: Molecular-Interaction Analysis
The docking results also indicated that among the tested ligands, delta-9-THC fit in the active site of human arginase with a binding energy of −6.  Moreover, among the selected phytochemical compounds of C. sativa, delta-9-tetrahydrocannabinol showed the best binding energy with both enzymes, followed by caryophyllene oxide for LmArg and cannabidiol for h-Arg.

Discussion
Cutaneous 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, Trop. Med. Infect. Dis. 2022, 7, 400 8 of 11 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 Mn 2+ [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].

Conclusions
Since CL is still a public health problem in low-income and developing countries, the discovery of an efficient, less toxic, and accessible therapy is a necessity. The present in silico study was the first to investigate C. sativa's selected constituents as selective inhibitory agents for parasitic as well as host arginases, which play an important role in this parasitic