Rational Design of Highly Selective Sialyllactose-Imprinted Nanogels.

We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE), to rapidly assess an affinity ranking. Finally, the best monomer 2-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant≈106 M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.

Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: Potential application in cancer and HIV-1 infection.

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure-activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-G-pseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Human carnosinases: A brief history, medicinal relevance, and in silico analyses.

Carnosine, an endogenous dipeptide, has been found to have a plethora of medicinal properties, such as antioxidant, antiageing, and chelating effects, but with one downside: a short half-life. Carnosinases and two hydrolytic enzymes, which remain enigmatic, are responsible for these features. Hence, here we emphasize why research is valuable for better understanding crucial concepts like ageing, neurodegradation, and cancerogenesis, given that inhibition of carnosinases might significantly prolong carnosine bioavailability and allow its further use in medicine. Herein, we explore the literature regarding carnosinases and present a short in silico analysis aimed at elucidating the possible recognition pattern between CN1 and its ligands.

Structure of human TRPM8 channel.

TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It is a therapeutic target for anticancer drug development, and its modulators can be utilized for several pathological conditions. Here, we present a cryo-electron microscopy structure of a human TRPM8 channel in the closed state that was solved at 2.7 Å resolution. Our structure comprises the most complete model of the N-terminal pre-melastatin homology region. We also visualized several lipids that are bound by the protein and modeled how the human channel interacts with icilin. Analyses of pore helices in available TRPM structures showed that all these structures can be grouped into different closed, desensitized and open state conformations based on the register of the pore helix S6 which positions particular amino acid residues at the channel constriction.

Chemoenzymatic approach towards the synthesis of the antitumor and antileishmanial marine metabolite (+)-Harzialactone A via the stereoselective, biocatalyzed reduction of a prochiral ketone.

As a rich source of biological active compounds, marine natural products have been increasingly screened as candidates for developing new drugs. Among the several marine products and metabolites, (+)-Harzialactone A has drawn considerable attention for its antitumor and antileishmanial activity. In this work a chemoenzymatic approach has been implemented for the preparation of the marine metabolite (+)-Harzialactone A. The synthesis involved a stereoselective, biocatalyzed reduction of the prochiral ketone 4-oxo-5-phenylpentanoic acid or the corresponding esters, all generated by chemical reactions. A collection of different promiscuous oxidoreductases (both wild-type and engineered) and diverse microorganism strains were investigated to mediate the bioconversions. After co-solvent and co-substrate investigation in order to enhance the bioreduction performance, T. molischiana in presence of NADES (choline hydrochloride-glucose) and ADH442 were identified as the most promising biocatalysts, allowing the obtainment of the (S)-enantiomer with excellent ee (97% to > 99% respectively) and good to excellent conversion (88% to 80% respectively). The successful attempt in this study provides a new chemoenzymatic approach for the synthesis of (+)-Harzialactone A.

Obesity and Type 2 Diabetes: Adiposopathy as a Triggering Factor and Therapeutic Options.

Obesity and type 2 diabetes (T2DM) are major public health concerns associated with serious morbidity and increased mortality. Both obesity and T2DM are strongly associated with adiposopathy, a term that describes the pathophysiological changes of the adipose tissue. In this review, we have highlighted adipose tissue dysfunction as a major factor in the etiology of these conditions since it promotes chronic inflammation, dysregulated glucose homeostasis, and impaired adipogenesis, leading to the accumulation of ectopic fat and insulin resistance. This dysfunctional state can be effectively ameliorated by the loss of at least 15% of body weight, that is correlated with better glycemic control, decreased likelihood of cardiometabolic disease, and an improvement in overall quality of life. Weight loss can be achieved through lifestyle modifications (healthy diet, regular physical activity) and pharmacotherapy. In this review, we summarized different effective management strategies to address weight loss, such as bariatric surgery and several classes of drugs, namely metformin, GLP-1 receptor agonists, amylin analogs, and SGLT2 inhibitors. These drugs act by targeting various mechanisms involved in the pathophysiology of obesity and T2DM, and they have been shown to induce significant weight loss and improve glycemic control in obese individuals with T2DM.

An integrated metabolomic and proteomic approach for the identification of covalent inhibitors of the main protease (Mpro) of SARS-COV-2 from crude natural extracts.

Mpro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen Mpro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many Mpro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant Mpro with natural extracts and identifying compounds adducted to the residues within the Mpro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of Mpro. Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 Mpro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process.

Discovery of a Potent and Highly Selective Dipeptidyl Peptidase IV and Carbonic Anhydrase Inhibitor as "Antidiabesity" Agents Based on Repurposing and Morphing of WB-4101.

The management of patients with type 2 diabetes mellitus (T2DM) is shifting from cardio-centric to weight-centric or, even better, adipose-centric treatments. Considering the downsides of multidrug therapies and the relevance of dipeptidyl peptidase IV (DPP IV) and carbonic anhydrases (CAs II and V) in T2DM and in the weight loss, we report a new class of multitarget ligands targeting the mentioned enzymes. We started from the known α1-AR inhibitor WB-4101, which was progressively modified through a tailored morphing strategy to optimize the potency of DPP IV and CAs while losing the adrenergic activity. The obtained compound 12 shows a satisfactory DPP IV inhibition with a good selectivity CA profile (DPP IV IC50: 0.0490 µM; CA II Ki 0.2615 µM; CA VA Ki 0.0941 µM; CA VB Ki 0.0428 µM). Furthermore, its DPP IV inhibitory activity in Caco-2 and its acceptable pre-ADME/Tox profile indicate it as a lead compound in this novel class of multitarget ligands.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Computational Insights into the Sequence-Activity Relationships of the NGF(1-14) Peptide by Molecular Dynamics Simulations.

The Nerve Growth Factor (NGF) belongs to the neurothrophins protein family involved in the survival of neurons in the nervous system. The interaction of NGF with its high-affinity receptor TrkA mediates different cellular pathways related to Alzheimer's disease, pain, ocular dysfunction, and cancer. Therefore, targeting NGF-TrkA interaction represents a valuable strategy for the development of new therapeutic agents. In recent years, experimental studies have revealed that peptides belonging to the N-terminal domain of NGF are able to partly mimic the biological activity of the whole protein paving the way towards the development of small peptides that can selectively target specific signaling pathways. Hence, understanding the molecular basis of the interaction between the N-terminal segment of NGF and TrkA is fundamental for the rational design of new peptides mimicking the NGF N-terminal domain. In this study, molecular dynamics simulation, binding free energy calculations and per-residue energy decomposition analysis were combined in order to explore the molecular recognition pattern between the experimentally active NGF(1-14) peptide and TrkA. The results highlighted the importance of His4, Arg9 and Glu11 as crucial residues for the stabilization of NGF(1-14)-TrkA interaction, thus suggesting useful insights for the structure-based design of new therapeutic peptides able to modulate NGF-TrkA interaction.

Hempseed (Cannabis sativa) Peptide H3 (IGFLIIWV) Exerts Cholesterol-Lowering Effects in Human Hepatic Cell Line.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC50 value of 59 µM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.

Antitumor activity of novel POLA1-HDAC11 dual inhibitors.

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72-77%). Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

MetaTREE, a Novel Database Focused on Metabolic Trees, Predicts an Important Detoxification Mechanism: The Glutathione Conjugation.

(1) Background: Data accuracy plays a key role in determining the model performances and the field of metabolism prediction suffers from the lack of truly reliable data. To enhance the accuracy of metabolic data, we recently proposed a manually curated database collected by a meta-analysis of the specialized literature (MetaQSAR). Here we aim to further increase data accuracy by focusing on publications reporting exhaustive metabolic trees. This selection should indeed reduce the number of false negative data. (2) Methods: A new metabolic database (MetaTREE) was thus collected and utilized to extract a dataset for metabolic data concerning glutathione conjugation (MT-dataset). After proper pre-processing, this dataset, along with the corresponding dataset extracted from MetaQSAR (MQ-dataset), was utilized to develop binary classification models using a random forest algorithm. (3) Results: The comparison of the models generated by the two collected datasets reveals the better performances reached by the MT-dataset (MCC raised from 0.63 to 0.67, sensitivity from 0.56 to 0.58). The analysis of the applicability domain also confirms that the model based on the MT-dataset shows a more robust predictive power with a larger applicability domain. (4) Conclusions: These results confirm that focusing on metabolic trees represents a convenient approach to increase data accuracy by reducing the false negative cases. The encouraging performances shown by the models developed by the MT-dataset invites to use of MetaTREE for predictive studies in the field of xenobiotic metabolism.

Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity.

Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

N-Acetyl-Cysteine Regenerates Albumin Cys34 by a Thiol-Disulfide Breaking Mechanism: An Explanation of Its Extracellular Antioxidant Activity.

In the present paper, the extracellular antioxidant activity of N-acetyl-cysteine (NAC) is explained by considering its ability to regenerate the free form of albumin Cys34 by breaking the disulfide bond of the cysteinylated form (HSA-Cys). NAC's capability to regenerate albumin Cys34 (HSA-SH) was studied by MS intact protein analysis in human plasma and in a concentration range of NAC easily achievable after oral and i.v. administration (5-50 µg/mL). NAC dose-dependently broke the HSA-Cys bond to form the dimer NAC-Cys thus regenerating Cys34, whose reduced state was maintained for at least 120 min. Cys was faster in restoring Cys34, according to the reaction constant determined with the glutathione disulfide (GSSG) reaction, but after 60 min the mixed disulfide HSA-Cys turned back due to the reaction of the dimer Cys-Cys with Cys34. The explanation for the different rate exchanges between Cys-Cys and Cys-NAC with Cys34 was given by molecular modeling studies. Finally, the Cys34 regenerating effect of NAC was related to its ability to improve the total antioxidant capacity of plasma (TRAP assay). The results well indicate that NAC greatly increases the plasma antioxidant activity and this effect is not reached by a direct effect but through the regenerating effect of Cys34.

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism.

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.