58 research outputs found
Evaluatie van de preproteïne-translocatiemotor SecA als doelwit voor de ontwikkeling van nieuwe antibiotica
No full textThe fight against bacterial infections is one of the major concerns in modern medicine due to the rapid rise of antibiotic-resistant bacteria. Therefore, there is an urgent need for new antibacterials – preferably directed against alternative bacterial targets – to combat hardly treatable infections caused by these resistant bacteria. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec-dependent protein translocation pathway, which is the major route for protein export across or into the bacterial cytoplasmic membrane. Since SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target.
In order to evaluate SecA as a target for the development of novel antibiotics with a new mode of action, first an in silico analysis of various SecA structures was performed to define potentially druggable pockets on the surface of SecA. Comparison of these potential binding sites for small-molecules in the different SecA structures showed that six of these pockets are highly conserved among SecA proteins of both Gram-negative and Gram-positive bacteria. To investigate whether three of these conserved pockets are essential for the function of SecA, various amino acid substitutions were introduced in each of the three predicted druggable pockets of both Escherichia coli SecA and Staphylococcus aureus SecA1, taken as representative SecAs for Gram-negative and Gram-positive bacteria, respectively. Subsequently the effect of the introduced mutations on the function of SecA was examined using an in vivo genetic complementation assay. This demonstrated that the three selected pockets are essential for the function of both E. coli SecA and S. aureus SecA1.
To examine whether these predicted druggable pockets may represent effective drug targets, structure-based virtual ligand screening was performed against one of the most attractive, essential pockets in E. coli SecA, the signal peptide-binding site. About 500,000 commercially available small-molecules from the ChemBridge compound library were virtually screened against this potentially druggable site in E. coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of SecA. Five inhibitors of the translocation ATPase, and not of the basal or membrane ATPase activity, were identified with IC50 values <65 µM. The most potent inhibitor revealed an IC50 of 24 µM. For the 5 most potent SecA inhibitors, the antimicrobial activity was determined. Two compounds were found to possess weak antibacterial activity against E. coli (IC50 ~198 µM), while others showed moderate antibacterial activity against S. aureus (IC50 ~100 µM). These molecules may be used as lead compounds leading to the design of more potent SecA inhibitors which block the SecA-preprotein interaction and specifically inhibit Sec-dependent protein translocation.
As an alternative approach to discover SecA and/or Sec-dependent protein translocation inhibitors, we developed target-based whole-cell screening assays. Therefore, recombinant E. coli strains were constructed for which inhibition of protein translocation can be easily detected using either enzyme- or fluorescence-based approaches. These developed assays were validated using the known SecA inhibitor sodium azide and can be used in the future for the discovery of Sec-dependent protein translocation inhibitors.
In this study, we demonstrated that SecA is an attractive target for the development of novel antibiotics since the bacterial protein translocation can be inhibited by small-molecules targeting the signal peptide-binding site of SecA, indicating that SecA is druggable. The identified SecA inhibitors, which are the first reported SecA-preprotein interaction inhibitors, should be further optimized to improve their potency and antibacterial activity. Alternatively, additional in silico, in vitro and/or target-based whole-cell screenings could be performed to identify more potent SecA and/or Sec-dependent protein translocation inhibitors. Hopefully, these inhibitors may contribute to the generation of a novel class of antibiotics which can be used to combat hardly treatable infections caused by multidrug-resistant bacteria.status: Publishe
Preclinical evaluation of [(11)C]NE40, a type 2 cannabinoid receptor PET tracer
No full textINTRODUCTION: Up-regulation of the type 2 cannabinoid receptor (CB(2)R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [(11)C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB(2)R. METHODS: The selectivity of NE40 for CB(2)R and its toxicity and mutagenicity were determined. [(11)C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [(11)C]NE40 to human CB(2)R (hCB(2)R) was studied in a rat model with hCB(2)R overexpression. RESULTS: [(11)C]NE40 shows specific CB(2)R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [(11)C]NE40 showed specific and reversible binding to hCB(2)R in vivo in a rat model with local hCB(2)R overexpression. CONCLUSIONS: [(11)C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB(2)R and is a promising candidate for hCB(2)R PET imaging.sponsorship: Research funded by a Ph.D. grant of IWT-Vlaanderen. We thank Peter Vermaelen and Ann Van Santvoort for their skillful help with the animal experiments. We thank Lieve Van Mellaert from the Laboratory for Microbiology (K.U. Leuven, Belgium) for performing the Ames test. We thank Johnson & Johnson Pharmaceutical Research and Development, LLC, for performing the CEREP assay. (IWT-Vlaanderen)status: Publishe
Immunoprophylaxis and immunotherapy of Staphylococcus epidermidis infections: challenges and prospects
No full textPreviously considered a human commensal, Staphylococcus epidermidis is a frequent cause of nosocomial infections and the most common cause of device-related infections. Because the expression of toxins and other obvious virulence factors is less in S. epidermidis, the biofilm-forming capacity is its major virulence factor. Biofilm growth is characterized by high resistance to antimicrobial agents and host immune responses, making biofilm eradication tremendously difficult. The increasing prevalence of multidrug-resistant S. epidermidis strains additionally hampers antimicrobial therapy. Therefore, immunoprophylaxis and immunotherapy targeting factors expressed at some point in biofilm formation might offer new tools to combat S. epidermidis infections. So far, a limited number of targets have been examined for their immunotherapeutic potential. In this review, we focus on the already tested and possible targets for vaccine development, discuss the accompanying challenges and speculate on future possibilities with respect to immunotherapeutic solutions to deal with S. epidermidis infections.status: Publishe
Characterization of the Streptomyces lividans PspA response
No full textPhage shock protein (Psp) is induced by extracytoplasmic stress that may reduce the energy status of the cell. It is encoded in Escherichia coli by the phage shock protein regulon consisting of pspABCDE and by pspF and pspG. The phage shock protein system is highly conserved among a large number of gram-negative bacteria. However, many bacterial genomes contain only a pspA homologue but no homologues of the other genes of the Psp system. This conservation indicates that PspA alone might play an important role in these bacteria. In Streptomyces lividans, a soil-borne gram-positive bacterium, the phage shock protein system consists only of the pspA gene. In this report, we showed that pspA encodes a 28-kDa protein that is present in both the cytoplasmic and the membrane fractions of the S. lividans mycelium. We demonstrated that the pspA gene is strongly induced under stress conditions that attack membrane integrity and that it is essential for growth and survival under most of these conditions. The data reported here clearly show that PspA plays an important role in S. lividans under stress conditions despite the absence of other psp homologues, suggesting that PspA may be more important in most bacteria than previously thought.sponsorship: K.V. is a research fellow of the Institute for the Promotion of Innovation by Science and Technology in Flanders. This research was supported by grants G.0389.05 from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen and QLK3-LSHG-CT-2007-037586 from the European Union.status: Publishe
Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug
No full textStandard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.sponsorship: HBV cccDNA assays were supported by the Division of Microbiology and Infectious Diseases (DMID), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) under contract number 75N93019D00015 "In Vitro Assessment for Antimicrobial Activity" Task Order B01 "Comprehensive Viral Screening Panel", under the direction of Dr. Mindy Davis as NIAID's Contracting Officer's Representative (COR), Ms. Amanda Ulloa as the Alternate COR, Dr. Roger Ptak as the Principal Investigator, and Dr. Raj Kalkeri as the Coinvestigator. Synthetic work was supported by the KU Leuven Research Fund via a C3 project (C32/17/045). (Division of Microbiology and Infectious Diseases (DMID), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)|75N93019D00015, KU Leuven Research Fund via a C3 project|C32/17/045)status: Publishe
Locust cellular defense against infections: Sites of pathogen clearance and hemocyte proliferation
No full textThe locust cellular defense is mediated by hemocytes and hematopoietic tissue. In Locusta migratoria, the hemocytes and hematopoietic tissue mutually assist each other in clearing invading pathogens from circulation. A β-1, 3-glucan infection induces nodule formation and apoptotic, TUNEL positive, cells in the hematopoietic tissue and massive loss of hemocytes in the circulation, calling for instant proliferation of hemocytes and hematopoietic tissue cells to assure continued host cellular defense. As the locust hematopoietic tissue persists at the adult stage, it was originally designated as being the major source for the replenishment process. Revisiting post infection hemocyte proliferation, using immunofluorescence based tests for DNA synthesis and mitosis, evidenced the lack of β-1, 3-glucan induced cell proliferation in the hematopoietic tissue. Instead these tests identified the circulating hemocytes as the major source for hemocyte replenishment in the circulation. The hematopoietic tissue, however, undergoes a continuous, slow and infection independent regeneration, thereby accumulating potential phagocytes despite infection, and might serve a prophylactic role in containing pathogens in this swarming insect.sponsorship: This work was supported by KU Leuven Research Foundation (GOA/11/002). The authors gratefully thank Roger Jonckers for caring for the locust breeding. We thank the lab of Prof. Lieve Van Mellaert (Rega Institute, KU Leuven) for providing us with heat-deactivated Sarcina lutea and Candida albicans. (KU Leuven Research Foundation|GOA/11/002)status: Publishe
Recombinant protein production and Streptomycetes
No full textThe biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories.sponsorship: FWO-G-08-00360status: Publishe
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