Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

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.

Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum.

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.

A fast virtual screening approach to identify structurally diverse inhibitors of trypanothione reductase.

Trypanothione reductase (TryR) is one of the favorite targets for those designing drugs for the treatment of Chagas disease. We present the application of a fast virtual screening approach for designing hit compounds active against TryR. Our protocol combines information derived from structurally known inhibitors and from the TryR receptor structure. Five structurally diverse hit compounds active against TryR and holding promise for the treatment of Chagas disease are reported.

Use of small angle neutron scattering to study the interaction of angiotensin II with model membranes.

Understanding biological processes assumes a detailed understanding of the interaction of all involved molecules. Here the effect of the peptide hormone angiotensin II (Ang II), an agonist of the angiotensin receptors, on the structure of unilamellar and multilamellar dimyristoyl phosphatidylcholine vesicles was studied by small angle neutron scattering, dynamic light scattering and differential scanning calorimetry. The calorimetry data indicate a weak interaction of Ang II with the surface of the membrane bilayer, as the pretransition persists during all experiments, and the main transition is only slightly shifted towards higher temperatures. From the SANS data we were able to confirm the calorimetric data and verify the interaction of the hormone with the membrane surface. At low temperatures, when the lipid molecules are in the gel phase, more precisely in the ripple phase, the peptide penetrates in the head group core, but due to the close packing of the acyl chains, the hydrophobic region is not affected. In a temperature region below but close to the region of the phase transition, the hydrophibic core starts to be affected by the peptide, and the same is true for the fluid phase. Upon binding of the peptide, the thickness of the head group increases, and the scattering length density of the head group starts to rise with increasing peptide concentrations. This interaction and binding to the membrane surface may be relevant for the relocation, binding and reconstitution of the angiotensin receptors into the membrane. Second, the peptide adsorption to the membrane surface may contribute to the binding of Ang II in the active site of the receptor.

Thiol peroxidase protects Salmonella enterica from hydrogen peroxide stress in vitro and facilitates intracellular growth.

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H(2)O(2)), that it has a reduced capacity to degrade H(2)O(2) compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.

Peroxiredoxin systems in mycobacteria.

Like other actinomycetes Mycobacterium tuberculosis lacks glutathione and, consequently, the glutathione peroxidases that dominate the antioxidant defence of its mammalian hosts. The hydrogen peroxide metabolism of the pathogen has for long been recognised to depend on a heme-containing catalase/peroxidase. Clinical isolates lacking the catalase were virulent and proved to be resistant to the first line tuberculostatic isoniazid, because the enzyme is evidently required to activate this drug. The survival and virulence of such strains are attributed to the peroxiredoxin-type peroxidases alkyl hydroperoxide reductase (AhpC) and thioredoxin peroxidase (TPx). The most common AhpC reductant in bacteria, the disulfide reductase AhpF, is deleted in M. tuberculosis. Instead, AhpC can be reduced by AhpD, a CXXC-motif-containing protein, or by one of the mycobacterial thioredoxins, TrxC. TPx is reduced by thioredoxins B and C. Mycobacteria contain three more peroxiredoxins, the 1-Cys-Prx AhpE, Bcp and BcpB, whose function and reductants are still unknown.

The thiol-based redox networks of pathogens: unexploited targets in the search for new drugs.

Hydroperoxide metabolism in diverse pathogens is reviewed under consideration of involved enzymes as potential drug targets. The common denominator of the peroxidase systems of Trypanosoma, Leishmania, Plasmodium, and Mycobacterium species is the use of NAD(P)H to reduce hydroperoxides including peroxynitrite via a flavin-containing disulfide reductase, a thioredoxin (Trx)-related protein and a peroxidase that operates with thiol catalysis. In Plasmodium falciparum, thioredoxin- and glutathione dependent systems appear to be linked via glutaredoxin and plasmoredoxin to terminal thioredoxin peroxidases belonging to both, the peroxiredoxin (Prx) and glutathione peroxidase (GPx) family. In Mycobacterium tuberculosis, a catalase-type peroxidase is complemented by the typical 2-C-Prx AhpC that, in contrast to most bacteria, is reduced by TrxC, and an atypical 2-C-Prx reduced by TrxB or C. A most complex variation of the scheme is found in trypanosomatids, where the unique redox metabolite trypanothione reduces the thioredoxin-related tryparedoxin, which fuels Prx- and GPx-type peroxidases as well as ribonucleotide reductase. In Trypanosoma brucei and Leishmania donovani the system has been shown to be essential for viability and virulence by inversed genetics. It is concluded that optimum efficacy can be expected from inhibitors of the most upstream components of the redox cascades. For trypanosomatids attractive validated drug targets are trypanothione reductase and trypanothione synthetase; for mycobacteria thioredoxin reductase appears most appealing, while in Plasmodium simultaneous inhibition of both the thioredoxin and the glutathione pathway appears advisable to avoid mutual substitution in co-substrate supply to the peroxidases. Financial and organisational needs to translate the scientific progress into applicable drugs are discussed under consideration of the socio-economic impact of the addressed diseases.

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.

Multiple thioredoxin-mediated routes to detoxify hydroperoxides in Mycobacterium tuberculosis.

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.