Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Introduction

The leishmaniases are a complex group of vector-borne diseases caused by protozoan parasites of genus Leishmania. Endemic in over 98 countries, placing in excess of 1 billion people at risk of infection [1, 2] and with an economic burden best represented by more than 2.4 million DALYs (disability-adjusted life years), leishmaniasis is considered by the WHO as one of the most important neglected tropical diseases [3]. Clinical manifestations vary according to the parasite species and the host immunological status [4]. These range from the more benign skin ulcers in cutaneous leishmaniasis (CL) to visceral leishmaniasis (VL), which infects the spleen, liver, and bone marrow and it is fatal in more than 95% of untreated cases [4]. With no human vaccine available, treatment relies on chemotherapy and the current pharmacopeia is reliant on a limited number of drugs: pentavalent antimonials, amphotericin B, pentamidine, miltefosine and paramomycin [5]. All of these possess drawbacks, including poorly-defined modes of action, invasive routes of administration, severe side effects, variable efficacy, high cost and growing resistance [6]. With approximately 1 million new cases of CL and 90,000 cases of VL each year worldwide, there is a major unmet need to develop new broad-spectrum chemotherapies based on new validated drug targets [7].

Chalcones have been described as a potential new class of antileishmanials. More than 300 chalcones have reported activity against Leishmania parasites [8-10]. We have previously reported the selective antileishmanial activity of a natural chalcone 2’,6’-dihydroxy-4’-methoxychalcone (DMC) 1a (Fig 1) extracted from the pepper plant Piper aduncum [11]. Subsequent synthetic refinement afforded the nitro-chalcone, 3-nitro-2’-hydroxy-4’,6’-dimethoxychalcone (CH8) 1b [12], which showed strong and selective anti-parasite activity in vitro and in vivo against both Leishmania amazonensis and Leishmania infantum [13-16]. Despite those promising results, further enhancement to activity were challenged by the fact the mechanism of action of CH8 1b remained to be elucidated. In this report, through a combination of chemical, molecular and cellular biology, we describe the use of a CH8-derived chemical probe 1c (3-nitro-2’,4’,6’- trimethoxychalcone, NAT22) to identify the cytosolic enzyme tryparedoxin peroxidase (cTXNPx) as a target for chalcones in a range of Leishmania species. Additionally, we demonstrated that the interaction between the chalcone and cTNXPx inhibits the capacity to detoxify reactive oxygen species (ROS) leading to parasite death. As such, this study demonstrates that this parasitic specific protein has significant potential as a drug target for the future treatment of leishmaniasis.

Fig 1

Chemical structures of compounds 1–4.

Experimental

Analogues and probe synthesis

Details concerning the chemical synthesis and compound characterization are described in the Supplementary Information (S1 Text).

Anti-promastigote activity

Promastigotes of L. amazonensis (strain MHOM/BR/75/Josefa, 5 x 105/mL), L. braziliensis (strain MHOM/BR/75/M2903, 2 x 106/mL) and L. infantum (strain MHOM/MA67ITMAP263, 2 x 106/mL) at the logarithmic phase of growth, were incubated at 26°C with different concentrations of compounds 1c, 2 and 3 for 72 h in M199 medium containing 5% of heat inactivated fetal bovine serum (HIFBS), 0.2% hemin (Sigma-Aldrich), 100 μg/mL streptomycin and 100 UI/mL penicillin (Sigma-Aldrich). Parasite viability was assessed in the last four hours of culture by Alamar blue (ThermoFisher Scientific). The concentrations that reduced cell viability by 50% (EC50) were calculated by non-linear regression analysis using GraphPad Prism 7 software.

For mutant cell lines, promastigotes of L. mexicana T7 Cas9 (Parental), Δ1040, Δ1160 and ΔcTXNPx (2 x 106/mL), were incubated at 26°C with different concentrations of compounds 1c for 48 h in Schneider’s medium (Sigma-Aldrich) containing 10% of heat inactivated fetal bovine serum (HIFBS), 100 μg/mL streptomycin and 100 UI/mL penicillin. Parasite viability and EC50 values were assessed as described above.

Anti-amastigote activity

Bone marrow-derived macrophages (BMDM) were differentiated from BALB/c mouse bone marrow [17] and plated at 5 x 105/well in 24-well culture plates onto circular glass coverslips in RPMI medium containing 10% of HIFBS at 37°C/5% CO2 for 24 h. Then the macrophage monolayers were infected with L. amazonensis promastigotes (5 x 106/well) at 34°C for 4 h. After removal of free parasites by washing with phosphate buffered saline (PBS), the cells were cultured at 37°C for further 24 h for parasite differentiation into amastigote forms. Infected cells were treated with different concentrations of compounds 1c, 2 or 3 for 48 h, washed with PBS and stained with Panotic stain (Newprov, Brazil) according to manufacturer instructions. At least 200 macrophages/coverslip and their intracellular amastigotes were counted at 400x magnification for the determination of EC50 values as described above.

Macrophage cytotoxicity

Uninfected BMDM were treated as above for infected cells. At the end of the 48 h incubation time, the specific release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in culture supernatants was measured using a commercial assay kit (Doles, Brazil), as previously described [15]. CC50 values were calculated as described for EC50 values. The selectivity index (SI) for each compound was calculated as the ratio between macrophage cytotoxicity (CC50) and anti-amastigote activity (EC50).

Mouse infection and oral treatment with chalcone 1c

All animal studies were approved by the Animal Care and Use Committee of the Health Sciences Center of the Federal University of Rio de Janeiro, Brazil, under the protocol number CEUA 030/17. All animal experiments were conducted in compliance with the principles stated in the Guide for the Care and Use of Laboratory Animals, 8 Edition.[18] BALB/c mice were randomly distributed into groups of 5 and intradermally infected in the right ear pinna with 2 × 106 L. amazonensis promastigotes at stationary phase of growth [16]. One week after infection, animals were orally treated daily for 3 weeks by intragastric gavage with compound 1c (40 mg/Kg/day) in propylene glycol (PPG) vehicle. Untreated controls received PPG vehicle alone. For clinical follow-up, ear thicknesses were periodically measured using a calliper gauge. For parasite burden, on day 30 of infection, the individual ear tissue homogenates were assayed by limiting dilution (LDA) [15]. Animal sample size was estimated by a power-based method using the following assumptions: α  =  0.05, 1 − β  =  0.9, and standard deviation 20% of mean for two groups [19].

Intracellular chalcone distribution by confocal microscopy

L. amazonensis promastigotes (1 x 106/mL) were incubated with compound 2 (5 μM) at 26°C in 1 mL of medium M199 for 5 h at 26°C. At the end of incubation time cells were washed 3 times with PBS, fixed with 4% of paraformaldehyde and permeabilized with 0.1% saponin in PBS for 5 min at room temperature (RT). Fixed/permeabilized parasites were then subjected to cycloaddition reaction (CuAAC) by incubation with fluorescent ‘trifunctional’ probe 4 (Fig 1; 5 μM) in the presence of CuSO4 (1 mM), sodium ascorbate (2 mM) and tris-hydroxypropyltriazolylmethylamine (THPTA, 2 mM) for 1h at room temperature. Then, cells were incubated for 15 min with Hoechst 33342 (8 μM) for nucleus and kinetoplast labelling. Cells were washed with PBS, placed onto microscope slides and imaged using confocal microscope (Leica TCS-SPE) at 63x (ACS APO 63x 1.30 oil objective) in the Hoechst (350 / 461 nm), TAMRA (555 / 580 nm) wavelengths and DIC (Differential interference contrast).

Intracellular protein tagging by SDS-PAGE and Western blotting

L. amazonensis promastigotes (6 × 107/mL), BMDM (2 x 106/well) and amastigotes isolated from infected macrophages [20], were treated with compound 2 (1 μM and 5 μM) for 4 h at 27°C (promastigotes) or 37°C (BMDM and amastigotes). Then, cells were washed twice with cold PBS, lysed by sonication in lysis buffer (1% sodium deoxycholate, 0.5% SDS, 50 mM Tris pH 7.4, 150 mM NaCl) containing EDTA-free protease inhibitor cocktail (Sigma-Aldrich). Proteins were precipitated with acetone (4 volumes at −20°C for 1 h), and quantified using Bicinchoninic Acid Kit (BCA1, Sigma Aldrich) according to manufacturer’s protocol.

For competition assay, promastigotes (6 × 107/mL) were incubated with 1c at different concentrations (1, 3, 9, 27 μM) for 4 h, washed twice with PBS, followed by incubation with 2 (1 μM) for additional 4 h in M199 medium at 26°C. Cells were then lysed, and parasite proteins precipitated and quantified as above.

For unidimensional electrophoresis (SDS-PAGE), proteins (1 mg/mL) were incubated with compound 4 (5 μM), CuSO4 (1 mM), sodium ascorbate (2 mM) and THPTA (2 mM). The click reaction was carried out for 1 hr at RT, then quenched by addition of 4x SDS-PAGE sample buffer with 50 mM of DTT (Bio-Rad). Proteins were then heated for 5 min at 95°C, run in 10% SDS-PAGE, and gel images taken at Gel Doc XR imager (Bio-Rad) using Comassie Blue and Oriole filter (270 / 604 nm) for compound 4 fluorescence detection. 2D electrophoresis analyses were performed as described by Higa et al [21].

Protein identification by mass spectrometry

Proteins from promastigotes treated with 2 and linked to 4 were separated by unidimensional SDS-PAGE as above. The gel section of interest (~22 kDa) was manually excised and incubated overnight with 25 mM NH4HCO3 in 50% acetonitrile to remove the stain. Then proteins were reduced for 1h at 56°C with 10 mM DTT (USB Corporation), alkylated for 45 min at room temperature with 55 mM iodoacetamide and digested by trypsin (modified sequencing grade; Promega) solution (0.01 μg/μL in 25 mM NH4HCO3) overnight at 37°C. Generated peptides were extracted with 5% trifluoroacetic acid (TFA) and 50% acetonitrile, followed by concentration in a Speed-Vac system. Each sample was then solubilized in 20 μL 0.1% formic acid and 3% acetonitrile in deionized water. The mass spectrometry analyses were performed as described previously [22]. In brief, the peptides were first desalted on-line using a Waters Symmetry C18 trap column, loaded on a Waters Nanoacquity UPLC system (Waters, Milford, MA) and finally sprayed into a Q-Tof quadrupole/orthogonal acceleration time-of-flight spectrometer (Waters, Milford, MA) interfaced with the Nanoacquity system capillary chromatograph. The exact mass was determined automatically using the Q-Tof LockSpray (Waters, Milford, MA). Data-dependent MS/MS acquisitions were performed on precursors with charge states of 2, 3 or 4 over a range of 50–2000 m/z and under a 2 m/z window.

Production and purification of recombinant cTXNPx

Expression plasmid pPOPINF containing ORF for cTXNPx from L. amazonensis (AY842247.1) tagged with N-terminal 6xHIS-tag was transformed into BL21 (DE3) competent cells (Agilent Technologies). One litre LB-ampicillin cultures were induced to express recombinant protein by addition of 1M isopropyl β-D-1-thiogalactopyranoside (IPTG). After 20 h of incubation at 30°C, cells were pelleted and resuspended in 50 mL of lysis buffer (20 mM Tris-HCI pH 7.5, 500 mM NaCI, 30 mM imidazole and 2 mM β-mercaptoethanol) with one tablet of Sigma-Fast protease inhibitors (Sigma-Aldrich). Cell pellets were further lysed by sonication and proteins were purified using His-tag affinity chromatography (HisTrap HP-1 mL column, GE healthcare). The cTXNPx peak fractions were pooled and concentrated to approximately 1 mg/mL as determined spectrophotometrically using a NanoDrop at 280 nm.

Chalcone and recombinant cTXNPx interaction by SDS-PAGE, LC-MS/MS and TSA

For SDS-PAGE analyses, cTXNPx (50μM) was incubated with different concentrations of compound 1c (5, 50, 500 μM) for 1 h, followed by incubation with compound 2 (50 μM) for an additional 1 h. The protein was then submitted to a click reaction with compound 4 (50 μM) as described above and analysed by SDS-PAGE.

For liquid chromatography–mass spectrometry (LC–MS/MS), cTXNPx (1 mg / mL) was incubated with compound 1c or 3 (1:1) and desalted using Waters MassPrep desalting cartridge. Then, intact proteins were analysed in UPLC system in tandem to LCT premier Acquity (Waters), as described above. Mass operating range was 100–2000 units, protein data was processed using Masslynx 4.1 and deconvoluted using MaxEnt 1 to show the nominal neutral mass of the protein. For thermal shift assay (TSA), 10 μL of cTXNPx (1 mg / mL) in 20 mM HEPES buffer, pH 6.9, 50 mM NaCl and 5 mM DTT were incubated with compound 1c or 3 at different ratios for 30 min. Then, proteins were transferred into 48-well PCR plates containing 10 μL of SYPRO Orange in water. Fluorescence data were collected using SYPRO Orange wavelengths, 472 / 570 nm on a StepOne qPCR machine (Thermo Fisher Scientific). Raw data was analyzed using NAMI software and Protein Thermal Shift software 1.3 (ThermoFisher Scientific) to calculate the derivative curve determined Tm values and to compare ΔTm values of different ligands [23, 24].

Molecular modeling

In the absence of a three-dimensional X-ray crystal structure of cTXNPx from L. amazonensis (LaTXNPx), homology modeling was employed based on the L. major crystal structure of the TXNPx homodecamer [25]. The two proteins are highly homologous with a sequence identity of 89%. The template search, selection, model building and quality estimation were conducted altogether with the SWISS-MODEL server [26].

To investigate the binding mode of compound 1a, 1b, 1c and 3, respectively within LaTXNPx, we performed molecular docking studies using the flexible docking approach GOLD [27]. First, the three-dimensional structures of ligands were built and optimized using the Molecular Mechanics Force Field (MMFF)[28] on Spartan’18 software (Wavefunction Inc, 2012) [29]. The protein model was prepared with GOLD with all H-atoms added and charges balanced. The search space was optimized to a radius of 10 A around the peroxidatic cysteine residue Cys 52. The docking process involved high accuracy settings first with GoldScore and rescoring with ChemScore, 30 genetic algorithm runs, and high ligand flexibility. The range of ChemScore fitness was 1.6 to 19.3 (1a),1.9 to 14.6 (1b), -1.6 to 17.4 (1c) and -14.4 to 14.8 (3). The best binding pose was identified as the solution with the highest ChemScore fitness value. All figures were created with PyMol (Schrodinger, LCC, 2010, The PyMol Molecular Graphics System, Version 2.1).

CRISPR-Cas9 cTXNPx gene knockouts

cTXNPx genes deletion was performed as described in Beneke et al [30]. The online primer design tool www.LeishGEdit.net was used to design primers for amplification of the 5′ and 3′ sgRNA templates and for amplification of donor DNA from pT plasmids (S3 Table). L. mexicana Cas9 T7 promastigotes [30] (1 x 107/mL), previously kept in the presence of nourseothricin sulphate (50 μg/mL) and hygromycin B (32 μg/mL), were transfected with 10 μg of each respective donor DNA and sgRNAs using 2 mm gap cuvettes (MBP) with program X-001 of the Amaxa Nucleofector IIb (Lonza Cologne AG, Germany).

Following transfection, parasites were transferred into 5 mL prewarmed medium in 25 cm2 flasks and left to recover overnight at 26°C. Then limiting dilution, in the presence of appropriate selection drugs, was used to generate clonal tagged cell lines. First, KO parasites for both copies of each gene were selected using different markers, LmxM.15.1040 KO (Δ1040) was selected with blasticidin-S deaminase (10 μg/mL), while LmxM.15.1160 KO (Δ1160) selected with puromycin dihydrochloride (20 μg/mL). Survival of selected transfectants became apparent 7–10 days after transfection. Deletion of both copies for each gene was confirmed by PCR (S9 Fig). Using the Δ1160 cell line, the remaining gene (LmxM.15.1040) was excluded through a new round of transfection using a neomycin resistance gene as the repair cassette. The final cell line, cTXNPx KO (ΔcTXNPx) was selected with G-418 disulfate (40 μg/mL).

Diagnostic PCR for knockout verification

Genomic DNA of drug-selected clones was isolated with Wizard Genomic DNA Purification Kit (Promega). To assess the loss of the target gene ORF and the presence of donor DNA in putative KO lines and the parental cell lines, a diagnostic PCR was performed using GoTaq G2 DNA Polymerase (Promega), within 100ng of gDNA as template and forward primer and reverse primer for each ORF or resistance gene.

The PCR conditions were 2 min at 95°C followed by 35 cycles of 30 s at 95°C, 45 s at 60°C, 1 min 72°C, and a final extension step of 5 min at 72°C. The presence of expected amplicon was confirmed by 1% agarose gel. Primer sequences are detailed in Supplementary Materials in S4 Table.

Mutant cell growth

Promastigotes of L. mexicana T7 Cas9 (Parental), Δ1040, Δ1160 and ΔcTXNPx, at the second passage, were cultured at 26°C in Schneider’s medium supplemented with 10% HIFBS, and the number of parasites was counted daily for four days using a Neubauer chamber. On day 3 of culture, parasite aliquots were placed onto microscope slides and cells imaged using fluorescence microscope (Nikon-EclipseTi) with a CFI Plan Apo Lambda 100X Oil objective and differential interference contrast (DIC) module.

Statistical analysis

Single-variance analysis (ANOVA) and t-test were used to compare differences between group samples followed by Dunett post-test, for comparisons of all groups to control group. For multiple comparison the post-test used was Bonferroni. All statistical analyses were performed using GraphPad Prism 7 software and differences were considered significant when p ≤ 0.05, in a number of at least 3 independent experiments.

Results

Chemical tools for chalcone target identification

Whilst CH8 (1b; Fig 1) showed good activity, drug development was challenged by a difficult synthesis in which flavone formation accompanied aldol condensation. Consequently, a trimethoxylated analogue NAT22 (1c; Fig 1), that could be prepared far more efficiently through simple aldol condensation of the commercially available trimethoxyacetophenone with 3-nitrobenzaldehyde, was used as the parent chalcone [31]. Importantly, this showed similar anti-leishmanial activity to chalcone CH8 (1b) in vitro and in vivo against L. amazonensis [31].

The highly electrophilic enone functional group within CH8 was predicted to be key to activity [32]. This hypothesis was verified by the lack of a significant antileishmanial effect of dehydrochalcone analogue (3) (Fig 2A). To further investigate the mode of action, chalcone 1c was converted into an activity-based probe 2 (S1 Fig). Briefly, commencing from commercially available 2,6-dimethoxyacetophenone, regioselective tandem Ir-catalysed C-H borylation—oxone oxidation followed by alkylation with 6-bromohex-1-yne and aldol condensation afforded the alkyne tagged chalcone probe 2. This modification did not alter the intrinsic chalcone activity against the promastigote parasite, with EC50 values (L. amazonensis 0.42 μM, L. braziliensis 1.45 μM and L. infantum 0.78 μM) comparable to compound 1c (Fig 2A–2C).

Fig 2

In vitro and in vivo antileishmanial activity.

(A) In vitro activity: Promastigotes of the indicated Leishmania species; intracellular amastigotes of L. amazonensis, and uninfected macrophages were incubated with different concentrations of 1c, 2 and 3 for 48 h. Then, cell viability was assessed as described in M&M for determination of EC50 and CC50 for parasites and macrophages, respectively. In vivo efficacy: Mice were infected in the ear with L. amazonensis. Seven days after infection, were given chalcone 1c (40 mg/Kg) or vehicle alone (100 μL of propylene glycol) daily for 3 weeks by intragastric gavage. (B) Ear thicknesses were measured in the indicated days with a digital caliper. Lesion sizes were the difference between infected and noninfected ears in each time point. (C) Ear parasite loads were determined by limiting dilution assay. Means ± SD (n = 5 animals/ group). *P < 0·05 in relation to Vehicle. NT- Not tested.

To interrogate the mechanism(s) by which chalcone 1c is able to kill Leishmania spp., live parasites were incubated with compound 2 (5 μM) and, following conjugation with the trifunctional reagent 4 [33, 34], visualized by confocal microscopy (Fig 3A). The images obtained indicated that the chalcone is widely dispersed in the parasite but is excluded from the nucleus. To further investigate this localisation, promastigotes were lysed, and the soluble protein fraction treated with 2 and then 4, as above. SDS-PAGE analysis revealed a fluorescent protein band at about 22 kDa (Fig 3B). Competitive dosing with chalcone 1c led to a reduction in labelling of this band in concentration-dependent manner (Figs 3C and S2). The labelled band was excised from the gel, digested and the tryptic peptides analysed by LC–MS/MS. The comparison of these peptide sequences with the reported genome for L. amazonensis [35], revealed 10 candidate proteins (S3 Fig and S1 Table). Comparison of molecular masses for these suggested that cytosolic tryparedoxin peroxidase (cTXNPx, NCBI entry: AAX47428.1) as the predominant chalcone target (Figs 3D and S3). Two-dimensional gel analysis followed by mass spectrometry (S4 Fig and S2 Table) and immunoblotting analysis with anti-cTXNPx which also indicated that cTXNPx was also labelled by chalcone 2 in intracellular amastigotes with no signal being observed in uninfected macrophage samples (S5 Fig).

Fig 3

Intracellular probe binding and identification of cytosolic tryparedoxin peroxidase as protein target.

(A) Confocal microscopy of L. amazonensis promastigotes treated (a5-a8) or not (a1-a4) with 2 (5 uM) for 5 h prior to labelling with red fluorescent probe 4 (5uM) by CuAAC. (B) In gel fluorescence, detection of proteins from L. amazonensis promastigote lysate pre-treated with 2 (5uM) for 4 h and linked to molecule 4 by CuAAC. White rectangle- fluorescent band at around 22 kDa. (C) In gel representative fluorescent image of competition assay between molecule 1c and 2. Promastigotes were incubated with molecule 1c (1, 3, 9 and 27 μM) for 4 h, washed, and incubated with 2 (1 μM) for additional 4 h. Parasites were lysed, and the lysates were incubated with 4 prior to SDS-PAGE (35 μg / lane).Qualitative decrease in fluorescence signal by addition of increasing amounts of 1c. (D) Identification of cytosolic tryparedoxin peroxidase (cTXNPx) as a best candidate by mass spectrometry analysis of peptides from excised 22kDa band, by ESI-Q-TOF.

Chalcones can form a strong and stable interaction with cTXNPx

To further explore the interaction between cTXNPx and antileishmanial chalcones, L. amazonensis cTXNPx (LaTXNPx) was expressed recombinantly in E. coli, purified and subsequently incubated with 2. SDS PAGE analysis following cycloaddition reaction with 4 (50 μM) showed a labelled band at 24 kDa (Fig 4A, L3). As observed for the live parasites, incubation with 1c inhibited the binding of 2 and the subsequent fluorescent signal of this band in a concentration dependent manner (Fig 4A). Thermal shift assay (TSA) revealed that binding of 1c led to a reduction in Tm in a dose dependent manner (Fig 4B) indicative of a significant degree of destabilisation of protein structure [36]. Mass spectrometric analysis of cTXNPx treated with chalcone 1c revealed a new peak at 24368 Da [24025 + 343] (Fig 4C and 4D), corresponding to a stable protein chalcone adduct that suggested that this interaction is based on a strong and stable interaction, very likely a covalent bond. Similar observations could be made using recombinant cTXNPx from L. major (LmTXNPx) incubated with compound 1c (S6 Fig). The degree of labelling is only partial and attempts to drive this to higher levels through longer incubation times were not successful. Evidence that this interaction is necessarily dependent on the enone group, was demonstrated through the use of dehydrochalcone 3. Using the same sequence of experiments, this compound, even at high concentrations (180 μM—4:1 (ligand:protein)) was not able to affect protein Tm values (Fig 4B) or lead to any evidence for a labelled protein by mass spectrometric analysis (Fig 4E).

Fig 4

Chalcone impairs the cTXNPx antioxidant effect in the parasite by a stable interaction mediated by enone group.

(A) Competition binding assay between molecules 1c and 2 employing recombinant cTXNPx. Recombinant L. amazonensis cTXNPx was incubated with the indicated concentrations of 1c for 60 min, then washed, incubated with 2 (50 μM), and linked to 4 by CuAAC. Representative fluorescence gel images signals of 4. (B) protein melting curve from thermal shift assay of cTXNPx protein incubated with the indicated concentrations of 1c (brown) or 3 (blue). (C-E) MS analysis of interaction between chalcone and cTXNPx. Purified proteins (0.6 mg/ mL) were incubated or not with compound 1c or 3 (1:1) for 30 min and their molecular weight analysed by MS. (C) cTXNPx of L. amazonensis [M-Met requires 24025 Da]. (D) cTXNPx of L. amazonensis plus 1 [M-Met+343 requires 24368 Da]. (E) cTXNPx of L. major plus 3 [M-2H-Met requires 24136 Da]. Means ± SD (n = 3 independent experiments). * p<0.05 in relation to untreated cells.

Chalcone 1c leads to ROS accumulation in parasites

cTXNPx belongs to the highly-conserved 2-cysteine peroxiredoxins that have a key role in the protection of the cell from hydroperoxides and peroxynitrites [25, 37]. Collectively, our results suggested that, in Leishmania parasites, compound 1c binds to and inhibits cTXNPx, leading to ROS accumulation and cell death. To confirm this hypothesis, L. amazonensis promastigotes were incubated with 1c or 3, and ROS production measured by fluorimetry using H2DCFDA (2,7-dichlorodihydrofluorescein diacetate) (S7A and S7B Fig) [38]. Compound 1c led to a decrease in the number of parasites in a concentration dependent fashion with parasite killing being accompanied by ROS accumulation (S7A Fig). In contrast, the dihydrochalcone 3, resulted in a lower parasite killing and, even at higher concentrations, no significant increase in ROS accumulation (S7B Fig). Similarly, in infected, but not in uninfected, macrophages 1c was able to induce ROS accumulation whilst 3 was unable to affect ROS levels in either infected or uninfected macrophages (S7C and S7D Fig). Importantly, the lack of a response on uninfected macrophages to treatment with 1c or 3 indicated that the increased ROS levels were attributable to intracellular amastigotes (S7D Fig).

Chalcones bind in the cTXNPx active site

To better understand the molecular mechanism of the interaction between the anti-leishmanial chalcones and cTXNPx, we examined the interaction by molecular modelling. Using the amino-acid sequence of LaTXNPx (UniProtKB database, access code Q4VKK8) [39, 40] and the crystal structure of LmTXNPx [41], which have 89% sequence identity, a homology model was built using the SWISS-MODEL server.

In common with most peroxiredoxins, this LaTXNPx model was obtained in the fully folded (FF) conformation as a homodecamer (Fig 5A) that can also be described as a pentamer of dimers (Fig 5B). The active site is formed by the N-proximal peroxidatic cysteine (Cys52) [42] from one subunit and a C-proximal cysteine from the two-fold symmetry related subunit (Cys173). These residues form a disulfide bridge in the oxidized state [43]. It is important to note that all crystal structures, as well as the homology model, are in the reduced form where the two cysteine residues are approximately 10 Å apart. Docking studies on the reduced homodimer, focussing on the critical Cys52, were performed to investigate possible binding modes of the chalcones. The dimer interface exhibits a narrow groove at the end of the helix harbouring the active Cys52 where all chalcones can bind as shown for 1a in Fig 5C. The chalcones 1b and 1c (Fig 5D and 5E) showed a similar binding pose with the phenyl and nitro-phenyl group on the same side of the tight groove where the enone moiety is located, approximately 7.7 Å from the sulphur atom of Cys52. Thus, only a relatively small rearrangement is required to allow the formation of the covalent adduct. This may explain why only partial covalent protein modification is observed. While compound 3 can also occupy the same pocket as shown in Fig 5F, its binding poses show significantly lower docking scores compared to the chalcones and the conformation is clearly more strained.

Fig 5

LaTXNPx 3D model and 3D/2D binding mode of 1c and 3.

(A) Ribbon diagram of the cTXNPx homology model. Each chain of the homodecamer is depicted in a single colour. The decamer can be described of pentamers of functional homodimers. (B) Ribbon diagram of one dimer with each chain depicted in green and cyan, respectively. The key cysteine residues 52 and 173 are depicted in CPK representation with C-atoms on grey, O-atoms in red, N-atoms in blue and S-atoms in yellow. Note, that the cysteines are present in their reduced forms. H-atoms are omitted for clarity. (C) Close-up of the proposed binding site with the highest scoring binding pose of compound 1a depicted in a stick-representation with C-atoms in grey and O-atoms in red. (D) highest scoring binding pose of 1b (E) 1c (F) 3.

Chalcone antipromastigote activity is dependent on cTXNPx

In order to confirm the cTXNPx enzyme as a chalcone target as well as to examine its importance for the parasite biology, we generated parasites lacking cTXNPx genes. Due to the availability of a well described and utilised CRISPR-Cas9 system giving a straightforward way to delete genes in L. mexicana, we choose this platform to explore these questions [30, 44].

cTXNPx is encoded by two genes (LmxM.15.1040 and LmxM.15.1160), located at chromosome 15, sharing 98% and 97% of similarity at the gene and protein level respectively (S8 Fig). To analyse the potential role of each in parasite fitness and chalcone sensitivity, using sgRNAs specific for the flanking regions (UTRs) we generated parasites with each gene knockout out separately. In this experiment LmxM.15.1040 alleles were replaced by a repair cassette encoding the blasticidin resistance maker (Δ1040) and, separately, LmxM.15.1160 by the puromycin resistance marker (Δ1160). Knockouts for each were selected following cloning and PCR analyses (S9 Fig). Subsequently, using the same approach, commencing from the Δ1160 cell-line, the remaining gene was replaced by a sequence encoding neomycin resistance to generate a line in which both LmxM.15.1040 and LmxM.15.1160 were ablated in this diploid parasite (ΔcTXNPx) (Fig 6A and 6B). The correct integration of the puromycin, blasticidin and neomycin-resistance markers in the cTXNPx locus, and the complete loss of cTXNPx, were confirmed by PCR analyses as shown in Figs 6B and S9. No growth defect or morphological alterations, in comparison to the parental line, were observed when only one cTXNPx gene (Δ1040 or Δ1160) was deleted (Fig 6C and 6D). However, the absence of both genes (ΔcTXNPx) caused a severe alteration in parasite growth in comparison to the other lines (Fig 6C) and significant phenotypic alterations when observed by optical microscopy (rounded cell body and short flagellum; Fig 6D).

Fig 6

Chalcone antiparasitic activity is partially dependent on cTXNPx.

(A) Diagrams showing the PCR strategy to confirm the replacement of LmxM.15.1040 with the neomycin (NEO) cassette in the LmxM.15.1160 double KO (puromycin [PUR] cassette; S9 Fig) to create ΔcTXNPx. (B) PCR analysis of the ΔcTXNPx cell line, PCR products visualized on agarose gels. Coding sequence for LmxM.15.1040 (CDS, grey arrow), puromycin (PUR, blue arrow) and neomycin (NEO, red arrow) in Parental and ΔcTXNPx cells. (C) Promastigotes were seeded at 1x105 parasites/mL and the number of parasites in culture were counted every day for 4 days using a Neubauer hemocytometer in triplicate from two independent experiments. Cell lines: Parental (black line), LmxM.15.1040 double KO (green line), LmxM.15.1160 double KO (blue line), and cTXNPx KO (red line) (D) Representative Differential interference contrast micrographs showing all cTXNPx mutant cell lines analysed in C and E. Scale bar, 5 μm. (E) cTXNPx mutant promastigotes (2x106/mL) were incubated with different concentrations of 1c for 48h. Cell viability was then assessed by the addition of resazurin solution for 4 h and EC50 values calculated by non-linear regression. Means ± SD (n = 3 independent experiments).

To investigate if the specific cTXNPx enzyme is the chalcone target in cellulo, promastigotes of parental and mutant cell lines (Δ1040, Δ1160 and ΔcTXNPx) were incubated with different concentrations of compound 1c for 48 h when the parasite viability was assessed using alamarBlue (Fig 6E). As expected, compound 1c showed a good activity against the parental line (EC50 1.04 μM), similar to the values found against other parasite species (Fig 2B). The lack of one of Δ1040 or Δ1160 cell lines reduced the EC50 2 and 1.6-fold respectively (EC50 1.99 μM and 1.67 μM; Fig 6E). When both genes were deleted in ΔcTXNPx cell line, the parasite was 3.8-fold more resistant to the chalcone, with EC50 = 3.87 μM (Fig 6E). Together, these results indicate that cTXNPx is important for the parasite biology and also likely to represent a major target of antileishmanial chalcones.

Discussion

Conventional chemotherapy employed in leishmaniasis treatment relies on only a few drugs, all of which have limitations including cost, toxic side effects, low or variable efficacy that depends on parasite species, difficult modes of administration and growing parasite resistance [5]. This current scenario clearly points to the urgent demand for new treatments and innovative approaches to control this neglected tropical disease (NTD) which affects more than one million people worldwide per year [2]. Strategies for the discovery of new drug leads can broadly be partitioned into target-based and phenotypic approaches [45]. For NTDs such as leishmaniasis, the latter has become the favoured approach with high-content high-throughput screening studies being described enabling the broad diversity of structures found in small molecule and natural product collections to be explored without preconceptions [46, 47]. However, determination of the molecular target and mode of action is then required to facilitate further drug development [48-50].

Previously, we described the discovery and development of the synthetic nitro-chalcone 1b (CH8), to effectively treat both CL and VL in an animal model [13-16]. However, as with many such studies, despite excellent results against the parasite, the molecular target and mechanism of action important for the development of new chalcone leads remained unknown [9]. To address this, we have employed a chalcone derived activity-based chemical probe (ABPP) based approach, to identify cytosolic tryparedoxin peroxidase (cTXNPx) as a target of this compound. Whilst ABPP has been employed quite widely in cell biology, application to Leishmania spp. is limited [51-57]. Similarly, whilst many bioactive natural products have been adapted for use as ABPP agents to identify and validate new drug targets [58], despite being intrinsically electrophilic and therefore able to label a protein target, the use of chalcones in this context is surprisingly limited [32, 59].

Belonging to the ubiquitous 2-Cys family of antioxidant enzymes that are highly conserved among trypanosomatids with no direct mammalian orthologue [60], the tryparedoxin peroxidases have key roles in parasite survival and virulence [37, 61]. Their primary function is to reduce reactive oxygen species (ROS), most notably generated in the oxidative burst that follows parasite internalisation by macrophages. The ultimate electron donor in this cycle is tryparedoxin which distinguishes Leishmania, and other kinetoplastids, from most other eukaryotes, including the mammalian host, where thioredoxin is employed. As such, TXNPx provides an essential mechanism for trypanosomatids to survive under these conditions of biotic stress [62, 63]. Moreover, TXNPx has been shown to be a major contributor to the growth of resistance to many of the existing drugs [64, 65], with over-expression in L. donovani and L. amazonensis providing protection against oxidative stress from macrophages as indicated by increased parasite infectivity [37, 66].

Reflecting this unique and species-specific role, TXNPx may be considered a potential drug target [67]. Significantly this hypothesis is supported by our results in which genes encoding cTXNPx were deleted, leading to parasites with a compromised phenotype. Moreover, the lower efficacy of the chalcone in these mutants clearly demonstrated the involvement of cTXNPx in the antileishmanial activity of these nitro chalcones. However, the fact that significant activity remains suggests that the chalcones have multiple modes of action that are working in concert. Significantly, in this context it is pertinent to note that Leishmania spp. possess two distinct tryparedoxin peroxidases, a mitochondrial and cytosolic form, sharing approximately 50% similarity at protein level and with a conserved tertiary structure (S8 Fig) [61]. Interaction of the chalcones with the mitochondrial tryparedoxin peroxidase would be consistent with the earlier reports of mitochondrial alterations on treatment with DMC (1a) and cannot be ruled out at this stage [11]. The fact that no labelling of this second tryparedoxin peroxidase was detected in our experiments may simply reflect its lower cellular abundance and the low levels of protein alkylation observed (Fig 4D) in our experiments.

The pull-down experiment reported here is consistent with a model in which the nitro chalcone and cTXNPx interplay occurs through a covalent bond, potentially the Michael addition of a cysteine thiol with the electrophilic enone group [32, 68]. Similar mechanisms of inhibition of thioredoxin reductases by chalcones [69] and other Michael acceptors, such as curcumins, cinnamaldehydes, and fungal metabolites [70-72] have been described. This proposal is supported by our studies with compound 3 where the lack of the α,β-unsaturation resulted in loss of chalcone interaction with cTXNPx protein in vitro and in situ, and consequently low antiprotozoal efficacy. However, despite the ability to isolate cTXNPx by pull-down, the labelling of the protein is not complete, and we were not able to identify the specific amino acid residues involved in this process. We suggest that this partial labelling is indicative of a two-stage process involving initial binding of the chalcone into active site followed by a slower non-specific alkylation event, potentially following disruption of the critical active dimer.

Support for this pathway comes from our molecular modelling and docking studies which reveal how 1c can access the enzyme active site, fitting between Cys-52 and Cys-173, and block disulfide bond formation. This orientation of 1c places the electrophilic carbon of the enone in close proximity (8.0 Å) to the reactive Cys-52 residue. Monitoring this interaction by thermal shift assay (TSA) revealed that binding of 1c leads to a lower melting temperature, suggesting that binding was accompanied by a partial unfolding of the protein [36, 73]. Again, this proposal is supported by the observation that the ‘inactive’ dihydro analogue 3 exhibited less favourable binding in the docking studies and did not affect protein stability. Consequently, whilst the α,β-unsaturated ketone is essential for ABPP activity, this suggests that the double bond has a conformational role in inhibition of enzyme activity. Importantly, this implies that alternative biosteres can be designed to avoid the potential problems associated with the generic electrophilicity and potential polypharmacology of the chalcone template.

Based on these findings, we propose a mechanism of action for the nitrochalcones. Chalcone 1c is taken up by the macrophage and is able to access the amastigotes. Here, 1c interacts with cTXNPx, enzyme recycling is impaired and ROS levels, especially H2O2 and ONOO−, rise within the amastigote leading to apoptosis-like cell death due to oxidative damage to lipids, proteins and nucleic acids [74-76]. In support of this hypothesis, many indications of apoptosis-like cell death, such as mitochondrial damage [11], DNA fragmentation and other morphological changes have been observed in Leishmania spp. on chalcone treatment.

Collectively, these results demonstrate that the nitrochalcone 1c interacts with cTXNPx leading to protein deformation and inactivation and ultimately parasite death due to cellular damage by non-scavenged ROS. Whilst many of the existing antileishmanial drugs, like pentavalent antimonials, also lead to elevated levels of ROS these effects are non-specific and contribute to the off-target toxicity associated with their use. Moreover, resistance to these chemotherapies is rising and this can be associated with increased tolerance to ROS. It is therefore significant that nitrochalcone 1c targets a parasite specific protein that contributes to this defence mechanism. As such 1c not only represents a valuable new drug lead, but also potentially enables the design of combination therapies with the added benefits of reducing therapeutic dose and related toxic effects, and retarding resistance mechanisms [77, 78]. Studies towards this goal are in progress and will reported in due course.

Conclusion

In conclusion, this work validates TXNPx as a much-needed drug target for the treatment of leishmaniasis. Moreover, the chalcone 1c represents a well-defined structural starting point in the search for new antileishmanials. As such, this work opens a new perspective in the search for treatments for leishmaniasis, an important but neglected tropical disease which impacts millions of people worldwide and has a high economic and social impact.

Synthetic routes for preparation of compounds 2–3.

(TIF)

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Uncropped SDS-PAGEs corresponding to Fig 3C.

(ai) SDS-PAGE of all tested conditions stained with comassie blue. (aii) Fluorescence image of all tested conditions.

(TIF)

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Uncropped SDS-PAGEs corresponding to Fig 3D.

(a) SDS-PAGE stained with Coomassie blue. (b) In-gel fluorescence image. Yellow rectangles- Protein bands excised for mass spectrometry analysis. (c) Proteins candidates identified from the band.

(TIF)

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Confirmation of cTXNPx as a chalcone target by two-dimensional gel analysis.

(a) Map of the dots excised from the 2D gel stained with comassie blue and submitted to mass spectrometry analysis. (b) In-gel fluorescence detection image. (c) Proteins candidates identified for each 2D dot.

(TIF)

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Immunoblotting analysis of chalcone labelling on cTXNPx in different parasite stages.

Proteins from promastigotes (Pro), isolated intracellular amastigotes (Ama), and macrophage (Mac) lysates that had been pre-incubated with 2 (5 μM) and linked to 4 (5 μM) prior to fluorescence revelation. Alternatively, the run lysates were revealed with mouse anti-cTXNPx antibody followed by anti-Mouse IgG-HRP (anti- cTXNPx). a. In-gel fluorescence detection image. b. Immunoblotting analysis with anti-cTXNPx. Proteins from promastigotes (P1), amastigotes (A1) and macrophages (M1) without 2. Proteins from promastigotes (P2), amastigotes (A2) and macrophages (M2) pre-incubated with 2.

(TIF)

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Mass spectrometry analysis of interaction between chalcone and Lm-cTXNPx.

Purified proteins (0.6 mg/ mL) were incubated or not with compound 1c (1:1) for 30 min and their molecular weight analysed by Mass Spectrometry. (a) cTXNPx of L. major molecular weight [M-Met requires 24138 Da]. (b) cTXNPx of L. major with 1 [M-Met+343 requires 24481 Da].

(TIF)

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Inhibition of cTXNPx by chalcone impairs the enzyme antioxidant effect in the parasite.

L. amazonensis promastigotes (1 x 106/mL) were incubated with compounds 1c or 3 at the indicated concentrations for 48 h, during which the numbers of cells were recorded. Then, 4 x 105 cells were transferred to black 96 well plates for fluorimetric assessment of ROS production with H2DCFDA (20 μM) for 30 min. (a-b) Parasite killing (dotted lines) and ROS production (continuous lines) induced by 1c (a) 3 (b). BMDM (1 x 105/well) infected or not with L. amazonensis promastigotes (10:1) for 72 h on 96 well-plate, then cells were incubated with 1c or 3 (0.07, 0.7 and 7.0 μM) for 1 h at 37°C and ROS production measured as described for promastigotes. (c) ROS production on infected macrophages. (d) ROS production on uninfected macrophages. Means ± SD (n = 3). * p<0.05 in relation to untreated cells.

(TIF)

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TXNPx sequence alignment.

Gene and protein sequences of mitochondrial (LmxM.23.0040) and cytosolic (LmxM.15.1040 and LmxM.15.1160) TXNPx were obtained from https://tritrypdb.org website and aligned using Jalview software (2.11.1.3). (a) Gene sequence alignment (b) Protein sequence alignment.

(TIF)

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Screening of KO clones for each gene.

(a) Diagrams showing PCR strategy for assessing presence of gene copies for LmxM.15.1040 and LmxM.15.1160 and their replacement with the blasticidin [BLA] or puromycin [PUR] cassette. (b) PCR of clonal lines following transfection with sgRNA targeting LmxM.15.1040 gene with BLA as the repair cassette. Yellow star highlights clonal double KO of LmxM.15.1040 (left; green arrow BLA and grey arrow LmxM.15.1040 CDS) and the retention of LmxM.15.1160 (right; yellow arrow LmxM.15.1160 CDS). (c) PCR of clonal lines following transfection with sgRNA targeting LmxM.15.1160 with PUR as the repair cassette. White star highlights clonal double KO of LmxM.15.1160 (right; blue arrow PUR and yellow arrow LmxM.15.1160 CDS) and the retention of LmxM.15.1040 (grey arrow LmxM.15.1040 CDS). P—positive control. Pop—population.

(TIF)

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Proteins candidates identified from the band on 1D-gel in S3 Fig.

(XLSX)

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Proteins candidates identified for each 2D dot from S4 Fig.

(XLSX)

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Primers used to knockout cTXNPx in L. mexicana using pT plasmids.

(XLSX)

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Primers used for the validation of cTXNPx knockouts.

(XLSX)

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Synthetic procedures.

(DOCX)

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10 Aug 2021

Dear Prof Steel,

Thank you very much for submitting your manuscript "Chalcones Identify cTXNPx as a Potential Antileishmanial Drug Target" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Tiago Donatelli Serafim

Associate Editor

PLOS Neglected Tropical Diseases

Laura-Isobel McCall

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods described in this study were appropriated and adequately described in the manuscript. The samples size (particularly for in vivo essays) was adequate and answered the hypothesis of the authors. Finally, there is no concern about ethical requirement.

Some minor points:

- In the Anti-promastigote activity, please include the growth phase of parasites used in the assays (page 6, lines 111-120).

- Please, indicate the number of mice used for in vivo experiment, using chalcone 1c (page 6, lines 148-160).

- Table S2, Neo-forward primer is missing in the Table.

Reviewer #2: The authors clearly designed a methodology that attended the main questions regarding the purpose of the work: the elucidation of a mechanism of action of chalcones that results in their antileishmanial effect. The in vivo analyses were made under ethic procedures validated by the ethic committee, as described in the methodology. At least 3 independent experiments were performed and the statistical analysis is proper, allowing to address the objectives. The use of competition assay, ABPP, and Leishmania cTXNPx knockouts supported by docking analysis were very important to confirm the hypothesis that TXNPx is a potential target for chalcones in the treatment of leishmaniasis.

Reviewer #3: Methods are sound and well explained. There is only one points I would like the authors to address: For mice infections and treatment, the authors must add to the method section who many animals were used per group/experiment, how many times the experiments were repeated and which statistical methods were used to decide the number of animals used to achieve statistical significance.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results are well presented throughout the manuscript. The figures, in general, are clear and have good and enough quality for the reader.

Some minor points:

- Figure 2A is a Table, so please replace it for a Table in the final version of the manuscript (page 27).

- Figure 2C, include the standard deviation of the limiting dilution assay (vehicle group) (page 27).

- Figures S3-C and S4-C should also be replaced by supplementary Tables.

Reviewer #2: The results are very clear and describe efficiently the analysis plan. The figures are sufficiently clear and well delineated.

Reviewer #3: Probe displacement assay - Are 2 and 4 permeable to life cells? If so, I would suggest to show the dose dependent probe displacement assay in live cells to prove target engagement of cTXNPx by 1c, and for future campaigns for the target. The assay will also allow for further calculations of inhibition as Ki and Kd of the compounds. The assay could be anyway developed for the purified recombinant protein to gain information on the kinetics of association/dissociation of the compound with the target. Although the gel-based assay is informative, it is hardly quantitative and is limited to the target binding.

Essentiality - The authors had shown that compound 1c binds to cTXNPx and defend it to be the molecular target responsible for its toxicity to Leishmania. These results go into conflict with the data showing that the genes composing the complex are dispensable in promastigotes. Also, the authors did not provide any evidence of the requirement of the genes to amastigotes. I highly recommend for the mutants to be also investigated as amastigotes in macrophages and/or mice infections.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions of the manuscript are clear and well supported by the results.

Reviewer #2: The authors conclude that the work validates TXNPx as a much-needed drug target for the treatment of leishmaniasis. Really, the current treatment possibilities have limitations, such as high prices, toxicity, and parasitic resistance. An important observation that could be better used to reinforce the importance of this work is that tryparedoxin peroxidases, such as TXNPx, are highly conserved among trypanosomatids, without direct mammalian orthologue. This suggests that chalcones could be a leishmanicidal drug with a low possibility of side effects in mammals.

As commented in the text, leishmaniasis is a disease that impacts millions of people worldwide and has a high economic and social impact. The public health relevance is adressed, considering the problems related to existing drugs and the fact that leishmaniasis are neglected diseases that can be disfiguring or fatal, depending on the clinical form presented.

The authors seem quite sure of the limitations of their analyses, and do not jump to conclusions about their findings.

Reviewer #3: Mode of action proposed - The authors discuss the role of cTXNPx in ROS resistance and suggest its inhibition as the mode of action for 1c, but do not provide any evidence for increase of ROS mediated cell death after treatment. These are well stablished assays that should be performed by the authors for better understanding and support of the mode of action here proposed.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Minor issues:

1. Line #38: “Instituo” – Instituto;

2. Line #86: The period is missing after “antileishmanials”;

3. Line #87: Remove the comma;

4. Lines #92-93: “...which showed strong and selective anti-parasite activity in vitro and in vivo against both CL and VL”. Replace CL and VL with the Leishmania species;

5. Line #165: cite the permeabilizing agent used;

6. Line #309: “Single-variance analysis (ANOVA) and t-test was used...” – Single-variance analysis (ANOVA) and t-test were used...;

7. Line #318: “Whilst CH8 (1b; Fig 1) showed good activity”. What good activity? Against what? Same for the line #327;

8. Line #934: Change Leishmania to Leishmania in italic.

- Revise the English with a mother tongue specialist.

- In general, the text has to be revised considering spacing of characters, punctuation, and some other spelling details. For example:

. Line #111: “5 x 105/mL” instead of “5 x 105 / mL”;

. Line #114: “72 h” instead of “72h”;

. Line #122: “26 °C” instead of “26° C”;

. Line #155: “of growth [16].” instead of “of growth.[16]”;

. Line #181: “1 h” instead of “1 hr”;

. Line #190: “1 h at room temperature” instead of “1 hr at RT”;

. Line #574: “leishmaniasis” instead of “leishmanaisis” ...

Reviewer #3: Line 86 - Missing the period at the end of the phrase.

Line 148 - Please indicate the number of animals used for mouse infection and treatment/parasite burden. Please provide the statistical method used to determine the number of animals necessary to achieve a reliable assessment. Also, in Fig. 2C provide a figure with results for each mouse and the exact p value obtained for the assay. Also, does the result represent independent assays? Does n=5 mean 5 animals/assay?

Line 284 - Please correct prefix (LmxM) of gene ID for LmxM.15.1040 and LmxM.15.1160 (also in other parts of the text, as line 432).

Line 335 and Figure 2A - The authors mix the use of IC50 and EC50 for cellular based assays in the text and Figure 2A, as well as in other parts of the text in experiments that have a similar read-out. Please consolidate the use of EC50 to report results based on cell death response to compound treatment.

Line 346 - Please insert space after Fig 3C.

Line 451 - The statement ‘To investigate if the specific cTXNPx enzyme is the chalcone target in cellulo’ must be rethought and rephrased since the method used by the authors only assess the cell viability after treatment, not the target engagement.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In this manuscript, the authors described the activity in vitro and in vivo of a nitroanalogue (compound NAT22, 1c) against Leishmania species. This compound is a derivative of a natural chalcone with previously reported activity against Leishmania in vitro and in vivo. The authors also described that the cytosolic tryparedoxin peroxidase (cTXNPx) is the main target of this compound, leading to ROS accumulation and parasite death. These findings were confirmed by deletion of cTXNPx genes by CRISPR-Cas9 technology. Finally, molecular docking studies confirmed that the compound 1c is able to bind the putative active site of cTXNPx.

In general, the manuscript is clear and well written; however some points should be considered for publication.

Main points:

- Concerning to the diagnostic PCR for mutant cell lines of cTXNPx genes, did the authors only investigate for LmXm.15.1160 coding sequence, as indicated in the Figure 6A (right panel)? What about the other copy (LmXm.15.1040 gene)? In Table S2, it is indicated that there are specific primers for each one of these genes.

- Please, include in the Figure 6A, the PCR product expected for LmXm.15.1040 coding sequence.

- It is also missing the diagnostic PCR for ORF of cTXNPx genes in each one of mutant cell lines (Δ1040 and Δ1160), using parental line as control. Please include it in the manuscript.

- Were mutant cell lines (Δ1040 and Δ1160) generated by loss of heterozogosity? These mutant cell lines only have one drug resistance marker. Please specify this point in the manuscript.

- In the agarose gel for ΔcTXNPx line, it is missing the PCR for blasticidin S deaminase gene.

- Please, include standard deviation of EC50 values of the mutant cell lines (page 31, Fig. 6D).

- I would also suggest to present the graphical abstract as a Figure in the manuscript.

- Line 1012 – Specify TOC.

Reviewer #2: Overall, the work was very well designed, clearly written, and presents a novelty regarding the mechanism of action of a promising drug against leishmaniasis. Considering some suggested spelling corrections and the need to reinforce the issue of possible drug selectivity in the treatment of infection in mammals, which reinforces the importance of this work, it is able to be accepted for publication in PLOSNTD.

Reviewer #3: cTXNPx null mutant - I found the way that the diagnostic PCRs were shown misleading, and did not understand well the strategy. The final cell line contains 1 copy of LmxM.15.1040 deleted by BSDr gene, and 1 copy of LmxM.15.1160 by PACr gene? But how did the second copies of both genes were deleted by only NEOr in one go? Please clarify the strategy in the text and include in Fig 6A the agarose gels that show presence/absence of both CDS in each step of the genetic manipulation for better understanding.

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Reviewer #2: No

Reviewer #3: No

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13 Sep 2021

Submitted filename: Response_to_Referees_Escrivani_et_al.docx

Click here for additional data file.

26 Oct 2021

Dear Prof Steel,

We are pleased to inform you that your manuscript 'Chalcones Identify cTXNPx as a Potential Antileishmanial Drug Target' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Tiago Donatelli Serafim

Associate Editor

PLOS Neglected Tropical Diseases

Laura-Isobel McCall

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #3: The authors provided the information requested during revision.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #3: I am satisfied with the quality of figures and the description of the results, all of which match what is presented by the authors.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #3: The authors made the appropriate corrections in text and figures and addressed the concerns. Text is correct and addresses the advances to the field and public health relevance of the work.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #3: No modifications needed.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #3: No comments.

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Reviewer #3: No

7 Nov 2021

Dear Prof Steel,

We are delighted to inform you that your manuscript, "Chalcones Identify cTXNPx as a Potential Antileishmanial Drug Target," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases