To Target or Not to Target Schistosoma mansoni Cyclic Nucleotide Phosphodiesterase 4A?

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages.

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

Discovery of novel Schistosoma mansoni PDE4A inhibitors as potential agents against schistosomiasis.

Aim: Due to the urgent need for effective drugs to treat schistosomiasis that act through a known molecular mechanism of action, we focused on a target-based approach with the aim to discover inhibitors of a cyclic nucleotide phosphodiesterase from Schistosoma mansoni (SmPDE4A). Materials & methods: To discover new inhibitors of SmPDE4A homology models of the enzyme structure were constructed based on known human and protozoan homologs. The best two models were selected for subsequent virtual screening of our in-house chemical library. Results & conclusion: A total of 25 library compounds were selected for experimental confirmation as SmPDE4A inhibitors and after dose-response experiments, three top hits were identified. The results presented validate the virtual screening approach to identify new inhibitors for clinically relevant phosphodiesterases.

Construction of Fluorescent Analogs to Follow the Uptake and Distribution of Cobalamin (Vitamin B12) in Bacteria, Worms, and Plants.

Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms, and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogs with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogs were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms, and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogs, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B12.

Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway.

Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In Staphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S. aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S. aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem.

The structure, function and properties of sirohaem decarboxylase--an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri AhbA/B have been produced and their physical properties compared. The D. vulgaris and M. barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D. desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed.

Recent advances in the biosynthesis of modified tetrapyrroles: the discovery of an alternative pathway for the formation of heme and heme d 1.

Hemes (a, b, c, and o) and heme d 1 belong to the group of modified tetrapyrroles, which also includes chlorophylls, cobalamins, coenzyme F430, and siroheme. These compounds are found throughout all domains of life and are involved in a variety of essential biological processes ranging from photosynthesis to methanogenesis. The biosynthesis of heme b has been well studied in many organisms, but in sulfate-reducing bacteria and archaea, the pathway has remained a mystery, as many of the enzymes involved in these characterized steps are absent. The heme pathway in most organisms proceeds from the cyclic precursor of all modified tetrapyrroles uroporphyrinogen III, to coproporphyrinogen III, which is followed by oxidation of the ring and finally iron insertion. Sulfate-reducing bacteria and some archaea lack the genetic information necessary to convert uroporphyrinogen III to heme along the "classical" route and instead use an "alternative" pathway. Biosynthesis of the isobacteriochlorin heme d 1, a cofactor of the dissimilatory nitrite reductase cytochrome cd 1, has also been a subject of much research, although the biosynthetic pathway and its intermediates have evaded discovery for quite some time. This review focuses on the recent advances in the understanding of these two pathways and their surprisingly close relationship via the unlikely intermediate siroheme, which is also a cofactor of sulfite and nitrite reductases in many organisms. The evolutionary questions raised by this discovery will also be discussed along with the potential regulation required by organisms with overlapping tetrapyrrole biosynthesis pathways.

In vitro and in vivo antibacterial evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.

Investigations of the mode of action and resistance development of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Cadazolid is a new oxazolidinone-type antibiotic currently in clinical development for the treatment of Clostridium difficile-associated diarrhea. Here, we report investigations on the mode of action and the propensity for spontaneous resistance development in C. difficile strains. Macromolecular labeling experiments indicated that cadazolid acts as a potent inhibitor of protein synthesis, while inhibition of DNA synthesis was also observed, albeit only at substantially higher concentrations of the drug. Strong inhibition of protein synthesis was also obtained in strains resistant to linezolid, in agreement with low MICs against such strains. Inhibition of protein synthesis was confirmed in coupled transcription/translation assays using extracts from different C. difficile strains, including strains resistant to linezolid, while inhibitory effects in DNA topoisomerase assays were weak or not detectable under the assay conditions. Spontaneous resistance frequencies of cadazolid were low in all strains tested (generally <10(-10) at 2× to 4× the MIC), and in multiple-passage experiments (up to 13 passages) MICs did not significantly increase. Furthermore, no cross-resistance was observed, as cadazolid retained potent activity against strains resistant or nonsusceptible to linezolid, fluoroquinolones, and the new antibiotic fidaxomicin. In conclusion, the data presented here indicate that cadazolid acts primarily by inhibition of protein synthesis, with weak inhibition of DNA synthesis as a potential second mode of action, and suggest a low potential for spontaneous resistance development.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.

The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Functional improvement in mouse models of familial amyotrophic lateral sclerosis by PEGylated insulin-like growth factor I treatment depends on disease severity.

Insulin-like growth factor I (IGF-I) has been successfully tested in the SOD1-G93A mouse model of familial amyotrophic lateral sclerosis (ALS) and proposed for clinical treatment. However, beneficial effects required gene therapy or intrathecal application. Circumventing the dosing issues we recently found that polyethylene glycol (PEG) modified IGF-I (PEG-IGF-I) modulated neuromuscular function after systemic application, and protected against disease progression in a motor neuron disease model. Here we investigated its effects in two SOD1-G93A mouse lines, the G1L with a milder and the G1H with a more severe phenotype. Results showed that in G1L mice, PEG-IGF-I treatment significantly improved muscle force, motor coordination and animal survival. In contrast, treatment of G1H mice with PEG-IGF-I or IGF-I even at high doses did not beneficially affect survival or functional outcomes despite increased signalling in brain and spinal cord by both agents. In conclusion, the data point towards further investigation of the therapeutic potential of PEG-IGF-I in ALS patients with less severe clinical phenotypes.

Local tolerance of intraarticular administration of lornoxicam into the rabbit knee joint.

The local tolerability of lornoxicam (Xefo) after single and repeated intraarticular administration was assessed in the rabbit and compared to established standard therapies (hyaluronic acid--Synvisc and the glucocorticoid triamcinolone--Triam), and the results are discussed in the context of the literature. Two local tolerance studies were performed using five male rabbits per group. Lornoxicam and competitor products were administered into the right knee joint in a volume of 500 µL. The contralateral left knee joint of the same animal was used as the control and was injected with water for injection. Three out of five animals were killed 72 h after the last administration, whereas the remaining two animals were subjected to a 2- or 6-week recovery period in the first and the second study, respectively. Findings revealed adaptive changes related to the mechanical irritation of the injection and to adaptive responses of the synoviocytes, but no signs of toxicity to bone or chondrotoxicity. Toxicokinetic analysis showed a fast and almost complete absorption of lornoxicam from the joints into the systemic circulation. As a conclusion, repeated intraarticular administration of lornoxicam was well tolerated in rabbits.

Integrated transcriptomic and proteomic evaluation of gentamicin nephrotoxicity in rats.

Gentamicin is an aminoglycoside antibiotic, which induces renal tubular necrosis in rats. In the context of the European InnoMed PredTox project, transcriptomic and proteomic studies were performed to provide new insights into the molecular mechanisms of gentamicin-induced nephrotoxicity. Male Wistar rats were treated with 25 and 75 mg/kg/day subcutaneously for 1, 3 and 14 days. Histopathology observations showed mild tubular degeneration/necrosis and regeneration and moderate mononuclear cell infiltrate after long-term treatment. Transcriptomic data indicated a strong treatment-related gene expression modulation in kidney and blood cells at the high dose after 14 days of treatment, with the regulation of 463 and 3241 genes, respectively. Of note, the induction of NF-kappa B pathway via the p38 MAPK cascade in the kidney, together with the activation of T-cell receptor signaling in blood cells were suggestive of inflammatory processes in relation with the recruitment of mononuclear cells in the kidney. Proteomic results showed a regulation of 163 proteins in kidney at the high dose after 14 days of treatment. These protein modulations were suggestive of a mitochondrial dysfunction with impairment of cellular energy production, induction of oxidative stress, an effect on protein biosynthesis and on cellular assembly and organization. Proteomic results also provided clues for potential nephrotoxicity biomarkers such as AGAT and PRBP4 which were strongly modulated in the kidney. Transcriptomic and proteomic data turned out to be complementary and their integration gave a more comprehensive insight into the putative mode of nephrotoxicity of gentamicin which was in accordance with histopathological findings.

EU framework 6 project: predictive toxicology (PredTox)--overview and outcome.

In this publication, we report the outcome of the integrated EU Framework 6 PROJECT: Predictive Toxicology (PredTox), including methodological aspects and overall conclusions. Specific details including data analysis and interpretation are reported in separate articles in this issue. The project, partly funded by the EU, was carried out by a consortium of 15 pharmaceutical companies, 2 SMEs, and 3 universities. The effects of 16 test compounds were characterized using conventional toxicological parameters and "omics" technologies. The three major observed toxicities, liver hypertrophy, bile duct necrosis and/or cholestasis, and kidney proximal tubular damage were analyzed in detail. The combined approach of "omics" and conventional toxicology proved a useful tool for mechanistic investigations and the identification of putative biomarkers. In our hands and in combination with histopathological assessment, target organ transcriptomics was the most prolific approach for the generation of mechanistic hypotheses. Proteomics approaches were relatively time-consuming and required careful standardization. NMR-based metabolomics detected metabolite changes accompanying histopathological findings, providing limited additional mechanistic information. Conversely, targeted metabolite profiling with LC/GC-MS was very useful for the investigation of bile duct necrosis/cholestasis. In general, both proteomics and metabolomics were supportive of other findings. Thus, the outcome of this program indicates that "omics" technologies can help toxicologists to make better informed decisions during exploratory toxicological studies. The data support that hypothesis on mode of action and discovery of putative biomarkers are tangible outcomes of integrated "omics" analysis. Qualification of biomarkers remains challenging, in particular in terms of identification, mechanistic anchoring, appropriate specificity, and sensitivity.

Dimers of nostocarboline with potent antibacterial activity.

OBJECTIVES: In this study, the in vitro antimicrobial activity and spectrum of new dimeric compounds derived from the cyanobacterial alkaloid nostocarboline were investigated. The mechanism of action and selectivity to bacteria were studied and compared to the cationic antiseptic chlorhexidine. METHODS: Minimal inhibitory concentrations were determined against clinical isolates and against a panel of microbial reference strains using the CLSI microdilution method. Bacterial membrane damage was addressed by measuring ATP leakage and the mode of action was investigated in Escherichia coli reporter strains. Selectivity was tested by a cytotoxicity assay using MTS. RESULTS: The antimicrobial potency of dimers varied with length of the hydrophobic linker. The most potent compounds, NCD9 and NCD10, had a C10 and C12 linker, respectively, and showed strong activity against Gram-positive bacteria, notably methicillin-resistant Staphylococcus aureus strains. Similar to chlorhexidine, these compounds showed a rapid concentration-dependent bactericidal effect, which correlated with membrane damage as indicated by ATP leakage. NCD9, in contrast to NCD10 and chlorhexidine, lacked activity against yeast strains and showed low cytotoxicity in CHO cells indicating a high degree of selectivity. In E. coli reporter strains, NCD9 induced the DegP response pathway as well as the SOS response, suggesting interaction with both the cell envelope and DNA metabolism. CONCLUSIONS: The results presented in this report indicate the potential of this new class of cationic antimicrobial compounds for the design of potent and selective antibacterials with low cytotoxicity.

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Creation of a digital slide and tissue microarray resource from a multi-institutional predictive toxicology study in the rat: an initial report from the PredTox group.

The widespread use of digital slides has only recently come to the fore with the development of high-throughput scanners and high performance viewing software. This development, along with the optimisation of compression standards and image transfer techniques, has allowed the technology to be used in wide reaching applications including integration of images into hospital information systems and histopathological training, as well as the development of automated image analysis algorithms for prediction of histological aberrations and quantification of immunohistochemical stains. Here, the use of this technology in the creation of a comprehensive library of images of preclinical toxicological relevance is demonstrated. The images, acquired using the Aperio ScanScope CS and XT slide acquisition systems, form part of the ongoing EU FP6 Integrated Project, Innovative Medicines for Europe (InnoMed). In more detail, PredTox (abbreviation for Predictive Toxicology) is a subproject of InnoMed and comprises a consortium of 15 industrial (13 large pharma, 1 technology provider and 1 SME) and three academic partners. The primary aim of this consortium is to assess the value of combining data generated from 'omics technologies (proteomics, transcriptomics, metabolomics) with the results from more conventional toxicology methods, to facilitate further informed decision making in preclinical safety evaluation. A library of 1709 scanned images was created of full-face sections of liver and kidney tissue specimens from male Wistar rats treated with 16 proprietary and reference compounds of known toxicity; additional biological materials from these treated animals were separately used to create 'omics data, that will ultimately be used to populate an integrated toxicological database. In respect to assessment of the digital slides, a web-enabled digital slide management system, Digital SlideServer (DSS), was employed to enable integration of the digital slide content into the 'omics database and to facilitate remote viewing by pathologists connected with the project. DSS also facilitated manual annotation of digital slides by the pathologists, specifically in relation to marking particular lesions of interest. Tissue microarrays (TMAs) were constructed from the specimens for the purpose of creating a repository of tissue from animals used in the study with a view to later-stage biomarker assessment. As the PredTox consortium itself aims to identify new biomarkers of toxicity, these TMAs will be a valuable means of validation. In summary, a large repository of histological images was created enabling the subsequent pathological analysis of samples through remote viewing and, along with the utilisation of TMA technology, will allow the validation of biomarkers identified by the PredTox consortium. The population of the PredTox database with these digitised images represents the creation of the first toxicological database integrating 'omics and preclinical data with histological images.

Structure-activity relationship in the oxazolidinone-quinolone hybrid series: influence of the central spacer on the antibacterial activity and the mode of action.

Oxazolidinone-quinolone hybrids, which combine the pharmacophores of a quinolone and an oxazolidinone, were synthesised and shown to be active against a variety of susceptible and resistant Gram-positive and Gram-negative bacteria. The nature of the spacer greatly influences the antibacterial activity by directing the mode of action, that is quinolone- and/or oxazolidinone-like activity. The best compounds in this series have a balanced dual mode of action and overcome all types of resistance, including resistance to quinolones and linezolid, in clinically relevant Gram-positive pathogens.

Design, synthesis and biological evaluation of oxazolidinone-quinolone hybrids.

Oxazolidinone-quinolone hybrids that combine the pharmacophores of a quinolone and an oxazolidinone were synthesised and shown to be active against a variety of resistant and susceptible Gram-positive and fastidious Gram-negative organisms. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens. The nature of the spacer greatly influences the antibacterial activity. The dual mode of action could be demonstrated for compounds having a piperazinyl spacer. Antibacterial activity was higher at acidic pH.