Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening.

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 µM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

Downstream processing of a plant-derived malaria transmission-blocking vaccine candidate.

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30-75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg-1 plant material after purification based on an initial yield of ∼49 mg kg-1 biomass after blanching.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 µM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 µM and 0.011 µM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry.

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.

Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas.

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.

Structure and organization of the peridinin-chlorophyll a-binding protein gene in Gonyaulax polyedra.

We have identified a major 32-kDa protein in the dinoflagellate Gonyaulax polyedra as a peridinin-chlorophyll a-binding protein (PCP), based on micro-sequence data and immunological cross-reaction with antibodies raised against PCP from another dinoflagellate species. A cDNA for this protein, identified by a PCR-based cloning strategy, encoded all 68 of the amino acids microsequenced, thus confirming the identity of the clone. The PCP gene is highly expressed at both the mRNA and protein levels, and only PCP transcripts corresponding in size to the cDNA sequence were detected. Slot blot analyses show that there are roughly 5000 copies of the PCP gene in Gonyaulax, making this gene one of the most highly repeated protein-coding genes ever reported, yet the sequence of the different gene copies in the genome appears extraordinarily well conserved as judged by Southern blot analyses. The gene, as indicated by Southern blot and PCR data, is suggested to be present in 5000 intronless copies arranged head to tail in the genome, separated by conserved 1-kb spacers. Based on the conserved sequence of the spacer region, its presence next to each of the PCP coding sequences, and the uniform size of the PCP transcript, we propose that this region represents a dinoflagellate transcriptional promoter. This putative promoter region contains none of the sequence elements for DNA-binding proteins involved in transcriptional initiation reported in other organisms.

Overexpression, purification and characterization of Dictyostelium calcineurin A.

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Molecular characterization of a third malic enzyme-like AP65 adhesin gene of Trichomonas vaginalis.

Adherence to the vaginal epithelium by the sexually transmitted parasite Trichomonas vaginalis is mediated by four trichomonad surface proteins (AP65, AP51, AP33 and AP23). We recently showed that the 65-kDa adhesin is a member of a multigene family comprised of two similar but distinct proteins, AP65-1 and AP65-2, encoded by the genes ap65-1 and ap65-2, respectively. An additional immuno-crossreactive clone, the 1.2 kb F11.1 cDNA, was isolated from a phagemid expression library and encoded a fusion protein of approximately 46,000 daltons (46 kDa) that bound to HeLa cell surfaces. A significant portion of the 5' end was missing which, using the 5'-RACE method, was obtained and combined with the F11.1 clone to give a full-length cDNA. The ap65-3 gene encoded for a protein of 567 amino acids with a molecular mass of 63.1 kDa. The gene showed 88% and 96% identity at the DNA level with ap65-1 and ap65-2, respectively. Restriction mapping confirmed that the three AP65 genes are different. Southern analysis revealed that the ap65-3 gene is present in the T. vaginalis genome in multiple copies. Experiments with agar clones of trichomonads showed that each gene of the multigene family is present in all parasites, and Northern analysis showed that ap65-3 is expressed and transcriptionally regulated by iron. The ap65-3 gene had a leader sequence and, as with ap65-1 and ap65-2, showed significant homology to malic enzyme. Finally, analysis of the 3'-untranslated regions revealed that the transcript of ap65-3 had a long poly (A) tail in comparison to ap65-1 and ap65-2. Even more intriguing, sequences were found that may relate to differential degradation of select AP65 transcripts, such as the sequence motifs AUUUA for ap65-1 mRNA and UUAUUUAU for the ap65-2 mRNA, which were not found for ap65-3.

Expression of a 32 kilodalton Theileria sergenti piroplasm surface protein by recombinant baculoviruses.

Previous studies detected a single amino acid substitution (Ala196 to Gly196) between cDNA clones encoding a 32 kDa antigen (p32) of Theileria sergenti (Chitose stock) obtained from a persistently infected calf. In this study, 2 different recombinant baculoviruses (pAc/p32-Ala196 and pAc/p32-Gly196) were constructed for the expression of p32. Molecular masses of the polypeptides produced in Spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of authentic p32. pAc/p32-Ala196 produced additional polypeptides, with molecular masses higher than 32 kDa, which resulted from differential N-glycosylation as revealed by endo N-glycosidase treatment. The results indicate that a single amino acid substitution may lead to a conformational change in p32 which affected post-translational modification of recombinant products.