[Show abstract][Hide abstract] ABSTRACT: Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy.
PLoS ONE 07/2015; 10(7):e0131456. DOI:10.1371/journal.pone.0131456 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
[Show abstract][Hide abstract] ABSTRACT: A number of synthetic pantothenate derivatives, such as pantothenamides, are known to inhibit the growth of the human malaria parasite Plasmodium falciparum, by interfering with the parasite Coenzyme A (CoA) biosynthetic pathway. The clinical use of pantothenamides is limited by their sensitivity to breakdown by ubiquitous human pantetheinases of the vanin family.
A number of pantothenate derivatives (pantothenones) with potent and specific inhibitory activity against mammalian vanins were tested in a proliferation assay of asexual P. falciparum blood stages alone, and in combination with pantothenamides.
The vanin inhibitors were found to protect pantothenamides against breakdown by plasma vanins, thereby preserving the in vitro anti-malarial activity. Moreover, some of the vanin inhibitors showed in vitro anti-malarial activity in the low micromolar range. The most potent antimalarial in this series of compounds (RR8), was found to compete with pantothenate in a combination proliferation assay. No correlation, however, was found between anti-vanin and anti-malarial activity, nor was pantetheinase activity detected in P. falciparum extracts.
Growth inhibition is most likely due to competition with pantothenate, rather than pantetheinase inhibition. As vanin inhibitors of the pantothenone class are stable in biological fluids and are non-toxic to mammalian cells, they may represent novel pantothenate-based anti-malarials, either on their own or in combination with pantothenamides.
[Show abstract][Hide abstract] ABSTRACT: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission reducing activity (TRA) of these agents is currently determined in the standard membrane feeding assay (SMFA) based on subjective microscopical read-outs and with limitations in up-scaling and throughput.
Utilising a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favour of a simple approach where whole mosquitoes are homogenised and examined directly for luciferase activity.
Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate.
This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates and sera from malaria exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.
The Journal of Infectious Diseases 05/2014; 210(9). DOI:10.1093/infdis/jiu271 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression.
Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2.
Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.
[Show abstract][Hide abstract] ABSTRACT: Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility.
Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA).
A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector.
The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an important epigenetic regulator of gene expression. Abnormalities in DNA methylation patterns have been associated with various developmental and proliferative diseases, particularly cancer. Targeting DNA methyltransferases (DNMTs) represents a promising strategy for the treatment of such diseases. Current DNMT inhibitors suffer important drawbacks with respect to their efficacy, specificity, and toxicity. In this study, we have set up a robust in vitro bacterial M.SssI DNMT activity assay to systematically screen a collection of 26 240 compounds that were predicted to compete with the S-adenosyl-L-methionine (SAM) substrate of DNMT. This resulted in the identification of a novel set of structurally distinct inhibitors of M.SssI DNMT activity. Although molecular docking studies using an M.SssI homology model suggest that these compounds might compete with SAM binding, mode of activity (MoA) assays are still needed to confirm this hypothesis. Our set of novel M.SssI DNMT inhibitors, once confirmed in an orthogonal DNMT assay, may thus serve as a starting point to identify and characterize suitable lead candidates for further drug optimization.
[Show abstract][Hide abstract] ABSTRACT: We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose
functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state
of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific
hydrogen-bond network including Glu723. This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of
Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular
interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR
in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic
activity compared with reference antagonists is due to an induced fit around Trp755, resulting in a decreased steric clash with Met909 but inducing a new internal clash with Val912 in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce
mixed profiles by altering the binding of 11β substituents. Together these structures further our understanding of the steric
and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future
[Show abstract][Hide abstract] ABSTRACT: Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS) not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.
PLoS ONE 10/2011; 6(10):e26678. DOI:10.1371/journal.pone.0026678 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The progesterone receptor is able to bind to a large number and variety of ligands that elicit a broad range of transcriptional
responses ranging from full agonism to full antagonism and numerous mixed profiles inbetween. We describe here two new progesterone
receptor ligand binding domain x-ray structures bound to compounds from a structurally related but functionally divergent
series, which show different binding modes corresponding to their agonistic or antagonistic nature. In addition, we present
a third progesterone receptor ligand binding domain dimer bound to an agonist in monomer A and an antagonist in monomer B,
which display binding modes in agreement with the earlier observation that agonists and antagonists from this series adopt
different binding modes.
Journal of Biological Chemistry 10/2011; 286(40):35079-35086. · 4.57 Impact Factor