Naphthoquinone as a New Chemical Scaffold for Leishmanicidal Inhibitors of Leishmania GSK-3

More than 1 billion people live in areas endemic for leishmaniasis, which is a relevant threat for public health worldwide. Due to the inadequate treatments, there is an urgent need to develop novel alternative drugs and to validate new targets to fight this disease. One appealing approach is the selective inhibition of protein kinases (PKs), enzymes involved in a wide range of processes along the life cycle of Leishmania. Several PKs, including glycogen synthase kinase 3 (GSK-3), have been validated as essential for this parasite by genetic or pharmacological methods. Recently, novel chemical scaffolds have been uncovered as Leishmania GSK-3 inhibitors with antiparasitic activity. In order to find new inhibitors of this enzyme, a virtual screening of our in-house chemical library was carried out on the structure of the Leishmania GSK-3. The virtual hits identified were experimentally assayed both for leishmanicidal activity and for in vitro inhibition of the enzyme. The best hits have a quinone scaffold. Their optimization through a medicinal chemistry approach led to a set of new compounds, provided a frame to establish biochemical and antiparasitic structure–activity relationships, and delivered molecules with an improved selectivity index. Altogether, this study paves the way for a systemic search of this class of inhibitors for further development as potential leishmanicidal drugs.


Introduction
More than 20 protozoan species of the genus Leishmania are responsible for the variety of clinical syndromes associated with human leishmaniasis. This heterogeneous pathology is grouped into three major clinical forms: cutaneous (in many cases self-healing), mucocutaneous, and visceral, that evolves fatally unless treated [1]. The incidence of leishmaniasis is estimated between 700,000 and 1 million new cases with 7000-15,000 annual deaths, and more than 1 billion people living in endemic areas. Its distribution encompasses a wide variety of ecological systems, mostly in tropical and subtropical areas, with an expanding distribution towards higher latitudes, driven by climate change and human displacement or migrations from or into endemic areas [2].

Computational Studies
The only crystal structure available for LmjGSK-3s was retrieved [31] (Protein Data Bank (PDB) code: 3E3P). Nevertheless, the crystallized protein did not display the decapeptide loop located at the upper part of the ATP binding pocket. Therefore, this loop was modelled prior to the analysis using the Modeller 9.20 software (University of San Francisco, San Francisco, CA, USA) [37].

Ligand Preparation
The preparation of the MBC chemical library and its 2D-to-3D conversion was carried out using the LigPrep [38] tool (Schrödinger Release 2015-4. Schrödinger, LLC: New York, NY, USA), with addition of hydrogen atoms and calculation of the ionization state of the molecules at physiological pH. All the molecules were desalted and minimized as default at the last step. The generation of possible tautomers and low-energy ring conformations, as well as a final energy minimization step were carried out using the OPLS-2005 force field [39,40].

Protein Preparation
The Protein Preparation Wizard tool [41] implemented on Maestro (Schrödinger Release 2015-4. Schrödinger, LLC: New York, NY, USA) [42] was used to pre-process and refine the structures of the proteins by H-bond assignment and calculation of the protonation state of the residues at physiological pH with a final restraint minimization.

Cavity Detection Analysis
The potential cavities on the different enzymes were identified using the Fpocket 2.0 software (Barcelona, Spain) [43] a pocket detection package, based on Voronoi tessellation and alpha spheres building. For that, the qhull package was used for Voronoi tessellation. After the structural analysis, the pockets of the protein were compiled and ranked according to the scores provided by the program. Further analyses were carried out based on the visual inspection of best ranked pockets. MDpocket, tool included in Fpocket 2.0, [44] was used to detect cavities for a set of PDB structures. A total number of 56 crystal structures of GSK-3 available at the moment of the study, mostly from human origin, were retrieved for the analysis (

Hotspots Maps
The Fragment Hotspot maps 0.11.0 software (CDCC Cambridge, UK) [45] was utilised to define the location and environment of the binding sites on the protein. After an initial calculation of atomic hotspots, the Fragment Hotspot maps were further produced using simple molecular probes. These maps uncover specifically fragment-binding sites and their respective pharmacophores. The interactions identified with the highest relevance were further employed to set up filters for virtual screening studies and the quest for molecules that fulfil these essential interactions.

Virtual Screening
Virtual screening was performed employing the Glide module [46] within the Schrödinger software package (Schrödinger Release 2017-1. Schrödinger, LLC: New York, NY, USA, with the corresponding 3D target structure and the MBC library [47]. In all cases, the centroid of the grid was taken as the centre of the catalytic or the specified pocket. For the grid generation a scaling factor of 1.0 in van der Waals radius scaling, and a partial charge cutoff of 0.25 were used. The virtual screening used either a standard precision (SP) or an extra precision (XP) mode [48]. The ligand sampling was flexible, epik state penalties were added, and a 2.5 kcal/mol energy window was used for ring sampling. The distance-dependent dielectric constant was 4.0 with a maximum number of minimization steps of 100,000 in the energy minimization step. In the clustering, poses were considered as duplicates and discarded if RMSD values were lower than 0.5 Å and the maximum atomic displacement lower than 1.3 Å.

Reagents
Commercial reagents were of the highest quality available and purchased from SIGMA-Aldrich Spain unless otherwise stated. Growth media (RPMI 1640 medium, RPMI 1640 without red phenol, and M199 medium) were obtained from Gibco (Life Technologies Europe, Bleiswij, The Netherlands).

Assessment of Inhibition of GSK-3 with Kinase Glo ®
Extraction, purification, and evaluation of compounds against recombinant LdGSK-3s was carried out as described. For that, a recombinant LdGSK-3s with a His-tag was obtained as inclusion bodies, that were further solubilized, and purified by chromatography first on a Ni 2+ column an in a second step on a DEAE (diethylaminoethyl) ionic exchange column [34]. Samples were made in duplicate, and assays were repeated at least three times.
Biomedicines 2022, 10, 1136 9 of 27 IC 50 (inhibitory concentration that decreases enzymatic activity by 50%) was calculated using the statistical module of SigmaPlot v11.0 software. Evaluation of compounds against hGSK-3β was performed using human recombinant GSK-3β purchased from Millipore as described previously [49]. Compounds were prepared as a 10 mM stock solution in DMSO.
The mechanism of inhibition of LdGSK-3s by 1 and 2 was studied as described for hGSK-3β [49]. Briefly, kinetic experiments were carried out at four different concentrations of ATP (10, 5, 2.5, and 1 µM), in the absence or presence of the inhibitors, at either 2.5 or 5 µM, while the phospho-glycogen synthase peptide-2 (GS2) substrate was kept constant at 25 µM. The data were processed using Microsoft Excel software and results presented as Eadie-Scatchard plots (v vs. v/[ATP]).

Cell Harvesting
Leishmania parasites were collected at late exponential growth phase by centrifugation at 1610× g at 4 • C. Elicited mouse peritoneal macrophages (MPM) were obtained from 8-week-old Balb/c mice through i.p. injection with 1 mL of 10% thioglycolate medium 72 h before extraction. Macrophages were obtained by peritoneal washing (10 mL PBS, 4 • C). After extraction, macrophages were cultured in RPMI 1640-HIFCS at 37 • C and 5% CO 2 .

Leishmanicidal and Cytotoxicity Assays of the Different Compounds
Both activities were assessed by inhibition of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) reduction, as described in [34]. Samples were made in triplicate and assays were repeated at least twice. DMSO content in the medium was adjusted to 0.25% v/v (final concentration), regardless of the final concentration of the drug, and for the control, the untreated parasites. ICx (inhibitory concentration that inhibits parasite growth or enzymatic activity by x%) was calculated using the statistical module of SigmaPlot v11.0 software.

Cytotoxicity against Intracellular Amastigotes
Mouse peritoneal macrophages resuspended in RPMI 1640-HIFCS were seeded in a 24-well plate (2 × 10 5 cells/well) with sterile 14 mm-diameter coverslides placed at the bottom of each well. Cells were allowed to adhere to the glass surface of the coverslides (24 h, 37 • C, 5% CO 2 ). Initial infection was carried out with mCherry-L. pifanoi (amastigote: macrophage ratio 3:1) at 32 • C for 4 h in RPMI 1640-HIFCS. After removal of the nonphagocytized amastigotes (3 times wash with 1 mL warm phosphate-buffered saline PBS) the infection was allowed to progress at 32 • C for another 24 h. Next, cells were incubated with each compound at 32 • C for 20 h. The infection index (number of amastigotes per macrophage) was evaluated by fluorescence microscopy (Leica DIALUX 20, Leica Microsystem, L'Hospitalet de Llobregat, Spain). At least 6 different fields gathering up to 100 macrophages were counted in each preparation. Statistical significance was calculated by p values using Student's t-test. Each sample was made by duplicate and experiments were repeated at least twice.

Mitochondrial Membrane Depolarization
Variation of the mitochondrial membrane polarization was evaluated by the intracellular accumulation of rhodamine 123 (Rh123) [50]. Leishmania parasites at 20 × 10 6 cells/mL in Hanks buffered salt solution supplemented with 10 mM D-glucose (HBSS-Glc) were incubated with the compounds for 4 h either at 26 • C or 32 • C for promastigotes or amastigotes, respectively. Then, cells were washed by centrifugation (4 min, 13,000× g, 4 • C) and the cells resuspended in 100 µL HBSS-Glc plus 0.3 µg/mL Rh123, incubated for 10 min in darkness. Extracellular Rh123 was removed by washing in cold HBSS-Glc. Lastly, the cells were resuspended in 900 µL HBSS-Glc and measured in a Coulter XL EPICS flow cytometer (λ EXC = 488 nm, λ EM = 520 nm).
Samples were prepared by duplicate and assays were repeated at least twice. The control for depolarization consisted of parasites incubated with 20 mM KCN for 40 min prior to Rh123 incubation.

Measurement of Sub G 0 /G 1 Population
L. donovani promastigotes (2 × 10 6 cells/mL) in RPMI-HIFCS were incubated with each compound in 24-well plates for 72 h at 26 • C by duplicate. Next, compounds were removed by washing with HBSS-Glc (15,700× g, 4 min, 4 • C) and cells were resuspended in 15 mL of HBSS-Glc, fixed and permeabilized by addition of 200 µL 70% cold EtOH and incubated overnight at 4 • C. Ethanol was removed by centrifugation and cellular pellets washed with HBSS-Glc. Finally, cells were resuspended in 500 µL HBSS-Glc with 20 µg/mL propidium iodide (PI) and 3 mg/mL Ribonuclease A, and incubated in darkness at r. t. for 30 min. PI fluorescence was measured in a Coulter XL EPICS flow cytometer (Beckman Coulter, Nyon, Switzerland) (λ EXC = 488 nm, λ EM = 620 nm) [50].
Each assay was repeated at least twice. Untreated cells were used as control for a standard cell cycle histogram, and Miltefosine (hexadecylphosphocholine) (15 µM) was used as control for an apoptosis-inducing respiratory inhibitor [51].

Computational Analysis of Leishmania GSK-3 Structure
In an initial step, identification of druggable cavities in the selected target was carried out to identify small-molecule ligands. The initial sequence alignment analysis of the human and the parasite enzyme (Supplementary Materials, Figure S1) showed that, despite a reasonable degree of dissimilarity between the Leishmania GSK-3 and its human orthologue, the identity in the ATP binding pocket is highly preserved, including the conserved Cys199 (Cys169 in LmjGSK-3s), critical for the binding of covalent inhibitors [52,53]. Remarkably, two residues differed in this region, Asp133 and Leu131; in the human enzyme, these were replaced, respectively, by Glu101 and Met100 in LmjGSK-3s, leading to a slight increase in the bulkiness of these residues located at the hinge region in the ATP-binding site. Based on this, and with the goal of identifying potential binding cavities in the parasite protein, we first proceeded to the surface analysis studies using the crystal structure available from LmjGSK-3s [31]. For this purpose, protein-druggable sites and their potential fragmentbinding sites for the protein kinase inhibitors (PKI) of each pocket were uncovered using the free geometry-based algorithm Fpocket [43] and MDpocket [44], as well as by the prediction of fragment hotspot maps [45].
Fpocket identified 16 potential cavities in the structure of the protein, that were ranked by score and volume based on the clustering of alpha spheres and Voroni tessellation, according to alpha sphere density, polarity, or hydrophobic density [43]. All of them underwent further visual inspection. The results pinpointed significant facts. Most of the cavities previously described for hGSK-3β [53] appeared also in the LmjGSK-3s protein.
The top-scoring pockets were the ATP-binding site, with its characteristic hinge region connecting the Cand N-lobes, the substrate-binding pocket next to the ATP-binding site, as well as several allosteric regions. Moreover, most of the residues that form the ATP-binding site (Pocket 0, depicted in Figure 1A) and the substrate pocket were highly conserved, while allosteric pockets showed higher differences respect to their homologues of the human enzyme. In order to analyse the conserved pockets in the different GSK-3 structures, the 56 structures available in the PDB [54] at the time of the study, including that from Leishmania, were retrieved. These structures, mostly from the human enzyme, were analysed by MDpocket [44]. The purpose is to identify potential differences between the conserved pockets of the structures, to be later compared with those of the Leishmania enzyme.  From these analyses, those pockets with high score in Fpocket were interrelated with those from MDpocket analysis in Figure 1A. According to Figure 1B, the ATP-binding site was by far the pocket with the highest conservation, named Pocket 0, in agreement with the aforementioned sequence comparison. In addition, four additional pockets were partially preserved in the different structures, but with higher divergences in volume and shape. Figure 1C shows the correlation between the top ranked cavities of LmjGSK-3s from Fpocket analysis with those pockets found in MDpocket.
After the analysis of the surface and clustering of all cavities in the search for potential allosteric pockets, Hotspots maps studies [45] identified the top pockets areas where key interactions driving ligand-protein binding might be established. As expected, the ATPbinding site accumulated most of the total hotspot maps. In addition, other areas within the pockets found in the cavity search analysis were spotted as potential sites for ligand binding by chemical probes (Figure 2). Altogether, four allosteric pockets, in addition to the ATP-binding site, were identified by this approach.

Searching for Novel Inhibitors Using Virtual Screening
The survey for new and highly selective active structures of Leishmania GSK-3 within the MBC chemical library [47] was performed by virtual screening on the pockets found in the LmjGSK-3s. A set of 24 compounds chemically diverse, were prioritised for biological evaluation according to the Glide XP Score and the hotspots maps calculations.
The inhibition of the enzymatic activity of the purified LdGSK-3s by the 24 selected Altogether, four allosteric pockets, in addition to the ATP-binding site, were identified by this approach.

Searching for Novel Inhibitors Using Virtual Screening
The survey for new and highly selective active structures of Leishmania GSK-3 within the MBC chemical library [47] was performed by virtual screening on the pockets found in the LmjGSK-3s. A set of 24 compounds chemically diverse, were prioritised for biological evaluation according to the Glide XP Score and the hotspots maps calculations.
The inhibition of the enzymatic activity of the purified LdGSK-3s by the 24 selected compounds, that represented a variety of chemical scaffolds, was evaluated using Kinase Glo ® (Promega Biotech Ibérica, Alcobendas, Spain) (Supplementary Material, Table S1) as previously described [34]. Out of the 24 compounds, only naphthoquinone MBC-10 showed a moderate inhibition of LdGSK-3s (45.7 ± 7.6% inhibition at 10 µM) ( Table 1). This result prompted us to evaluate additional molecules related to it within our in-house chemical library [47]. A set of related structures bearing a benzo-or a naphthoquinone core with different substituent patterns were prioritised and subsequently tested. The quinone MBC-132, with a carbamate group in position 2 and a chlorine atom in position 3 has an IC 50 of 2.5 ± 0.1 µM on LdGSK-3s inhibition, with 4-fold improvement over MBC-10 (Table 1). Then, the leishmanicidal activity of these two molecules were subsequently assessed on axenic promastigotes and amastigotes. Both molecules resulted as appealing candidates under the phenotypic screening. MBC-10 showed IC 50 s of 10.5 ± 1.2 µM and 11.2 ± 2.5 µM on promastigotes and axenic amastigotes, respectively, in the same trend as their inhibitory enzymatic activity. MBC-132 showed IC 50 s in the low micromolar and sub-micromolar range: 1.51 ± 0.02 µM and 0.51 ± 0.01 µM for promastigotes and axenic amastigotes, respectively (Table 1), improving by 10-fold the MBC-10 values, with a selectivity index of 5.1 on mouse peritoneal macrophages.  (Table 1). Then, the leishmanicidal activity of these two molecules were subsequently assessed on axenic promastigotes and amastigotes. Both molecules resulted as appealing candidates under the phenotypic screening. MBC-10 showed IC50s of 10.5 ± 1.2 μM and 11.2 ± 2.5 μM on promastigotes and axenic amastigotes, respectively, in the same trend as their inhibitory enzymatic activity. MBC-132 showed IC50s in the low micromolar and submicromolar range: 1.51 ± 0.02 μM and 0.51 ± 0.01 μM for promastigotes and axenic amastigotes, respectively (Table 1), improving by 10-fold the MBC-10 values, with a selectivity index of 5.1 on mouse peritoneal macrophages. Noteworthy, quinone scaffolds such as p-benzoquinone or naphthoquinone [55,56] were considered as privileged structure in drug discovery [57]. Moreover, several quinone derivatives are drugs approved for different pathologies [58]. However, to the best of our knowledge, inhibition of GSK-3 enzymes by quinones has not been reported yet, although it was for other PKs through binding to the ATP binding pocket, such as anthraquinones for human PIM1 kinase [59]. Other modes of PK inhibition by quinones include binding of naphtho(hydro)quinone into the allosteric sites of the p21-activated kinase PAK [60], or the covalent allosteric inhibition of Akt by 5,7-dimethoxy-1,4-phenanthrenequinone [61].
The biological properties of quinones are mostly based either on their extensive redox metabolism, frequently inducing ROS production, or as Michael acceptors to form adducts with highly nucleophilic residues [62]. The chemical properties, and hence their potential as therapeutics, are modulated by the substituents of the scaffold [63,64]. Therefore, following a ligand-based drug design strategy a new set of derivatives from MBC-132

MBC-132
Biomedicines 2022, 10, 1136 14 of 27 (Table 1). Then, the leishmanicidal activity of these two molecules were subsequently assessed on axenic promastigotes and amastigotes. Both molecules resulted as appealing candidates under the phenotypic screening. MBC-10 showed IC50s of 10.5 ± 1.2 μM and 11.2 ± 2.5 μM on promastigotes and axenic amastigotes, respectively, in the same trend as their inhibitory enzymatic activity. MBC-132 showed IC50s in the low micromolar and submicromolar range: 1.51 ± 0.02 μM and 0.51 ± 0.01 μM for promastigotes and axenic amastigotes, respectively (Table 1), improving by 10-fold the MBC-10 values, with a selectivity index of 5.1 on mouse peritoneal macrophages. Noteworthy, quinone scaffolds such as p-benzoquinone or naphthoquinone [55,56] were considered as privileged structure in drug discovery [57]. Moreover, several quinone derivatives are drugs approved for different pathologies [58]. However, to the best of our knowledge, inhibition of GSK-3 enzymes by quinones has not been reported yet, although it was for other PKs through binding to the ATP binding pocket, such as anthraquinones for human PIM1 kinase [59]. Other modes of PK inhibition by quinones include binding of naphtho(hydro)quinone into the allosteric sites of the p21-activated kinase PAK [60], or the covalent allosteric inhibition of Akt by 5,7-dimethoxy-1,4-phenanthrenequinone [61].
The biological properties of quinones are mostly based either on their extensive redox metabolism, frequently inducing ROS production, or as Michael acceptors to form adducts with highly nucleophilic residues [62]. The chemical properties, and hence their potential as therapeutics, are modulated by the substituents of the scaffold [ Noteworthy, quinone scaffolds such as p-benzoquinone or naphthoquinone [55,56] were considered as privileged structure in drug discovery [57]. Moreover, several quinone derivatives are drugs approved for different pathologies [58]. However, to the best of our knowledge, inhibition of GSK-3 enzymes by quinones has not been reported yet, although it was for other PKs through binding to the ATP binding pocket, such as anthraquinones for human PIM1 kinase [59]. Other modes of PK inhibition by quinones include binding of naphtho(hydro)quinone into the allosteric sites of the p21-activated kinase PAK [60], or the covalent allosteric inhibition of Akt by 5,7-dimethoxy-1,4-phenanthrenequinone [61].
The biological properties of quinones are mostly based either on their extensive redox metabolism, frequently inducing ROS production, or as Michael acceptors to form adducts with highly nucleophilic residues [62]. The chemical properties, and hence their potential as therapeutics, are modulated by the substituents of the scaffold [63,64]. Therefore, following a ligand-based drug design strategy a new set of derivatives from MBC-132 were synthesized to explore structure-activity relationships (SAR).

Design, Synthesis, Biological Evaluation, and SAR Analysis of a Second Generation of Naphthoquinone Derivatives
MBC-132 improved substantially the LdGSK-3s inhibition and its associated leishmanicidal values with respect to the initial compound MBC-10. To further optimize this new hit in terms of activity and selectivity, a medicinal chemistry approach was developed to explore the chemical space of the naphthoquinone scaffold. For this, a dedicated synthesis of a new generation of naphthoquinones was carried out with a double aim; to assess the role of the chemical nature of the substituent at position 2, and to define the importance of the chlorine at position 3. The first objective was tackled by the synthesis of a subset of compounds that maintained the chlorine atom at position 3, but with different functional groups at position 2, such as amines, urea, or amides, as well as the initial carbamate itself. For the second objective, a new series of naphthoquinone derivatives compounds without the chlorine in position 3 was synthesized to determine its influence on the activity ( Figure 3).  A previously described procedure [35] was followed to synthesize a series of carbamate derivatives 1-5 generated with a moderate yield, by the use of the corresponding aliphatic and aromatic alcohols. The fast kinetics of this reaction minimized the hydrolysis of the intermediate isocyanate, but the formation of the 2-amino-3-chloro-1,4-naphthoquinone was occasionally observed, as previously reported [35]. Secondly, the influence of the carbamate moiety in the biological activity was explored with a new set of naphthoquinone derivatives in which the carbamate was replaced either by an amine group, or by a urea moiety at the same position. In the first case, derivatives (6)(7)(8)(9)(10)(11)(12)(13) were obtained by reaction of the 2,3-dichloro-1,4-naphthoquinone with different aliphatic and aromatic amines in anhydrous DMSO at r. t. with good yields (Scheme 1). The replacement of carbamate by urea followed a similar chemical strategy employed for the carbamate synthesis, but with the use of primary amines differently substituted, either aliphatic or aromatic. Nevertheless, a final urea derivative was obtained only for tert-butylamine (14). For the other cases, instead of the expected urea formation, the direct addition of the amine occurred due to the low nucleophilic properties of the potassium cyanate compared with the respective amine. A previously described procedure [35] was followed to synthesize a series of carbamate derivatives 1-5 generated with a moderate yield, by the use of the corresponding aliphatic and aromatic alcohols. The fast kinetics of this reaction minimized the hydrolysis of the intermediate isocyanate, but the formation of the 2-amino-3-chloro-1,4-naphthoquinone was occasionally observed, as previously reported [35]. Secondly, the influence of the carbamate moiety in the biological activity was explored with a new set of naphthoquinone derivatives in which the carbamate was replaced either by an amine group, or by a urea moiety at the same position. In the first case, derivatives (6)(7)(8)(9)(10)(11)(12)(13) were obtained by reaction of the 2,3-dichloro-1,4-naphthoquinone with different aliphatic and aromatic amines in anhydrous DMSO at r. t. with good yields (Scheme 1). The replacement of carbamate by urea followed a similar chemical strategy employed for the carbamate synthesis, but with the use of primary amines differently substituted, either aliphatic or aromatic. Nevertheless, a final urea derivative was obtained only for tert-butylamine (14). For the other cases, instead of the expected urea formation, the direct addition of the amine occurred due to the low nucleophilic properties of the potassium cyanate compared with the respective amine. For this, amide and carbamate naphthoquinone derivatives were synthesized from 2-amino-1,4-naphthoquinone as the starting material under a two-step protocol. In the first step, 2-amino-1,4-naphthoquinone (15) was obtained by treatment of 2-bromo-1,4naphthoquinone with aqueous ammonia, 32% [65]. The moderate yields and the formation of secondary products were substantially improved by the replacement of the aqueous ammonia as solvent by THF (r. t., 24 h) (Scheme 2). In the second step, the reaction of the quinone 15 with the respective chloroformate or acid halide led to the formation of the corresponding carbamates 16-17 or amide derivatives 18-20 [66]. The nucleophilic substitution was performed in the presence of sodium hydride, obtaining the final compounds with moderate yields. The identification and characterization of the compounds were included within the Materials and Methods Section.
In the next step, inhibition of the LdGSK-3s was evaluated for the 19 newly synthesized quinones (compounds 1-14 and 16-20, Table 2). As with the parent compound MBC-132, its carbamate derivatives (1-5) maintained their respective IC50s at the low micromolar range. However, a 4-fold increase in IC50s (>10 μM) occurred when either the carbamate moiety was replaced by urea, amide, or amine, or the chlorine in position 3 was absent. Finally, the role of the chlorine atom at position 3 in the biological activity of this chemical class of compounds was assessed by synthesis of 1,4-naphthoquinone derivatives devoid of this substituent.
For this, amide and carbamate naphthoquinone derivatives were synthesized from 2-amino-1,4-naphthoquinone as the starting material under a two-step protocol. In the first step, 2-amino-1,4-naphthoquinone (15) was obtained by treatment of 2-bromo-1,4naphthoquinone with aqueous ammonia, 32% [65]. The moderate yields and the formation of secondary products were substantially improved by the replacement of the aqueous ammonia as solvent by THF (r. t., 24 h) (Scheme 2). In the second step, the reaction of the quinone 15 with the respective chloroformate or acid halide led to the formation of the corresponding carbamates 16-17 or amide derivatives 18-20 [66]. The nucleophilic substitution was performed in the presence of sodium hydride, obtaining the final compounds with moderate yields. The identification and characterization of the compounds were included within the Materials and Methods Section. Finally, the role of the chlorine atom at position 3 in the biological activity of th chemical class of compounds was assessed by synthesis of 1,4-naphthoquinone deriv tives devoid of this substituent.
For this, amide and carbamate naphthoquinone derivatives were synthesized fro 2-amino-1,4-naphthoquinone as the starting material under a two-step protocol. In th first step, 2-amino-1,4-naphthoquinone (15) was obtained by treatment of 2-bromo-1, naphthoquinone with aqueous ammonia, 32% [65]. The moderate yields and the fo mation of secondary products were substantially improved by the replacement of th aqueous ammonia as solvent by THF (r. t., 24 h) (Scheme 2). In the second step, the rea tion of the quinone 15 with the respective chloroformate or acid halide led to the formatio of the corresponding carbamates 16-17 or amide derivatives 18-20 [66]. The nucleophil substitution was performed in the presence of sodium hydride, obtaining the final com pounds with moderate yields. The identification and characterization of the compound were included within the Materials and Methods Section. In the next step, inhibition of the LdGSK-3s was evaluated for the 19 newly synth sized quinones (compounds 1-14 and 16-20, Table 2). As with the parent compound MBC 132, its carbamate derivatives (1-5) maintained their respective IC50s at the low microm lar range. However, a 4-fold increase in IC50s (>10 μM) occurred when either the carbama moiety was replaced by urea, amide, or amine, or the chlorine in position 3 was absent. In the next step, inhibition of the LdGSK-3s was evaluated for the 19 newly synthesized quinones (compounds 1-14 and 16-20, Table 2). As with the parent compound MBC-132, its carbamate derivatives (1-5) maintained their respective IC 50 s at the low micromolar range. However, a 4-fold increase in IC 50 s (>10 µM) occurred when either the carbamate moiety was replaced by urea, amide, or amine, or the chlorine in position 3 was absent.               In a step ahead, the leishmanicidal activity of the compounds on L. pifanoi axenic amastigotes and L. infantum promastigotes was evaluated. Axenic Leishmania amastigotes afford the appraisal of the leishmanicidal activity without the constrains imposed on the access of the compounds to intracellular amastigotes within the parasitophorous vacuole, as well as to preclude the interference of the compounds via host-parasite interface in the final activity. For that, the axenic line of L. pifanoi amastigotes belonging to the mexicana complex was chosen, as in this species the infectivity, antigenicity, and metabolic identity between axenic and intracellular amastigotes have been consistently validated [67].
The IC50s for compounds 1-5 on both axenic forms of the parasite fell within the low micromolar and sub-micromolar ranges. Compounds with an aliphatic substitution of the carbamate (1 and 2), improved slightly the activity of the MBC-132 on the amastigote, while on promastigote, values were rather similar. The replacement of an aliphatic carbamate (1-2) by an aromatic one (3-5) produced a dissimilar effect on the leishmanicidal activity; whereas on promastigotes IC50s were maintained at low micromolar values, re- In a step ahead, the leishmanicidal activity of the compounds on L. pifanoi axenic amastigotes and L. infantum promastigotes was evaluated. Axenic Leishmania amastigotes afford the appraisal of the leishmanicidal activity without the constrains imposed on the access of the compounds to intracellular amastigotes within the parasitophorous vacuole, as well as to preclude the interference of the compounds via host-parasite interface in the final activity. For that, the axenic line of L. pifanoi amastigotes belonging to the mexicana complex was chosen, as in this species the infectivity, antigenicity, and metabolic identity between axenic and intracellular amastigotes have been consistently validated [67].
The IC50s for compounds 1-5 on both axenic forms of the parasite fell within the low micromolar and sub-micromolar ranges. Compounds with an aliphatic substitution of the carbamate (1 and 2), improved slightly the activity of the MBC-132 on the amastigote, while on promastigote, values were rather similar. The replacement of an aliphatic carbamate (1-2) by an aromatic one (3-5) produced a dissimilar effect on the leishmanicidal In a step ahead, the leishmanicidal activity of the compounds on L. pifanoi axenic amastigotes and L. infantum promastigotes was evaluated. Axenic Leishmania amastigotes afford the appraisal of the leishmanicidal activity without the constrains imposed on the access of the compounds to intracellular amastigotes within the parasitophorous vacuole, as well as to preclude the interference of the compounds via host-parasite interface in the final activity. For that, the axenic line of L. pifanoi amastigotes belonging to the mexicana complex was chosen, as in this species the infectivity, antigenicity, and metabolic identity between axenic and intracellular amastigotes have been consistently validated [67].
The IC50s for compounds 1-5 on both axenic forms of the parasite fell within the low micromolar and sub-micromolar ranges. Compounds with an aliphatic substitution of the carbamate (1 and 2), improved slightly the activity of the MBC-132 on the amastigote, while on promastigote, values were rather similar. In a step ahead, the leishmanicidal activity of the compounds on L. pifanoi axenic amastigotes and L. infantum promastigotes was evaluated. Axenic Leishmania amastigotes afford the appraisal of the leishmanicidal activity without the constrains imposed on the access of the compounds to intracellular amastigotes within the parasitophorous vacuole, as well as to preclude the interference of the compounds via host-parasite interface in the final activity. For that, the axenic line of L. pifanoi amastigotes belonging to the mexicana complex was chosen, as in this species the infectivity, antigenicity, and metabolic identity between axenic and intracellular amastigotes have been consistently validated [67].
The IC50s for compounds 1-5 on both axenic forms of the parasite fell within the low micromolar and sub-micromolar ranges. Compounds with an aliphatic substitution of the carbamate (1 and 2), improved slightly the activity of the MBC-132 on the amastigote, In a step ahead, the leishmanicidal activity of the compounds on L. pifanoi axenic amastigotes and L. infantum promastigotes was evaluated. Axenic Leishmania amastigotes afford the appraisal of the leishmanicidal activity without the constrains imposed on the access of the compounds to intracellular amastigotes within the parasitophorous vacuole, as well as to preclude the interference of the compounds via host-parasite interface in the final activity. For that, the axenic line of L. pifanoi amastigotes belonging to the mexicana complex was chosen, as in this species the infectivity, antigenicity, and metabolic identity between axenic and intracellular amastigotes have been consistently validated [67].
The IC 50 s for compounds 1-5 on both axenic forms of the parasite fell within the low micromolar and sub-micromolar ranges. Compounds with an aliphatic substitution of the carbamate (1 and 2), improved slightly the activity of the MBC-132 on the amastigote, while on promastigote, values were rather similar. The replacement of an aliphatic carbamate (1-2) by an aromatic one (3-5) produced a dissimilar effect on the leishmanicidal activity; whereas on promastigotes IC 50s were maintained at low micromolar values, regardless of the aromatic or aliphatic nature of the substituents, on amastigotes, the IC 50 s were higher for aromatic than for aliphatic derivatives. The selectivity index of 1 and 2 increased 2-and 4-fold with respect to MBC-132 (Table 2), also driven by their lower toxicity on peritoneal macrophages. Compound 1, the carbamate with an ethyl substituent, resulted with the highest selectivity index (SI = 20.6). In contrast, the slightly higher toxicity on macrophages, and the lower activity on amastigotes, decreased the SI for 3-5 below MBC-132.
Replacement of the carbamate by an amine at position 2 (compounds 6-13), plummeted IC 50 values for both forms of the parasite, even for aliphatic substituents (6)(7)(8). These compounds showed, in vitro, a nil inhibition of LdGSK-3s in agreement with their poor leishmanicidal activity. The substitution of the carbamate by urea (compound 14) led to an activity on promastigotes comparable to the initial hit MBC-132; although, their higher cytotoxicity on macrophages and lower activity on amastigotes ruined their selectivity index.
The elimination of the chlorine at position 3 (compounds 16-20) maintained the leishmanicidal activity of the reference MBC-132. However, they showed a lower selectivity index because of their higher toxicity on macrophages.
The structural modifications on the quinone scaffold led to active derivatives whose substitution pattern widely differed from the initial hit MBC-10. In order to decipher the mechanism of inhibition, experimental enzymatic kinetic studies were performed. Two of the most active LdGSK-3s inhibitors, quinones 1 and 2, were chosen to study their competition with ATP. Kinetic experiments with variation of the concentration of either ATP (from 1 to 10 µM) or the inhibitors 1 and 2 (from 2.5 to 5 µM) were performed. An Eadie-Scatchard plot (v vs. v/[ATP]) of the data is depicted in Figure 4. The intercept of the lines just below the x-axis endorsed that both compounds act as ATP competitive inhibitors. This mode of inhibition could be presumably extrapolated to similar compounds as MBC-132 or 3-5. The elimination of the chlorine at position 3 (compounds 16-20) maintained the leishmanicidal activity of the reference MBC-132. However, they showed a lower selectivity index because of their higher toxicity on macrophages.
The structural modifications on the quinone scaffold led to active derivatives whose substitution pattern widely differed from the initial hit MBC-10. In order to decipher the mechanism of inhibition, experimental enzymatic kinetic studies were performed. Two of the most active LdGSK-3s inhibitors, quinones 1 and 2, were chosen to study their competition with ATP. Kinetic experiments with variation of the concentration of either ATP (from 1 to 10 μM) or the inhibitors 1 and 2 (from 2.5 to 5 μM) were performed. An Eadie-Scatchard plot (v vs. v/[ATP]) of the data is depicted in Figure 4. The intercept of the lines just below the x-axis endorsed that both compounds act as ATP competitive inhibitors. This mode of inhibition could be presumably extrapolated to similar compounds as MBC-132 or 3-5.
Noteworthy, the best LdGSK-3s inhibitors (MBC-132, 1-3) showed a poor inhibition of hGSK-3β inhibition (at 10 μM the percentages of inhibition were 19, 30, 25, and 19%, respectively). To the best of our knowledge, these are the first LdGSK-3s inhibitors with modest affinity for hGSK-3β reported to date, with compounds 1 (SI = 20.6) and 2 (SI = 11.2) being the lead molecules of these series of naphthoquinone in the phenotypic assay, as they showed the highest SI values. In this regard, the specific inhibition of LdGSK-3s over hGSK-3β has a special relevance in leishmaniasis. The resolution of the disease relies on the induction of a proinflammatory process, where activation of the macrophages and elimination of intracellular parasites ensued. The pleiotropic function of GSK-3β in macrophages was recently re- The elimination of the chlorine at position 3 (compounds 16-20) maintained the leishmanicidal activity of the reference MBC-132. However, they showed a lower selectivity index because of their higher toxicity on macrophages.
The structural modifications on the quinone scaffold led to active derivatives whose substitution pattern widely differed from the initial hit MBC-10. In order to decipher the mechanism of inhibition, experimental enzymatic kinetic studies were performed. Two of the most active LdGSK-3s inhibitors, quinones 1 and 2, were chosen to study their competition with ATP. Kinetic experiments with variation of the concentration of either ATP (from 1 to 10 μM) or the inhibitors 1 and 2 (from 2.5 to 5 μM) were performed. An Eadie-Scatchard plot (v vs. v/[ATP]) of the data is depicted in Figure 4. The intercept of the lines just below the x-axis endorsed that both compounds act as ATP competitive inhibitors. This mode of inhibition could be presumably extrapolated to similar compounds as MBC-132 or 3-5.
Noteworthy, the best LdGSK-3s inhibitors (MBC-132, 1-3) showed a poor inhibition of hGSK-3β inhibition (at 10 μM the percentages of inhibition were 19, 30, 25, and 19%, respectively). To the best of our knowledge, these are the first LdGSK-3s inhibitors with modest affinity for hGSK-3β reported to date, with compounds 1 (SI = 20.6) and 2 (SI = 11.2) being the lead molecules of these series of naphthoquinone in the phenotypic assay, as they showed the highest SI values. In this regard, the specific inhibition of LdGSK-3s over hGSK-3β has a special relevance in leishmaniasis. The resolution of the disease relies on the induction of a proinflammatory process, where activation of the macrophages and elimination of intracellular parasites ensued. The pleiotropic function of GSK-3β in macrophages was recently reported [68], as well as the ambiguous and complex modulation of inflammation by GSK- The elimination of the chlorine at position 3 (compounds 16-20) maintained the leishmanicidal activity of the reference MBC-132. However, they showed a lower selectivity index because of their higher toxicity on macrophages.
The structural modifications on the quinone scaffold led to active derivatives whose substitution pattern widely differed from the initial hit MBC-10. In order to decipher the mechanism of inhibition, experimental enzymatic kinetic studies were performed. Two of the most active LdGSK-3s inhibitors, quinones 1 and 2, were chosen to study their competition with ATP. Kinetic experiments with variation of the concentration of either ATP (from 1 to 10 μM) or the inhibitors 1 and 2 (from 2.5 to 5 μM) were performed. An Eadie-Scatchard plot (v vs. v/[ATP]) of the data is depicted in Figure 4. The intercept of the lines just below the x-axis endorsed that both compounds act as ATP competitive inhibitors. This mode of inhibition could be presumably extrapolated to similar compounds as MBC-132 or 3-5.
Noteworthy, the best LdGSK-3s inhibitors (MBC-132, 1-3) showed a poor inhibition of hGSK-3β inhibition (at 10 μM the percentages of inhibition were 19, 30, 25, and 19%, respectively). To the best of our knowledge, these are the first LdGSK-3s inhibitors with modest affinity for hGSK-3β reported to date, with compounds 1 (SI = 20.6) and 2 (SI = 11.2) being the lead molecules of these series of naphthoquinone in the phenotypic assay, as they showed the highest SI values. In this regard, the specific inhibition of LdGSK-3s over hGSK-3β has a special relevance in leishmaniasis. The resolution of the disease relies on the induction of a proinflammatory process, where activation of the macrophages and elimination of intracellular parasites ensued. The pleiotropic function of GSK-3β in macrophages was recently reported [68], as well as the ambiguous and complex modulation of inflammation by GSK- The elimination of the chlorine at position 3 (compounds 16-20) maintained the leishmanicidal activity of the reference MBC-132. However, they showed a lower selectivity index because of their higher toxicity on macrophages.
The structural modifications on the quinone scaffold led to active derivatives whose substitution pattern widely differed from the initial hit MBC-10. In order to decipher the mechanism of inhibition, experimental enzymatic kinetic studies were performed. Two of the most active LdGSK-3s inhibitors, quinones 1 and 2, were chosen to study their competition with ATP. Kinetic experiments with variation of the concentration of either ATP (from 1 to 10 μM) or the inhibitors 1 and 2 (from 2.5 to 5 μM) were performed. An Eadie-Scatchard plot (v vs. v/[ATP]) of the data is depicted in Figure 4. The intercept of the lines just below the x-axis endorsed that both compounds act as ATP competitive inhibitors. This mode of inhibition could be presumably extrapolated to similar compounds as MBC-132 or 3-5.
Noteworthy, the best LdGSK-3s inhibitors (MBC-132, 1-3) showed a poor inhibition of hGSK-3β inhibition (at 10 μM the percentages of inhibition were 19, 30, 25, and 19%, respectively). To the best of our knowledge, these are the first LdGSK-3s inhibitors with modest affinity for hGSK-3β reported to date, with compounds 1 (SI = 20.6) and 2 (SI = 11.2) being the lead molecules of these series of naphthoquinone in the phenotypic assay, as they showed the highest SI values. In this regard, the specific inhibition of LdGSK-3s over hGSK-3β has a special relevance in leishmaniasis. The resolution of the disease relies on the induction of a proinflammatory process, where activation of the macrophages and elimination of intracellular parasites ensued. The pleiotropic function of GSK-3β in macrophages was recently reported [68], as well as the ambiguous and complex modulation of inflammation by GSK-) µM, respectively. The phospho-glycogen synthase peptide-2 (GS2) was used as a substrate at a fixed concentration of 25 µM. Each kinetic point was made by duplicate, and represented as the mean of two independent experiments. Noteworthy, the best LdGSK-3s inhibitors (MBC-132, 1-3) showed a poor inhibition of hGSK-3β inhibition (at 10 µM the percentages of inhibition were 19, 30, 25, and 19%, respectively). To the best of our knowledge, these are the first LdGSK-3s inhibitors with modest affinity for hGSK-3β reported to date, with compounds 1 (SI = 20.6) and 2 (SI = 11.2) being the lead molecules of these series of naphthoquinone in the phenotypic assay, as they showed the highest SI values.
In this regard, the specific inhibition of LdGSK-3s over hGSK-3β has a special relevance in leishmaniasis. The resolution of the disease relies on the induction of a proinflammatory process, where activation of the macrophages and elimination of intracellular parasites ensued. The pleiotropic function of GSK-3β in macrophages was recently reported [68], as well as the ambiguous and complex modulation of inflammation by GSK-3β [69,70]. Works addressing the role of hGSK-3β on leishmaniasis support the beneficial activity of this enzyme for parasite elimination [71,72]. Consequently, these LdGSK-3s inhibitors, themselves lethal to the parasites, partially spare the inhibition of hGSK-3β, with a presumed synergic leishmanicidal effect, caused by the activity of the human enzyme, while that of the parasite was selectively inhibited.

Leishmanicidal Activity on Intracellular Amastigotes of LdGSK-3s Quinone Inhibitors
The assay of drugs on intracellular parasites is a model closer to the natural infection, due to the inclusion of traffic and accumulation of the drug into the parasitophorous vacuole, as well as feasible effects at the host-parasite interface. Consequently, the six quinones with inhibitory activity on LdGSK-3s and leishmanicidal activity on axenic amastigotes (compounds MBC-132 and 1-5) were tested on macrophages infected with L. pifanoi amastigotes.
Compounds MBC-132 and 2 showed the higher decrease in parasite load ( Figure 5). Quinone 2 at 2 µM induced the highest decrease (76.3%), whereas quinone MBC-132 at 1 µM caused a decrease of 39.1%. In all, we surmise that the loss of effectiveness in intracellular infections for 2 of the 6 quinones relative to axenic amastigotes is likely due to a faulty access of the quinone to the intracellular parasite. Figure 6 showed representative images of infections treated with compound 2. The assay of drugs on intracellular parasites is a model closer to the natural infection, due to the inclusion of traffic and accumulation of the drug into the parasitophorous vacuole, as well as feasible effects at the host-parasite interface. Consequently, the six quinones with inhibitory activity on LdGSK-3s and leishmanicidal activity on axenic amastigotes (compounds MBC-132 and 1-5) were tested on macrophages infected with L. pifanoi amastigotes.
Compounds MBC-132 and 2 showed the higher decrease in parasite load ( Figure 5). Quinone 2 at 2 μM induced the highest decrease (76.3%), whereas quinone MBC-132 at 1 μM caused a decrease of 39.1%. In all, we surmise that the loss of effectiveness in intracellular infections for 2 of the 6 quinones relative to axenic amastigotes is likely due to a faulty access of the quinone to the intracellular parasite. Figure 6 showed representative images of infections treated with compound 2. Figure 5. Variation of the parasite load of BALB/c murine peritoneal macrophages infected with mCherry-L. pifanoi axenic amastigotes after treatment with selected LdGSK-3s inhibitors. Macrophages were infected with a 3:1 parasite:macrophage ratio for 4 h. Afterwards, infection was allowed to progress for 24 h. Then, macrophages were challenged with the corresponding concentration of each compound for 24 h, and changes in the parasite:macrophage ratio was obtained by fluorescence microscopy. Compounds were added from stock solutions in DMSO. The same final concentration of DMSO (0.25% v/v) was present in all the samples, including control untreated infected macrophages). Data were referred as the ratio percentage of treated vs. untreated macrophages. Student's t-test (*: p < 0.05, ***: p < 0.001). Macrophages were infected with a 3:1 parasite:macrophage ratio for 4 h. Afterwards, infection was allowed to progress for 24 h. Then, macrophages were challenged with the corresponding concentration of each compound for 24 h, and changes in the parasite:macrophage ratio was obtained by fluorescence microscopy. Compounds were added from stock solutions in DMSO. The same final concentration of DMSO (0.25% v/v) was present in all the samples, including control untreated infected macrophages). Data were referred as the ratio percentage of treated vs. untreated macrophages. Student's t-test (*: p < 0.05, ***: p < 0.001). lowed to progress for 24 h. Then, macrophages were challenged with the corresponding co tion of each compound for 24 h, and changes in the parasite:macrophage ratio was obt fluorescence microscopy. Compounds were added from stock solutions in DMSO. The sa concentration of DMSO (0.25% v/v) was present in all the samples, including control untr fected macrophages). Data were referred as the ratio percentage of treated vs. untreate phages. Student's t-test (*: p < 0.05, ***: p < 0.001).

Energy Metabolism of Leishmania as an Off-Target Effect of LdGSK-3s Inhibitors
The redox chemistry of quinones makes them suitable candidates to mimic ubiquinone, a natural quinone working as electron carrier within the respiratory chain, thus with a potential interference with the electron transport of the respiratory chain of trypanosomatids, including Leishmania [73,74]. Therefore, appraisal of the effects of the quinones described here on the energy metabolism of promastigotes was undertaken.

Inhibition of the Electrochemical Potential of the Leishmania Mitochondrion (∆Ψ m )
In Leishmania, especially in the promastigote, oxidative phosphorylation is the main source for ATP biosynthesis [75], dependent on the maintenance of the electrochemical potential (∆Ψ m ) created by the respiratory chain.
Variation of the ∆Ψ m of Leishmania parasites were monitored through the preferential accumulation of rhodamine 123 (Rh123) within the mitochondrion [50], driven by the Nernst equation. Quinones MBC-132, 1, and 2 were incubated for 4 h at their respective IC 80 s on promastigotes prior to Rh123 accumulation. A 40 min incubation with 20 mM KCN was used as a control inhibitor of oxidative phosphorylation (Figure 7). Quinone MBC-132 (3 µM) induced the highest decrease in Rh123 accumulation, similar to the KCN-treated control (ca 70%). Quinones 1 and 2 showed similar effects at 2 µM, with decrease of 33.0 and 26.4% Rh123 fluorescence, respectively, supporting an inhibitory activity on the oxidative phosphorylation of L. donovani, also described for other quinones in Leishmania [76][77][78].
IC80s on promastigotes prior to Rh123 accumulation. A 40 min incubation with 20 mM KCN was used as a control inhibitor of oxidative phosphorylation (Figure 7). Quinone MBC-132 (3 μM) induced the highest decrease in Rh123 accumulation, similar to the KCNtreated control (ca 70%). Quinones 1 and 2 showed similar effects at 2 μM, with decrease of 33.0 and 26.4% Rh123 fluorescence, respectively, supporting an inhibitory activity on the oxidative phosphorylation of L. donovani, also described for other quinones in Leishmania [76][77][78].

Inhibition of Oxygen Consumption
To confirm the dysfunction of the respiratory chain achieved by the different compounds, the inhibition of respiration was assessed by polarographic methods using a Clark oxygen electrode. The cellular density required for this technique is five times higher than that used for the other bioenergetic assays. Consequently, quinones MBC-132, 1, and 2 were assayed at 3.3-5-fold their IC80 (10 μM) (Figure 8) for inhibition of the respiration of L. donovani promastigotes. Quinones MBC-132, 1 and 2 induced an initial acceleration of the O2 consumption rates in the L. donovani promastigotes, followed by a deceleration, which, only for quinone 2, was still higher than the initial respiration rate, prior to quinone addition. The same process of deceleration, although in a lesser extent occurred in control parasites ( Figure 8A), due to the slight deviation underwent by the electrode at low oxygen concentrations, even in the absence of cells using chemical reducing agents.

Inhibition of Oxygen Consumption
To confirm the dysfunction of the respiratory chain achieved by the different compounds, the inhibition of respiration was assessed by polarographic methods using a Clark oxygen electrode. The cellular density required for this technique is five times higher than that used for the other bioenergetic assays. Consequently, quinones MBC-132, 1, and 2 were assayed at 3.3-5-fold their IC 80 (10 µM) (Figure 8) for inhibition of the respiration of L. donovani promastigotes. Quinones MBC-132, 1 and 2 induced an initial acceleration of the O 2 consumption rates in the L. donovani promastigotes, followed by a deceleration, which, only for quinone 2, was still higher than the initial respiration rate, prior to quinone addition. The same process of deceleration, although in a lesser extent occurred in control parasites ( Figure 8A), due to the slight deviation underwent by the electrode at low oxygen concentrations, even in the absence of cells using chemical reducing agents. Thus, a dual quinone effect of the quinones with a poor and late inhibition of the oxygen consumption rate may be reasonably discarded. Thus, a dual quinone effect of the quinones with a poor and late inhibition of the oxygen consumption rate may be reasonably discarded.  The role of mitochondrion is mandatory for the intrinsic pathway of the programmed cell death, the only pathway available for trypanosomatids [79]. The induction of apoptoticlike processes in promastigotes was evaluated for quinones MBC-132, 1, and 2 at their respective IC 80 s on L. donovani promastigotes, selected as inducers of mitochondrial depolarization, and evaluated by the induction of subG 0 /G 1 population in flow cytometry studies by DNA staining with propidium iodide (PI). Miltefosine at 15 µM was used as a positive control for this process [50].
As shown in Figure 9, the three selected quinones induced programmed cell death in L.donovani promastigotes, with increase in the subG 0 /G 1 population in treated promastigotes. Therefore, the three naphthoquinones studied induced a bioenergetic collapse in the promastigote of Leishmania by targeting the respiratory chain of the parasite leading to apoptosis, thus adding a new off-target mechanism of action to these LdGSK-3s inhibitors.

Conclusions
In all, quinones have been added as a new and appealing template for leishmanicidal LdGSK-3s inhibitors, active on two quite different Leishmania species, from a clinical perspective. These naphthoquinones showed a multitarget leishmanicidal mechanism that  80 . Parasites were incubated with the corresponding drug for 72 h, followed by EtOH permeabilization, incubated with 3 mg/mL Ribonuclease A and stained with 20 µg/mL of propidium iodide (PI) before cytofluorometric analysis. (λ EXC = 488 nm, λ EM = 620 nm). The percentage of subG 0 /G 1 population (enclosed within green bars) stands for degraded chromatin, associated with the apoptotic-like process. Miltefosine (hexadecylphosphocholine, HePc) at 15 µM was added as a control for induction of apoptosis.

Conclusions
In all, quinones have been added as a new and appealing template for leishmanicidal LdGSK-3s inhibitors, active on two quite different Leishmania species, from a clinical perspective. These naphthoquinones showed a multitarget leishmanicidal mechanism that may also encompass the respiratory chain, as it is the case here. Far from being detrimental for its pharmacological development, this fact presents an immediate advantage to curtail resistance induction by target mutation. As such, multitarget drugs have been hailed as relevant approach for neglected tropical diseases [80] with miltefosine as a paradigm [81], in tune with a current trend that advocate for a polypharmacology approach for neglected diseases.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10051136/s1, Figure S1: Sequence alignment of the GSK-3 structures of Leishmania major (Q4QE15) and Homo sapiens (P49841), Table S1: Chemical structures of the 24 compounds from the MBC library selected by virtual screening and their biological evaluation.

Data Availability Statement:
The data presented in this study are available in the article and Supplementary Materials and upon request from the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.