ABSTRACT
Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.
Subject(s)
Amide Synthases , Glutathione , NADH, NADPH Oxidoreductases , Trypanosoma , NADH, NADPH Oxidoreductases/metabolism , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Humans , Amide Synthases/metabolism , Amide Synthases/antagonists & inhibitors , Trypanosoma/drug effects , Trypanosoma/metabolism , Glutathione/metabolism , Glutathione/analogs & derivatives , Animals , Spermidine/analogs & derivatives , Spermidine/metabolism , Leishmania/drug effects , Leishmania/metabolism , Trypanocidal Agents/pharmacology , Trypanocidal Agents/therapeutic use , Leishmaniasis/drug therapy , Leishmaniasis/metabolism , Leishmaniasis/parasitology , Trypanosomatina/metabolism , Trypanosomatina/drug effects , Protozoan Proteins/metabolism , Protozoan Proteins/antagonists & inhibitors , Chagas Disease/drug therapy , Chagas Disease/parasitology , Chagas Disease/metabolismABSTRACT
Leishmaniasis is an infectious disease caused by obligate intracellular protozoa of the genus Leishmania with high infection and death rates in developing countries. New drugs with better pharmacological performance with regards to safety, efficacy, toxicity, and drug resistance than those/the ones currently used are urgently needed. Trypanothione synthetase (TryS) is an attractive target for the development of drugs against leishmaniasis because it is specific and essential to kinetoplastid parasites. In this study, Leishmaniamajor TryS was expressed and purified, and the kinetic parameters of purified TryS were determined. To identify novel inhibitors of LmTryS, a high-throughput screening (HTS) assay was developed and used to screen a library of 35,040 compounds. In the confirmatory assay, 42 compounds displayed half maximal inhibitory concentration (IC50) values < 50 µM and six of them corresponded to novel structures with IC50 ranging from 9 to 19 µM against LmTryS enzyme activity. Of the six inhibitors, TS001 showed the highest activity against growth of L. major promastigotes, L. donovani promastigotes, and Trypanosoma brucei brucei Lister 427 with IC50 values of 17, 26, and 31 µM, respectively. An in silico docking study using a homology model of LmTryS predicted the molecular interactions between LmTryS and the inhibitors.
Subject(s)
Amide Synthases , Antiprotozoal Agents , Leishmania major , Amide Synthases/antagonists & inhibitors , Gene Library , High-Throughput Screening Assays , Leishmania major/drug effects , Leishmania major/enzymology , Antiprotozoal Agents/pharmacologyABSTRACT
Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Antiprotozoal Agents/pharmacology , Leishmania infantum/drug effects , Trypanosoma cruzi/drug effects , Amide Synthases/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leishmania infantum/enzymology , Macrophages/drug effects , Molecular Structure , Structure-Activity Relationship , Trypanosoma cruzi/enzymologyABSTRACT
Trypanosomatid-caused diseases are among the neglected infectious diseases with the highest disease burden, affecting about 27 million people worldwide and, in particular, socio-economically vulnerable populations. Trypanothione synthetase (TryS) is considered one of the most attractive drug targets within the thiol-polyamine metabolism of typanosomatids, being unique, essential and druggable. Here, we have compiled a dataset of 401 T. brucei TryS inhibitors that includes compounds with inhibitory data reported in the literature, but also in-house acquired data. QSAR classifiers were derived and validated from such dataset, using publicly available and open-source software, thus assuring the portability of the obtained models. The performance and robustness of the resulting models were substantially improved through ensemble learning. The performance of the individual models and the model ensembles was further assessed through retrospective virtual screening campaigns. At last, as an application example, the chosen model-ensemble has been applied in a prospective virtual screening campaign on DrugBank 5.1.6 compound library. All the in-house scripts used in this study are available on request, whereas the dataset has been included as supplementary material.
Subject(s)
Amide Synthases/chemistry , Drug Discovery/methods , Enzyme Inhibitors/chemistry , Machine Learning , Algorithms , Amide Synthases/antagonists & inhibitors , Amide Synthases/metabolism , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Databases, Pharmaceutical , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/standards , Enzyme Inhibitors/pharmacology , Humans , Metabolic Networks and Pathways , Models, Theoretical , ROC Curve , Structure-Activity RelationshipABSTRACT
Leishmania and Trypanosoma parasites are responsible for the challenging neglected tropical diseases leishmaniases, Chagas disease, and human African trypanosomiasis, which account for up to 40,000 deaths annually mainly in developing countries. Current chemotherapy relies on drugs with significant limitations in efficacy and safety, prompting the urgent need to explore innovative approaches to improve the drug discovery pipeline. The unique trypanothione-based redox pathway, which is absent in human hosts, is vital for all trypanosomatids and offers valuable opportunities to guide the rational development of specific, broad-spectrum and innovative anti-trypanosomatid agents. Major efforts focused on the key metabolic enzymes trypanothione synthetase-amidase and trypanothione reductase, whose inhibition should affect the entire pathway and, finally, parasite survival. Herein, we will report and comment on the most recent studies in the search for enzyme inhibitors, underlining the promising opportunities that have emerged so far to drive the exploration of future successful therapeutic approaches.
Subject(s)
Enzyme Inhibitors/pharmacology , Glutathione/analogs & derivatives , Spermidine/analogs & derivatives , Trypanocidal Agents/pharmacology , Amide Synthases/antagonists & inhibitors , Amidohydrolases/antagonists & inhibitors , Animals , Chagas Disease/drug therapy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Glutathione/metabolism , Humans , Leishmania/drug effects , Leishmania/enzymology , Leishmaniasis/drug therapy , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Spermidine/metabolism , Trypanocidal Agents/chemistry , Trypanocidal Agents/therapeutic use , Trypanosoma/drug effects , Trypanosoma/enzymologyABSTRACT
NAD (nicotinamide adenine dinucleotide) synthase catalyses the biochemical synthesis of NAD, from nicotinic acid adenine dinucleotide (NAAD). NAD may be synthesized through the de novo pathways and/or the salvage pathways in cells. However, in Leishmania parasite, the synthesis of NAD solely depends on the salvage pathways. NAD synthetase is widely explored as a drug target in various microorganisms. In Bacillus anthracis, a group of sulphonamides 5599, 5617 and 5824 and complex amide 5833 were reported to have activity at micromolar range against NAD synthetase. Hence, in the present study, the same group of sulphonamides and complex amide were validated through in silico and in vitro studies for its efficiency towards Leishmania donovani NAD synthase. In silico study revealed the ligands 5824 and 5833 to have better docking score. Molecular dynamics simulation for a duration of 50 ns of all the ligand-protein complexes suggested that the complexes with the ligands 5824 and 5833 were stable and interacting. In vitro and ex vivo studies have shown that 5824 and 5833 inhibit the cell viability of the organism at a lower concentration than 5599 and 5617. Hence, with further in vivo validation, 5824 (or its synthetic analogues) and 5833 could be the choice that may work synergistically with other potential drugs in treating drug-resistant cases of leishmaniasis. Communicated by Ramaswamy H. Sarma.
Subject(s)
Amide Synthases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Leishmania donovani/enzymology , Amide Synthases/metabolism , Animals , Cell Death/drug effects , Cell Survival/drug effects , Hydrogen Bonding , Leishmania donovani/drug effects , Ligands , Mice, Inbred BALB C , Molecular Docking Simulation , Molecular Dynamics Simulation , Principal Component AnalysisABSTRACT
Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.
Subject(s)
Buthionine Sulfoximine/pharmacology , Glutathione/analogs & derivatives , Spermidine/analogs & derivatives , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/metabolism , Amide Synthases/antagonists & inhibitors , Amide Synthases/chemistry , Amide Synthases/genetics , Animals , Enzyme Inhibitors/pharmacology , Glutamate-Cysteine Ligase/antagonists & inhibitors , Glutamate-Cysteine Ligase/genetics , Glutathione/biosynthesis , Glutathione/metabolism , Glutathione Synthase/antagonists & inhibitors , Glutathione Synthase/genetics , Humans , Kinetics , Metabolic Networks and Pathways/drug effects , Molecular Docking Simulation , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spermidine/biosynthesis , Trypanosoma cruzi/geneticsABSTRACT
Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50=1000µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antitubercular Agents/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Amide Synthases/metabolism , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Molecular Structure , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Structure-Activity RelationshipABSTRACT
Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.
Subject(s)
Antiprotozoal Agents/pharmacology , Benzazepines/pharmacology , Indoles/pharmacology , Trypanosoma brucei brucei/drug effects , Amide Synthases/antagonists & inhibitors , Animals , Antiprotozoal Agents/chemistry , Benzazepines/chemistry , Humans , Indoles/chemistry , Spectrum Analysis/methods , Trypanosoma brucei brucei/enzymologyABSTRACT
BACKGROUND: The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. METHODOLOGY/PRINCIPAL FINDING: A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low µM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 µM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. CONCLUSIONS/SIGNIFICANCE: Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antiprotozoal Agents/isolation & purification , Enzyme Inhibitors/isolation & purification , Leishmania infantum/drug effects , Trypanosoma brucei brucei/drug effects , Trypanosoma cruzi/drug effects , Antiprotozoal Agents/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , Leishmania infantum/enzymology , Trypanosoma brucei brucei/enzymology , Trypanosoma cruzi/enzymologyABSTRACT
Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.
Subject(s)
Amide Synthases/antagonists & inhibitors , Chagas Disease/drug therapy , Drug Repositioning , Polyamines/chemistry , Amide Synthases/chemistry , Antiprotozoal Agents/chemistry , Chagas Disease/parasitology , Computer Simulation , Glutathione/analogs & derivatives , Glutathione/chemistry , Glutathione/therapeutic use , Humans , Imidazoles/chemistry , Imidazoles/therapeutic use , Nitroimidazoles/chemistry , Nitroimidazoles/therapeutic use , Polyamines/therapeutic use , Spermidine/analogs & derivatives , Spermidine/chemistry , Spermidine/therapeutic use , Thiophenes/chemistry , Thiophenes/therapeutic use , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/pathogenicity , User-Computer InterfaceABSTRACT
Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different forms of leishmaniasis. This family of protozoan parasite possesses a trypanothione-based redox metabolism that provides the reducing equivalents for various vital processes such as the biosynthesis of DNA precursors and the detoxification of hydroperoxides. Almost all enzymes of the redox pathway proved to be essential and therefore fulfil one crucial prerequisite for a putative drug target. Trypanothione synthetase and trypanothione reductase are present in all trypanosomatids but absent from the mammalian host which, in addition to the essentiality, renders them highly specific. The chemotherapy research on both enzymes is further supported by the availability of high-throughput screening techniques and crystal structures. In this review we focus on the recent advances and limitations in the development of lead compounds targeting trypanothione synthetase and trypanothione reductase. We present an overview of the available inhibitors and discuss future perspectives including other components of the parasite-specific redox pathway.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antiprotozoal Agents/pharmacology , Enzyme Inhibitors/pharmacology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Amide Synthases/genetics , Amide Synthases/metabolism , Animals , Antiprotozoal Agents/chemical synthesis , Chagas Disease/drug therapy , Chagas Disease/parasitology , Enzyme Inhibitors/chemical synthesis , Gene Expression , High-Throughput Screening Assays , Leishmania/drug effects , Leishmania/enzymology , Leishmania/genetics , Leishmania/growth & development , Leishmaniasis/drug therapy , Leishmaniasis/parasitology , Molecular Targeted Therapy , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Structure-Activity Relationship , Sulfhydryl Compounds/metabolism , Trypanosoma/drug effects , Trypanosoma/enzymology , Trypanosoma/genetics , Trypanosoma/growth & development , Trypanosomiasis, African/drug therapy , Trypanosomiasis, African/parasitologyABSTRACT
Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps(-/-) knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys(-/-)/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.
Subject(s)
Amide Synthases/antagonists & inhibitors , Amide Synthases/genetics , Antiprotozoal Agents/pharmacology , Leishmania infantum/enzymology , Amide Synthases/biosynthesis , Animals , Gene Knockout Techniques , Glutathione/analogs & derivatives , Glutathione/biosynthesis , Glutathione/chemistry , Leishmania infantum/genetics , Leishmaniasis, Visceral/drug therapy , Male , Mice , Mice, Inbred BALB C , Spermidine/analogs & derivatives , Spermidine/biosynthesis , Spermidine/chemistryABSTRACT
A great number of sesquiterpenes are reported in the available literature as good antileishmanial leads. However, their mode of action at the molecular level has not been elucidated. The lack of molecular studies could be considered an impediment for studies seeking to improve sesquiterpene-based drug design. The present in silico study allows us to make important observations about the molecular details of the binding modes of a set of antileishmanial sesquiterpenes against four drug-enzyme targets [pteridine reductase-1 (PTR1), N-myristoyl transferase (NMT), cysteine synthase (CS), trypanothione synthetase (TryS)]. Through molecular docking it was found that two sesquiterpene coumarins are promising leads for the PTR1 and TryS inhibition purposes, and some xanthanolides also exhibited better affinity towards PTR1 and CS binding. In addition, the affinity values were clustered by Principal Component Analysis and drug-like properties were analyzed for the strongest-docking sesquiterpenes. The results are an excellent starting point for future studies of structural optimization of this kind of compounds.
Subject(s)
Drug Design , Leishmania/drug effects , Leishmaniasis/drug therapy , Sesquiterpenes/administration & dosage , Amide Synthases/antagonists & inhibitors , Computer Simulation , Humans , Leishmania/enzymology , Oxidoreductases/antagonists & inhibitors , Sesquiterpenes/chemistryABSTRACT
UNLABELLED: Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by (13)C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE: To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antitubercular Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/enzymology , NAD/biosynthesis , Nicotinamide-Nucleotide Adenylyltransferase/antagonists & inhibitors , Drug Discovery/methods , Gene Knockdown Techniques , Genes, Essential , Microbial Viability , NAD/antagonists & inhibitorsSubject(s)
Amide Synthases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Biofilms/growth & development , Drug Discovery/methods , Drug Tolerance/physiology , Gene Expression Regulation, Enzymologic/physiology , Mycobacterium tuberculosis/drug effects , Proteolysis/drug effects , Serine Endopeptidases/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Tuberculosis/prevention & control , Animals , FemaleABSTRACT
Tuberculosis is considered to be one of the major health problem not only in the less developed countries but in the economically developed countries as well. Roughly one third of the world's population are infected with Mycobacterium tuberculosis and a significant part of them are carriers of latent tuberculosis. From ten percent of these latent infections are developing the active TB disease and fifty percent of them die from the illness without appropriate treatment. The drug-resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) and TB-HIV co-infection attracted attention to the most serious infectious disease. Inhibition of alternative signaling pathways were an important part of the research strategies for cancer and inflammatory diseases in recent years. In case of Mycobacterium tuberculosis such pathways were also identified, for example, three serine-threonine kinases (PknA, PknB, PknG) which are necessary and essential for bacterial growth. In this paper we summarize our best anti-TB active compounds, their biological effects and structure-activity relationships using in silico modeling, biochemical measurements and tests on active bacteria.
Subject(s)
Amide Synthases/antagonists & inhibitors , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Computer Simulation , Models, Chemical , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapy , Amides/chemistry , Amides/pharmacology , Coinfection/epidemiology , HIV Infections/epidemiology , Humans , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Protein Serine-Threonine Kinases/metabolism , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Tuberculosis/epidemiology , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dual-control genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies.
Subject(s)
Amide Synthases/antagonists & inhibitors , Drug Discovery/methods , Drug Tolerance/physiology , Gene Expression Regulation, Enzymologic/physiology , Mycobacterium tuberculosis/drug effects , Tuberculosis/prevention & control , Animals , Carrier Proteins , Escherichia coli Proteins , Genetic Engineering/methods , Luciferases , Mice , Mycobacterium tuberculosis/growth & developmentABSTRACT
In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,ß-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.
Subject(s)
Lactones/chemistry , Lactones/pharmacology , Protozoan Proteins/antagonists & inhibitors , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/enzymology , Amide Synthases/antagonists & inhibitors , Amide Synthases/metabolism , Enzyme Activation/drug effects , Humans , Lactones/therapeutic use , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/metabolism , Ornithine Decarboxylase/metabolism , Ornithine Decarboxylase Inhibitors , Oxidation-Reduction , Protozoan Proteins/metabolism , Sesquiterpenes/therapeutic use , Sulfhydryl Compounds/chemistry , Trypanosomiasis, African/drug therapy , Trypanosomiasis, African/metabolism , Trypanosomiasis, African/pathologyABSTRACT
Leishmaniasis is a neglected disease that kills 60,000 people worldwide, and which is caused by the protozoa Leishmania. The enzymes of the trypanothione pathway: trypanothione synthetase-amidase, trypanothione reductase (TR) and tryparedoxin-dependent peroxidase are absent in human hosts, and are essential for parasite survival and druggable. The most promising target is trypanothione synthetase-amidase, which has been also chemically validated. However, the structural data presented in this review show that TR also should be considered as a good target. Indeed, it is strongly inhibited by silver- and gold-containing compounds, which are active against Leishmania parasites and can be used for the development of novel antileishmanial agents. Moreover, TR trypanothione-binding site is not featureless but contains a sub-pocket where inhibitors bind, potentially useful for the design of new lead compounds.