Systematic Exploration of Functional Group Relevance for Anti-Leishmanial Activity of Anisomycin

Assessment of structure–activity relationships for anti-protozoan activity revealed a strategy for preparing potent anisomycin derivatives with reduced host toxicity. Thirteen anisomycin analogs were synthesized by modifying the alcohol, amine, and aromatic functional groups. Examination of anti-protozoal activity against various strains of Leishmania and cytotoxicity against leucocytes with comparison against the parent natural product demonstrated typical losses of activity with modifications of the alcohol, amine, and aromatic meta-positions. On the other hand, the para-phenol moiety of anisomycin proved an effective location for introducing substituents without significant loss of anti-protozoan potency. An entry point for differentiating activity against Leishmania versus host has been uncovered by this systematic study.


Introduction
Anisomycin (1) is an antibiotic isolated from Streptomyces [1].First demonstrated to inhibit protein translation with eukaryote selectivity [2], anisomycin has since been shown to target various organisms by multiple mechanisms of action [3].In complex with the 80S ribosome, anisomycin reorients the 25S rRNA residue 2397 (2055) influencing the conformation of the nearby U2873 (U2504) residue and preventing aminoacyl-tRNA binding to the peptidyl transferase site [4].The 2397 (2055) rRNA nucleotide has been suggested to dictate binding selectivity between bacterial-and eukaryotic-specific inhibitors because cytosine and adenine are typically found, respectively, in bacteria and most eukaryotes [4].At concentrations below those inhibitive of protein synthesis, anisomycin elicited specific and strong activation of mitogen-activated protein kinases (MAPKs, e.g., JNK and p38) and induced c-fos gene expression [5,6].Initiation of p38 MAP kinase followed by inhibition of protein synthesis by anisomycin in mammalian cells was shown to lead to activation of the glucose transporter GLUT1, an early event in the adaptive response of mammalian cells to metabolic stress [7].
Anisomycin has exhibited activity against protozoa and viruses, as well as mammalian macrophages and cancer cells.Activation of p38 signaling by anisomycin reduced L. donovani survival within human macrophages in vitro by a mechanism blocked by SB203580, a p38-specific inhibitor [8].Anisomycin reversed Japanese encephalitis virus-induced downregulation of extracellular signal-regulated protein kinase (ERK) phosphorylation and protected murine neuroblastoma (N18) cells against infection [9].The anti-viral activity of anisomycin was further demonstrated against dengue virus (DENV) and Zika virus (ZIKV) strains in Vero cells by a mode of action implicating likely blockage of macromolecular synthesis instead of p38 MAPK activation.In ZIKV-infected mice, a low dose of anisomycin caused a significant reduction in viral levels and enhanced survival [10].In rabbit atheromalike lesions in vivo, anisomycin decreased selective macrophage content by a mechanism implicating p38 MAPK [11].In addition, as an inducer of apoptosis in tumor cells, anisomycin inhibited angiogenesis, proliferation, and invasion in ovarian cancer cells [12].Moreover, anisomycin inhibited T cell behavior, curbing transplantation rejection without significant side effects at effective therapeutic doses; however, over-dosage led to pulmo-, nephro-, and hepato-toxicity, slight micronucleus formation, and sperm aberration [13].
Interest in anisomycin has led to various syntheses of the densely functionalized chiral pyrrolidine structure and derivatives using a variety of starting materials, such as various carbohydrate and amino acid precursors [14].Early studies of relationships between anisomycin structure and activity on protein translation demonstrated that removal of the 3-position acetoxy group (desacetylanisomycin, 2), nitrogen acetylation and quaternization, as well as aromatic bromination, all resulted in analogs (e.g., 3-5) exhibiting significant reductions of activity [2].Movement of the acetyl group from the 3-to the 4-position alcohol gave isomer 6, which exhibited 3-fold less inhibitory activity on protein synthesis but comparable cytotoxicity as anisomycin in four tumor cell lines [15].Activity against protozoa and fungi dropped mildly on replacement of the para-methoxy substituent by a methyl group or proton but was significantly reduced on moving the methoxy group from the para to the meta and ortho positions, as well as upon replacement of the benzyl methylene protons by methyl and phenyl substituents [16].In the triple-negative breast cancer cell line MDA-MB-468, both anisomycin and (2R,3S,4S)-1-benzyl-4-benzyloxy-3hydroxy-2-(4-methoxybenzyl)-pyrrolidine (7) inhibited protein synthesis and exhibited cytotoxicity at equal concentrations [17].In examinations of their ability to activate, respectively, JNK and p38 MAPK, replacement of the (4S)-alcohol by hydrogen or a methyl group gave analogs exhibiting similar and significantly reduced activity relative to anisomycin and desacetylanisomycin (2); however, Nand O-benzyl analogs (e.g., 7) did not exhibit kinase activating activity [18].The combination of N-acylation in various amide, carbamate, and urea components and replacement of the acetyl group by different carbamoyl residues gave a library of anisomycin analogs exhibiting activity against Staphylococcus aureus and Candida albicans in whole-cell assays, with reduced cytotoxicity against HEK293 mammalian cells [19].In sum, the structure-activity data for anisomycin is relatively dispersed and somewhat challenging to interpret due in part to the range of targeted indications and mechanisms of action, the latter of which include inhibitory activity on protein synthesis and the potential to activate various kinases (Figure 1).
In the interest of furthering the utility of anisomycin for treating Leishmania, a systematic structure-activity relationship study has been performed to identify positions for modification to ideally improve selectivity by increasing anti-parasite activity and reducing toxicity.Modifications were performed on the secondary amine, hydroxyl, and acetyl groups.Moreover, the aromatic ring was modified at the meta-and para-positions.Although the structural elements for maintaining activity were illustrated, modifications were typically unfruitful in providing active and selective analogs.Replacement of the methoxy group by other ethers was, however, found to offer a point of entry for altering anisomycin in a way that could maintain activity yet improve anti-protozoan selectivity.

In Vitro Inhibition of Translation Assays
Cytosolic ribosome susceptibility and selectivity were assessed through three different cell-free transcription-translation assays: (1) An extract of E. coli (S30, Promega, Madison, WI, USA), (2) in rabbit reticulocytes (Promega), and (3) in L. tarentolae lysate (Jena Bioscience, Jena, Germany).Three plasmids, compatible with each extract system, were used in this study: pBESTlucTM (Promega) for the prokaryotic translation assay, Luciferase T7 DNA (Promega) for the reticulocytes, and pLEXSY-invitro2-EGFP (Jena Bioscience) for Leishmania.Firefly luciferase was used as a reporter gene for the bacterial and reticulocyte systems, and EGFP for Leishmania.Reaction mixtures were prepared as suggested by the manufacturer, except that the final reaction volume was adjusted to 15 µL to which 1.5 µL (ten-fold) of the relevant compound concentration was supplemented.Assays were performed in white polystyrene 96-well flat-bottom plates (Nunc, Roskilde, Denmark) for luciferase and black polystyrene 384-well flat-bottom plates (Greiner, Kremsmunster, Austria) for EGFP.Incubation times were 60 min at 37 °C, 90 min at 30 °C, and 120 min at 26 °C for the bacterial, reticulocyte, and Leishmania systems, respectively.Reactions were stopped by quick snap cooling followed by a 5 min incubation on ice.Luciferase activity was measured for each well following the addition of 50 µL of Luciferase Assay reagent (Promega, Madison, WI, USA) with an automatic reagent injector (Tecan, Mannedorf, In the interest of furthering the utility of anisomycin for treating Leishmania, a systematic structure-activity relationship study has been performed to identify positions for modification to ideally improve selectivity by increasing anti-parasite activity and reducing toxicity.Modifications were performed on the secondary amine, hydroxyl, and acetyl groups.Moreover, the aromatic ring was modified at the meta-and para-positions.Although the structural elements for maintaining activity were illustrated, modifications were typically unfruitful in providing active and selective analogs.Replacement of the methoxy group by other ethers was, however, found to offer a point of entry for altering anisomycin in a way that could maintain activity yet improve anti-protozoan selectivity.

In Vitro Inhibition of Translation Assays
Cytosolic ribosome susceptibility and selectivity were assessed through three different cell-free transcription-translation assays: (1) An extract of E. coli (S30, Promega, Madison, WI, USA), (2) in rabbit reticulocytes (Promega), and (3) in L. tarentolae lysate (Jena Bioscience, Jena, Germany).Three plasmids, compatible with each extract system, were used in this study: pBESTlucTM (Promega) for the prokaryotic translation assay, Luciferase T7 DNA (Promega) for the reticulocytes, and pLEXSY-invitro2-EGFP (Jena Bioscience) for Leishmania.Firefly luciferase was used as a reporter gene for the bacterial and reticulocyte systems, and EGFP for Leishmania.Reaction mixtures were prepared as suggested by the manufacturer, except that the final reaction volume was adjusted to 15 µL to which 1.5 µL (ten-fold) of the relevant compound concentration was supplemented.Assays were performed in white polystyrene 96-well flat-bottom plates (Nunc, Roskilde, Denmark) for luciferase and black polystyrene 384-well flat-bottom plates (Greiner, Kremsmunster, Austria) for EGFP.Incubation times were 60 min at 37 • C, 90 min at 30 • C, and 120 min at 26 • C for the bacterial, reticulocyte, and Leishmania systems, respectively.Reactions were stopped by quick snap cooling followed by a 5 min incubation on ice.Luciferase activity was measured for each well following the addition of 50 µL of Luciferase Assay reagent (Promega, Madison, WI, USA) with an automatic reagent injector (Tecan, Mannedorf, Switzerland) by recording the chemical-luminescence signal.EGFP fluorescence (λex = 488 nm; λem = 507 nm) was recorded on a Tecan Infinite R ® F200 microplate reader (Tecan).Extracts lacking the circular DNA template were used as a negative control to calculate the fluorescence/chemical luminescence background.Reaction mixtures without compounds were used as positive controls and were regarded as 100% translation.Paromomycin was used as a control reagent in all assays.At least six different concentrations were used to plot each translation inhibition curve; experiments were performed in three independent repeats in duplicate.Half maximal inhibition concentrations (IC 50 ) values were calculated from the concentration-response fitting curves using GraFit7.0.3 software [28].

Drug Susceptibility Assays in Infected BMDM
The effect of isopropyl ether 22a on the amastigote intracellular form of Leishmania was determined by the measurement of EC 50 values in cells, as previously described [34].Briefly, 2.5 × 10 5 bone-marrow-derived macrophages (BMDMs) were plated into the wells of 12-well chamber slides (Ibidi) with complete DMEM medium.A culture of L. infantum or L. major WT promastigotes in stationary phase in a ratio of 1:5 BMDM to parasites was used to infect cells.Infection was carried out for 3 h at 37 • C in DMEM medium with 5% CO 2 without drug.After 24 h, increasing concentrations of ether 22a were added to the medium containing the infected macrophages.After 48 h, the slides were fixed in methanol and stained with Diff-Quick solution to facilitate parasite visualization.The number of infecting amastigotes per 100 cells was determined by examining 300 macrophages per triplicate assay and normalized to the untreated control.EC 50 values in the amastigote form were calculated based on dose-response curves that were analyzed by non-linear regression using the GraphPad Prism 9.0 software.An average of three independent biological replicates was used to perform the analyses.

Chemistry
Anhydrous solvents (THF, DMF, MeCN) were obtained by passage through solvent filtration systems (GlassContour, Irvine, CA, USA).Deionized water was used in the purifications.Unless specified otherwise, all reagents were from commercial sources and used as received.Anisomycin and di-tert-butyl decarbonate (Boc 2 O) were purchased from ChemImpex; acetic anhydride was purchased from Alfa Aesar; pyridine, 4-dimethylaminopyridine (DMAP), sodium hydride, iodomethane, boron tribromide, trifluoroacetic acid (TFA), Niodosuccinimide (NIS), dimethylamine (40% wt. in water), formaldehyde (37% wt. in water), N-methylmorpholine, cesium carbonate, 2-iodopropane, 1-iodooctane, benzyl bromide, N,N-dimethylethylenediamine, formic acid (FA), and triethylamine, all were purchased from Sigma-Aldrich; acetic acid, sodium hydroxide, potassium carbonate and solvents were obtained from Fisher.Sodium hydride (60% dispersion in mineral oil) was washed with hexane three times to remove oil prior to use.Chromatography was on 230−400 mesh silica gel.Analytical thin-layer chromatography (TLC) was performed on glass-backed silica gel plates (Merck 60 F254).Visualization of the developed chromatogram was performed by UV absorbance or staining with ninhydrin. 1H and 13 C NMR spectra were measured respectively in CD 3 OD at 500 MHz and 126 MHz and referenced to CD 3 OD (3.31 ppm and 49.0 ppm).Coupling constant J values were measured in Hertz (Hz) and chemical shift values in parts per million (ppm).Specific rotations, [α] D were measured at 25 • C at the specified concentrations (c in g/100 mL) using a 1 dm cell on a PerkinElmer Polarimeter 589 and expressed using the general formula: [α] D 25 = (100 × α)/(d × c).High resolution mass spectrometric analyses were obtained by the Centre Régional de Spectrométrie de Masse de l'Université de Montréal.Protonated molecular ions [M + H] + and sodium adducts [M + Na] + were used for empirical formula confirmation.
A mixture of anisomycin (1, 10 mg, 0.038 mmol, 1 eq.) and NaOH 1N (0.25 mL) was heated and stirred for 3 h at 80 • C.After cooling to room temperature, the resulting mixture was extracted with ethyl acetate.The organic layer was washed with brine, dried over MgSO 4 , filtered, and evaporated.The residue was purified by HPLC on a C18 column using 5 to 50% MeOH in H 2 O. Evaporation of the collected fractions provided diol 2 as white solid (5.9 mg, 70%), which was shown to be >99% pure by LC-MS analysis [

Chemistry
Employing anisomycin (1) as a starting material, a set of amine and alcohol-modified analogs (e.g., 8-11) were first synthesized to examine the importance of the polar substituents for anti-protozoan activity (Scheme 1).Desacetyl and diacetylanisomysin (2 and 9) were prepared to evaluate the relevance of the acyl functionality [38].N-methyl anisomycin (8) has been previously synthesized by a fifteen-step synthesis from L-diethyl tartrate [39].Employing anisomycin, N-methyl pyrrolidine 8 was prepared in 58% yield using Eschweiler-Clarke reaction conditions with formic acid and formaldehyde and microwave heating [40].
Modifications of the hydroxyl groups were accomplished by protocols commencing with Nand N,O-protection with the Boc group using di-tert-butyl dicarbonate in THF and in MeCN with 4-dimethylaminopyridine (DMAP) to give, respectively, N-(Boc)anisomycin (12) and N,O-bis-Boc-anisomycin (14) [41].Subsequently, alcohol 12 was acylated using acetic anhydride in pyridine, and the Boc group was removed with HCl gas to furnish diacetylanisomycin (9) in 66% yield.O,O-bis-dimethyl ether 10 was also prepared from alcohol 12 in 67% yield by a route featuring saponification of the acetyl group, alkylation of the resulting diol with iodomethane and sodium hydride in DMF, followed by removal of the Boc group with HCl gas.Employing a similar protocol on N,O-bis-Boc-anisomycin (14) gave methyl ether 11 in 59% yield.

Chemistry
Employing anisomycin (1) as a starting material, a set of amine and alcohol-modified analogs (e.g., 8-11) were first synthesized to examine the importance of the polar substituents for anti-protozoan activity (Scheme 1).Desacetyl and diacetylanisomysin (2 and 9) were prepared to evaluate the relevance of the acyl functionality [38].N-methyl anisomycin (8) has been previously synthesized by a fifteen-step synthesis from L-diethyl tartrate [39].Employing anisomycin, N-methyl pyrrolidine 8 was prepared in 58% yield using Eschweiler-Clarke reaction conditions with formic acid and formaldehyde and microwave heating [40].

Scheme 1. Synthesis of methylated anisomycin analogs.
Modifications of the hydroxyl groups were accomplished by protocols commencing with N-and N,O-protection with the Boc group using di-tert-butyl dicarbonate in THF and in MeCN with 4-dimethylaminopyridine (DMAP) to give, respectively, N-(Boc)anisomycin (12) and N,O-bis-Boc-anisomycin (14) [41].Subsequently, alcohol 12 was acylated using acetic anhydride in pyridine, and the Boc group was removed with HCl gas to furnish diacetylanisomycin (9) in 66% yield.O,O-bis-dimethyl ether 10 was also prepared from alcohol 12 in 67% yield by a route featuring saponification of the acetyl group, alkylation of the resulting diol with iodomethane and sodium hydride in DMF, followed by removal of the Boc group with HCl gas.Employing a similar protocol on N,O-bis-Bocanisomycin ( 14) gave methyl ether 11 in 59% yield.
The aromatic ring of anisomycin (1) was next modified at the meta-position (e.g., 16, 19-20) by two different protocols (Scheme 2).Electrophilic iodination was accomplished with N-iodosuccinimide and trifluoroacetic acid in dichloromethane to provide iodide 16 in 53% yield.Substituents were also added to the aromatic ring of phenol 17, which was obtained from anisomycin using boron tribromide in dichloromethane at -78 °C in 56% yield [42].The amine of phenol 17 was protected using Boc2O in THF to provide N-Boc phenol 18. Mannich reactions on phenol 18 using, respectively, dimethylamine and morpholine with formaldehyde provided bis-aminomethyl phenols 19 and 20 in 38% and 41% yields after Boc group removal with HCl gas.Moreover, phenol 18 was O-alkylated by alkoxide formation with cesium carbonate in DMF at 65 °C, followed by reaction with a set of alkyl halides: 2-iodopropane, 1-iodooctane, benzyl bromide, and tert-butyl bromoacetate.Ethers 22a-c were prepared to respectively examine the effects of alkyl branching, long chain hydrophobicity, and aromaticity at the phenol position and obtained in 65-73% yields after Boc group removal with HCl gas.Carboxymethyl ether 22d was isolated in 67% yield after Boc group and tert-butyl ester removal using trifluoroacetic acid in dichloromethane, followed by ion exchange with HCl gas to give the hydrochloride salt.The aromatic ring of anisomycin (1) was next modified at the meta-position (e.g., 16, 19-20) by two different protocols (Scheme 2).Electrophilic iodination was accomplished with N-iodosuccinimide and trifluoroacetic acid in dichloromethane to provide iodide 16 in 53% yield.Substituents were also added to the aromatic ring of phenol 17, which was obtained from anisomycin using boron tribromide in dichloromethane at -78 • C in 56% yield [42].The amine of phenol 17 was protected using Boc 2 O in THF to provide N-Boc phenol 18. Mannich reactions on phenol 18 using, respectively, dimethylamine and morpholine with formaldehyde provided bis-aminomethyl phenols 19 and 20 in 38% and 41% yields after Boc group removal with HCl gas.Moreover, phenol 18 was O-alkylated by alkoxide formation with cesium carbonate in DMF at 65 • C, followed by reaction with a set of alkyl halides: 2-iodopropane, 1-iodooctane, benzyl bromide, and tert-butyl bromoacetate.Ethers 22a-c were prepared to respectively examine the effects of alkyl branching, long chain hydrophobicity, and aromaticity at the phenol position and obtained in 65-73% yields after Boc group removal with HCl gas.Carboxymethyl ether 22d was isolated in 67% yield after Boc group and tert-butyl ester removal using trifluoroacetic acid in dichloromethane, followed by ion exchange with HCl gas to give the hydrochloride salt.

Anti-Leishmanial Activity of Anisomycin Derivatives
Anisomycin and derivatives possessing modifications on the polar functional groups (e.g., 2 and 8-11), aromatic meta-position (16, 19, and 20), and phenol ( 17 and 22a-c) were initially examined for inhibitory activity on protein synthesis in active lysates of L. tarentolae, Escherichia coli, and rabbit reticulocytes (Table 1).Anisomycin activity (IC50 = 0.55 µM) on Leishmania was typically eliminated by modification of the amine and alcohol

Anti-Leishmanial Activity of Anisomycin Derivatives
Anisomycin and derivatives possessing modifications on the polar functional groups (e.g., 2 and 8-11), aromatic meta-position (16, 19, and 20), and phenol (17 and 22a-c) were initially examined for inhibitory activity on protein synthesis in active lysates of L. tarentolae, Escherichia coli, and rabbit reticulocytes (Table 1).Anisomycin activity (IC 50 = 0.55 µM) on Leishmania was typically eliminated by modification of the amine and alcohol functional groups (e.g., 2 and 8-10).Notably, replacement of the 3-acetoxy group by a methyl ether gave a 44-fold drop in activity against Leishmania and a 700-fold loss in reticulocyte cytotoxicity, with a net improvement in the selectivity index.Furthermore, the introduction of substituents to the aromatic meta-position in analogs 16, 19, and 20 abolished anti-protozoan activity.Removal of the methyl ether at the aromatic para-position caused a nearly 100-fold loss of anti-protozoan activity, which was partially restored by replacing the methyl group with other ethers: O-i-Pr, O-n-octyl, and O-benzyl analogs 22a-c.Ethers 22a-c were, respectively, 2-, 67-, and 14-fold less active than anisomycin against Leishmania.Relative to the clinically used paromomycin, anisomycin (1) and iso-propyl ether 22a exhibited better anti-protozoan activity, no effect on E. coli, but lower selectivity indices.Inhibitory activity on L. donovani promastigotes was subsequently measured according to the reported protocol [43] and reflected results from the inhibition of protein translation assay.
Table 1.Inhibition of translation in active cell lysates derived from L. tarentolae, bacteria (E.coli), and rabbit reticulocytes (RR) and inhibition of L. donovani promastigote growth by anisomycin and derivatives.
Scheme 2. Synthesis of anisomycin analogs modified on the aromatic ring.

Anti-Leishmanial Activity of Anisomycin Derivatives
Anisomycin and derivatives possessing modifications on the polar functional groups (e.g., 2 and 8-11), aromatic meta-position (16, 19, and 20), and phenol (17 and 22a-c) were initially examined for inhibitory activity on protein synthesis in active lysates of L. tarentolae, Escherichia coli, and rabbit reticulocytes (Table 1).Anisomycin activity (IC50 = 0.55 µM) on Leishmania was typically eliminated by modification of the amine and alcohol functional groups (e.g., 2 and 8-10).Notably, replacement of the 3-acetoxy group by a methyl ether gave a 44-fold drop in activity against Leishmania and a 700-fold loss in reticulocyte cytotoxicity, with a net improvement in the selectivity index.Furthermore, the introduction of substituents to the aromatic meta-position in analogs 16, 19, and 20 abolished anti-protozoan activity.Removal of the methyl ether at the aromatic para-position caused a nearly 100-fold loss of anti-protozoan activity, which was partially restored by replacing the methyl group with other ethers: O-i-Pr, O-n-octyl, and O-benzyl analogs 22a-c.Ethers 22a-c were, respectively, 2-, 67-, and 14-fold less active than anisomycin against Leishmania.Relative to the clinically used paromomycin, anisomycin (1) and isopropyl ether 22a exhibited better anti-protozoan activity, no effect on E. coli, but lower selectivity indices.Inhibitory activity on L. donovani promastigotes was subsequently measured according to the reported protocol [43] and reflected results from the inhibition of protein translation assay.Considering the in vitro protein translation inhibition activities of the different ether analogs as well as activity on L. donovani prosmatigotes, the O-i-Pr, O-octyl, and Ocarboxymethyl analogs (22a, 22c, and 22d) were further tested against promastigotes of various Leishmania strains, including L. major and L. infantum, as well as strains that developed resistance to current anti-leishmanial drugs such as pentavalent antimonials, miltefosine, and amphotericin B (Table 2).The most active O-i-Pr analog, 22a, in the translation assay exhibited similar inhibitory activity against the different Leishmania promastigotes and slightly improved activity against the resistant strains as that displayed by anisomycin (1).The most active analog in the promastigote assays, O-iso-propyl ether 22a, was tested on intracellular forms of Leishmania in macrophages.Against the amastigotes of L. infantum and L. major, ether 22a exhibited, respectively, 3.3 and 3.6-fold higher activity (EC50 33.31 nM and 53.66 nM) than that observed against the promastigote (Figure 2).In addition, ether 22a exhibited 5-10-fold greater selectivity against the different amastigotes relative to host macrophage toxicity, which was examined using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) survival assay.The most active analog in the promastigote assays, O-iso-propyl ether 22a, was tested on intracellular forms of Leishmania in macrophages.Against the amastigotes of L. infantum and L. major, ether 22a exhibited, respectively, 3.3 and 3.6-fold higher activity (EC 50 33.31nM and 53.66 nM) than that observed against the promastigote (Figure 2).In addition, ether 22a exhibited 5-10-fold greater selectivity against the different amastigotes relative to host macrophage toxicity, which was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) survival assay.

Discussion
Focused on a better understanding of the structure-activity relationships of anisomycin against Leishmania, a systematic study was performed to validate the importance of the pyrrolidine and aromatic ring substituents for anti-parasitic activity and reticulocyte toxicity.
Previously, crystal structures of anisomycin bound to eukaryotic ribosomes (Saccharomyces cerevisiae) and archaeal ribosomes (Haloarcula marismortui) demonstrated high

Discussion
Focused on a better understanding of the structure-activity relationships of anisomycin against Leishmania, a systematic study was performed to validate the importance of the pyrrolidine and aromatic ring substituents for anti-parasitic activity and reticulocyte toxicity.
Previously, crystal structures of anisomycin bound to eukaryotic ribosomes (Saccharomyces cerevisiae) and archaeal ribosomes (Haloarcula marismortui) demonstrated high conservation in the binding orientation [4].Anisomycin is positioned in the peptidyl transferase center of the ribosome through the hydrogen bonds formed by the hydroxy group (with nucleotides of LSU rRNA Y2504, G2505 (E. coli numbering)) and the nitrogen (C2452) [44].Considering that the binding site of anisomycin is highly conserved between bacteria, yeast, humans, and Leishmania, a graphic model was constructed to rationalize the observed results (Figure 3).

Discussion
Focused on a better understanding of the structure-activity relationships of anisomycin against Leishmania, a systematic study was performed to validate the importance of the pyrrolidine and aromatic ring substituents for anti-parasitic activity and reticulocyte toxicity.
Previously, crystal structures of anisomycin bound to eukaryotic ribosomes (Saccharomyces cerevisiae) and archaeal ribosomes (Haloarcula marismortui) demonstrated high conservation in the binding orientation [4].Anisomycin is positioned in the peptidyl transferase center of the ribosome through the hydrogen bonds formed by the hydroxy group (with nucleotides of LSU rRNA Y2504, G2505 (E. coli numbering)) and the nitrogen (C2452) [44].Considering that the binding site of anisomycin is highly conserved between bacteria, yeast, humans, and Leishmania, a graphic model was constructed to rationalize the observed results (Figure 3).Decreases in anti-leishmanial activity upon modification of the pyrrolidine ring substituents may likely be due to losses of key hydrogen-bond interactions.For example, deacetyl, 3-methoxy, and 3,4-dimethoxy analogs 2, 10, and 11 were inactive against L. Decreases in anti-leishmanial activity upon modification of the pyrrolidine ring substituents may likely be due to losses of key hydrogen-bond interactions.For example, deacetyl, 3-methoxy, and 3,4-dimethoxy analogs 2, 10, and 11 were inactive against L. tarentolae translation and confirmed the importance of the acetyl group as previously reported using an anti-yeast assay [45].The affinity and specificity of anisomycin for the ribosome A-site are also determined by the interactions of the aromatic ring with the nucleotide bases of C2452 and U2506 [43].The loss of activity of the mono-and di-meta-substituted analogs (e.g., 20) suggests that steric hindrance may prevent the entrance of aromatic rings in the A-site.
Upon binding to the ribosome, the p-methoxyphenyl group inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, causing the disruption of peptide bond formation [46].The inactivity of the demethylated analog on the inhibition of translation may be explained by a loss of hydrophobicity.On the other hand, alternative phenolic ethers were tolerated.Notably, O-i-Pr, O-Octyl, O-benzyl, and O-CH 2 CO 2 H phenolic ethers 22a-22d all retained varying degrees of anti-parasitic activity in the Leishmania translation assay and against live promastigotes.Moreover, certain phenolic ethers (e.g., O-i-Pr 22a) exhibited subtle but significantly lower inhibition of reticulocyte translation, indicating the potential to differentiate toxicity from anti-leishmanial activity.Against the intracellular parasite stage (amastigotes), the anti-leishmanial activity of O-i-Pr 22a was higher than against the extracellular stage (promastigotes) [47].This phenomenon demonstrated that O-i-Pr 22a was able to enter the host BMDM cells to act on the intracellular amastigote infection without host cell toxicity [48].

Conclusions
The anti-protozoal activity of anisomycin has been known since the 1950s.In spite of intriguing mechanisms of action involving inhibitory activity on protein synthesis and modulatory activity on various kinases, the therapeutic utility of anisomycin has been compromised due to its limited selectivity.A systematic study of the importance of the pyrrolidine and aromatic ring functional groups for the anti-protozoal activity of anisomycin has revealed a high sensitivity to chemical modification.Earlier substitutions of the para-methoxy substituent by methyl and protons gave analogs with reduced antiprotozoal activity [16].Conversion of the methoxyphenyl group to alternative ethers has, however, illuminated an entry point for designing anisomycin analogs that differentiate anti-leishmanial activity from host cytotoxicity.Future efforts to improve anti-parasitic potency and selectivity are under investigation and will be reported in due time.

Figure 2 .
Figure 2. Effect of O-iso-propyl ether 22a on BMDM macrophages infected with (A) L. infantum WT and (B) L. major WT for 48 h.

Figure 2 .
Figure 2. Effect of O-iso-propyl ether 22a on BMDM macrophages infected with (A) L. infantum WT and (B) L. major WT for 48 h.

Figure 3 .
Figure 3. Graphical depiction of the conserved binding site of anisomycin in L. major ribosome.The 80S ribosome (8OVJ) of L. major was superimposed on the 50S ribosome of H. marismortui (PDB ID: 3CC4), and nucleotides proposed to contact anisomycin (colored in cyan) are highlighted in orange; the corresponding homologs in E. coli rRNA are presented in parentheses; pseudouridine nucleotide is indicated with Ψ symbol.

Figure 3 .
Figure 3. Graphical depiction of the conserved binding site of anisomycin in L. major ribosome.The 80S ribosome (8OVJ) of L. major was superimposed on the 50S ribosome of H. marismortui (PDB ID: 3CC4), and nucleotides proposed to contact anisomycin (colored in cyan) are highlighted in orange; the corresponding homologs in E. coli rRNA are presented in parentheses; pseudouridine nucleotide is indicated with Ψ symbol.

Author
Contributions: C.R.-T.and A.V.I.-M.formal analysis, investigation; C.F.-P.and W.D.L. conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, visualization, writing-review and editing; M.S.-B.conducted the in vitro experiments; A.M.T.N. chemical syntheses, formal analysis, investigation, writing; M.S.-B., A.Y., A.B., C.L.J., M.O., C.F.-P.and W.D.L. proofread and edited the manuscript; A.Y., A.B., C.L.J., C.F.-P., M.O. and W.D.L. supervised the progress of the project.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by a New Frontiers in Research Fund Exploration Grant NFRFE-2018-00766 and Canadian Institutes of Health Research grant (#469305) "Safe and selective anti-parasite therapy targeting ribosome and glycosome function" awarded to CFP, MO, and WL; the Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant Program Projects #06647 (WL) and RGPIN-2017-04480 (CFP); the Fonds de recherche du Québec-Nature et technologies (FRQNT) Centre in Green Chemistry and Catalysis, Project #171310 (WL).The Canada Foundation for Innovation (www.innovation.ca),grant number 37324.CRT was supported by the FRQNT and the NSERC studentship programs.This work was supported by the UM-Israel Partnership for Research, the Weizmann Abroad Postdoctoral Program for Advancing Women in Science (to M.S.-B); and the Michael and Penny Feiwel Chair for Research in Dermatology (C.L.J).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicanle.

Table 1 .
Inhibition of translation in cell lysates derived from L. tarentolae, bacteria (E.coli), and rabbit reticulocytes (RR) and inhibition of L. donovani promastigote growth by anisomycin and derivatives.