Iron affects localization of Ght5 in fission yeast.

Iron is an essential cofactor for eukaryotic cells, as well as a toxic metal under certain conditions. On the other hand, glucose is the preferred energy and carbon source by most organisms and is an important signaling molecule in the regulation of biological processes. In Schizosaccharomyces pombe, the Ght5 hexose transporter, known as a high affinity glucose transporter, is required for cell proliferation in low glucose concentrations. Herein, we aimed to investigate the effects of iron stress on the Ght5 hexose transporter under glucose repression and derepression conditions. The effect of iron stress on the expression profile of the ght5 gene was analyzed by RT-qPCR and western blot. The localization of the Ght5-mNeonGreen fusion protein examined with confocal microscopy. Our results revealed that iron stress had an inhibitory effect on ght5 expression, and it altered Ght5 localization on the cell surface, causing it to accumulate in the cytoplasm.

Mitochondrial Spermidine Synthase is Essential for Blood-stage growth of the Malaria Parasite.

Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei, the inhibition of the synthesis of intermediate polyamine Putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescence imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion.

Novel thienopyrimidine analogues as potential metabotropic glutamate receptors inhibitors and anticancer activity: Synthesis, In-vitro, In-silico, and SAR approaches.

There is a continuous need in drug development approach for synthetic anticancer analogues with new therapeutic targets to diminish chemotherapeutic resistance of cancer cells. This study presents new group of synthetic thienopyrimidine analogues (1-9) aims as mGluR-1 inhibitors with anticancer activity. In-vitro antiproliferative assessment was carried out using viability assay against cancer cell lines (MCF-7, A-549 and PC-3) compared to WI-38 normal cell line. Analogues showed variable anticancer activity with IC50 ranging from 6.60 to 121 µg/mL with compound 7b is the most potent analogue against the three cancer cell lines (MCF-7; 6.57 ± 0.200, A-549; 6.31 ± 0.400, PC-3;7.39 ± 0.500 µg/mL) compared to Doxorubicin, 5-Flurouracil and Riluzole controls. Selected compounds were tested as mGluR-1 inhibitors in MCF-7 cell line and results revealed compound 7b induced significant reduction in extracellular glutamate release (IC50; 4.96 ± 0.700 µM) compared to other analogues and next to Riluzole (IC50; 2.80 ± 0.500 µM) of the same suggested mode of action. Furthermore, both cell cycle and apoptosis assays confirmed the potency of compound 7b for early apoptosis of MCF-7 at G2/M phase and apoptotic positive cell shift to (91.4%) compared to untreated control (19.6%) and Raptinal positive control (51.4%). On gene expression level, compound 7b induced over-expression of extrinsic (FasL, TNF-α and Casp-8), intrinsic (Cyt-C, Casp-3, Bax) apoptotic genes with down-regulation of anti-apoptotic Bcl-2 gene with boosted Bax/Bcl-2 ratio to 2.6-fold increase. Molecular docking and dynamic studies confirmed the biological potency through strong binding and stability modes of 7b where it was faster in reaching the equilibrium point and achieving the stability than Riluzole over 20 ns MD. These results suggest compound 7b as a promising mGluR inhibitory scaffold with anticancer activity that deserves further optimization and in-depth In-vivo and clinical investigations.

Synthesis and Antimicrobial Activity of Novel 1, 2, 4-Triazolopyrimidofuroquinazolinones from Natural Furochromones (Visnagenone and Khellinone).

BACKGROUND: Previous and recent scientific research has shown that triazolopyrimidine and furochromones have a wide range of pharmacological activities for the treatment of numerous diseases, including anticancer, antiviral, anti-depressant, anti-microbial, anti-inflammatory, and analgesic activities. OBJECTIVE: Preparation of new drugs derived from a natural furochromones as (1-hydrazinyl or methylthio),-furopyrimidoquinazolinone, 1, 2, 4-triazolopyrimidofuroquinazolin-5-one, and quinazoline- pyrimidofuro- quinazoline-8, 10-dione and the study of their biological activity as antimicrobial agents. METHODS: A series of novel N'-furopyrimidoquinazoline-hydrazide; 1, 2, 4-triazolopyrimidofuroquinazolin- 5-one; furopyrimidoquinazolin-3-one and quinazoline-pyrimidofuroquinazoline-8, 10- dione derivatives were synthesized from substituted (methylthio)-furopyrimidoquinazolinone (3ab) and 1-hydrazinyl-furopyrimido- quinazolinone (4a-b) as the starting material. RESULTS: All compounds were synthesized in good yields (71-95%) in a gradually efficient system under mild condition and some of the procedures were used such as microwave oven. The new compounds have been confirmed by means of different spectroscopic methods such as IR, 1D and 2D -NMR techniques and mass spectrum. The in vitro antimicrobial activities were evaluated for the prepared compounds using many types of bacteria (Gram-positive and Gram-negative) and fungi. CONCLUSION: 1, 2, 4-triazolopyrimidofuroquinazolin-5-one derivatives (10a-f, 8a-b, 7a-b and 6a-d) showed the most efficient antimicrobial activities compared with the cefotaxime sodium and nystatin as standard drugs.

Synthetic DNA Vaccines Adjuvanted with pIL-33 Drive Liver-Localized T Cells and Provide Protection from Plasmodium Challenge in a Mouse Model.

The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%-88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.

Genetic Characterization of Coenzyme A Biosynthesis Reveals Essential Distinctive Functions during Malaria Parasite Development in Blood and Mosquito.

Coenzyme A (CoA) is an essential universal cofactor for all prokaryotic and eukaryotic cells. In nearly all non-photosynthetic cells, CoA biosynthesis depends on the uptake and phosphorylation of vitamin B5 (pantothenic acid or pantothenate). Recently, putative pantothenate transporter (PAT) and pantothenate kinases (PanKs) were functionally characterized in P. yoelii. PAT and PanKs were shown to be dispensable for blood stage development, but they were essential for mosquito stages development. Yet, little is known about the cellular functions of the other enzymes of the CoA biosynthesis pathway in malaria parasite life cycle stages. All enzymes of this pathway were targeted for deletion or deletion/complementation analyses by knockout/knock-in plasmid constructs to reveal their essential roles in P. yoelii life cycle stages. The intermediate enzymes PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase) were shown to be dispensable for asexual and sexual blood stage development, but they were essential for oocyst development and the production of sporozoites. However, the last two enzymes of this pathway, PPAT (Phosphopantetheine Adenylyltransferase) and DPCK (Dephospho-CoA Kinase), were essential for blood stage development. These results indicate alternative first substrate requirement for the malaria parasite, other than the canonical pantothenate, for the synthesis of CoA in the blood but not inside the mosquito midgut. Collectively, our data shows that CoA de novo biosynthesis is essential for both blood and mosquito stages, and thus validates the enzymes of this pathway as potential antimalarial targets.

Plasmodium AdoMetDC/ODC bifunctional enzyme is essential for male sexual stage development and mosquito transmission.

Polyamines are positively-charged organic molecules that are important for cellular growth and division. Polyamines and their synthesizing enzymes are particularly abundant in rapidly proliferating eukaryotic cells such as parasitic protozoa and cancer cells. Polyamine biosynthesis inhibitors, such as Elfornithine, are now being considered for cancer prevention and have been used effectively against Trypanosoma brucei Inhibitors of polyamine biosynthesis have caused growth arrest of Plasmodium falciparum blood stages in vitro, but in P. berghei only partial inhibition has been observed. While polyamine biosynthesis enzymes are characterized and conserved in Plasmodium spp., little is known on the biological roles of these enzymes inside malaria parasite hosts. The bifunctional polyamine biosynthesis enzyme S-adenosyl methionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) was targeted for deletion in P. yoelii Deletion of AdoMetDC/ODC significantly reduced blood stage parasitemia but Anopheles transmission was completely blocked. We showed that male gametocytogenesis and male gamete exflagellation were abolished and consequently no ookinetes or oocyst sporozoites could be generated from adometdc/odc(-) parasites. Supplementation of putrescine and spermidine did not rescue the defective phenotypes of male gametocytes and gametes of the knockout parasites. These results highlight the crucial role of polyamine homeostasis in the development and functions of Plasmodium erythrocytic stages in the blood and in the mosquito vector and validate polyamine biosynthesis pathway enzymes as drug targeting candidates for malaria parasite transmission blocking.

Synthesis of new pyrazole, triazole, and thiazolidine-pyrimido [4, 5-b] quinoline derivatives with potential antitumor activity.

2-Hydrazinyltetrahydropyrimido [4, 5-b] quinolin-4(3H)-one (3) was prepared by desulfurization reaction of S- and N-dimethyl derivatives 2 with hydrazine hydrate. Reactions of (3) with malonitrile, carbondisulfide, potassium thiocyanate, phthalic anhydride and aromatic aldehydes afforded 3, 5-di aminopyrazolopyrimido [4, 5-b] quinoline (4), triazolotetrahydropyrimido [4, 5-b] quinoline (5), aminotriazolopyrimido [4, 5-b] quinoline (6), aminophthalimidopyrimido [4, 5-b] quinoline (7) and N-arylidene hydrazinepyrimido [4, 5-b] quinoline 8a-d, respectively. Furthermore, 8a-d reacted with mercaptoacetic acid gave the thiazolidinonepyrimido [4, 5-b] quinoline 9a-d, which afforded the thiazolotriazolopyrimido [4, 5-b] quinolinone 10a-d upon treatment with ethanolic potassium hydroxide. The newly synthesized compounds were characterized by elemental analyses, IR, 1H-NMR, 13C-NMR and mass spectrometer. The investigated compounds were screened for their cytotoxicity. Compounds 4, 6 and 5 exhibited potent antitumor activity.

Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites.

While subunit vaccines have shown partial efficacy in clinical trials, radiation-attenuated sporozoites (RAS) remain the "gold standard" for sterilizing protection against Plasmodium infection in human vaccinees. The variability in immunogenicity and replication introduced by the extensive, random DNA damage necessary to generate RAS could be overcome by genetically attenuated parasites (GAP) designed via gene deletion to arrest at defined points during liver-stage development. Here, we demonstrate the principle that late liver stage-arresting GAP induce larger and broader CD8 T cell responses that provide superior protection in inbred and outbred mice compared to RAS or early-arresting GAP immunizations. Late liver stage-arresting GAP also engender high levels of cross-stage and cross-species protection and complete protection when administered by translationally relevant intradermal or subcutaneous routes. Collectively, our results underscore the potential utility of late liver stage-arresting GAP as broadly protective next-generation live-attenuated malaria vaccines and support their potential as a powerful model for identifying antigens to generate cross-stage protection.

Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.

Malaria parasite sporozoites prepare for transmission to a mammalian host by upregulation of UIS (Upregulated in Infectious Sporozoites) genes. A number of UIS gene products are essential for the establishment of the intrahepatocytic niche. However, the factors that regulate the expression of genes involved in gain of infectivity for the liver are unknown. Herein, we show that a conserved Plasmodium sporozoite low-complexity asparagine-rich protein, SAP1 (Sporozoite Asparagine-rich Protein 1), has an essential role in malaria parasite liver infection. Targeted deletion of SAP1 in the rodent malaria parasite Plasmodium yoelii generated mutant parasites that traverse and invade hepatocytes normally but cannot initiate liver-stage development in vitro and in vivo. Moreover, immunizations with Pysap1(-) sporozoites confer long-lasting sterile protection against wild-type sporozoite infection. Strikingly, lack of SAP1 abolished expression of essential UIS genes including UIS3, UIS4 and P52 but not the constitutively expressed genes encoding, among others, sporozoite proteins CSP and TRAP. SAP1 localization to the cell interior but not the nucleus of sporozoites suggests its involvement in a post-transcriptional mechanism of gene expression control. These findings demonstrate that SAP1 is essential for liver infection possibly by functioning as a selective regulator controlling the expression of infectivity-associated parasite effector genes.

Preerythrocytic malaria vaccine development.

PURPOSE OF REVIEW: This review examines the potential of current preerythrocytic stage malaria vaccine approaches to reduce the global burden of malaria. RECENT FINDINGS: Radiation-attenuated parasite vaccines induce lasting sterile protection in all models tested. Inherent safety concerns in conjunction with challenges to produce and deliver a radiation-attenuated parasite vaccine have prevented its mass production and application. Recent advances in genetic engineering and initiatives in production process development of live attenuated malaria vaccines, however, will overcome roadblocks that currently prevent their large-scale application. Development of preerythrocytic subunit vaccines has focused on the circumsporozoite protein and the thrombospondin related anonymous protein, yet the most advanced circumsporozoite protein-based vaccine confers limited protection against infection in malaria endemic areas. Work in rodent malaria models demonstrated that circumsporozoite protein-based immunity is not required for to achieve sterile protection. SUMMARY: We conclude that preerythrocytic malaria vaccine efforts should focus on two major areas: development of a safe live attenuated sporozoite vaccine with its accelerated testing in malaria endemic areas and identification of as yet unknown antigens that reproduce sterilizing immune responses induced by vaccination with whole parasites. The sporozoite challenge model provides a unique opportunity to rapidly test preerythrocytic vaccine candidates.

A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts.

The Plasmodium life cycle is a sequence of alternating invasive and replicative stages within the vertebrate and invertebrate hosts. How malarial parasites exit their host cells after completion of reproduction remains largely unsolved. Inhibitor studies indicated a role of Plasmodium cysteine proteases in merozoite release from host erythrocytes. To validate a vital function of malarial cysteine proteases in active parasite egress, we searched for target genes that can be analyzed functionally by reverse genetics. Herein, we describe a complete arrest of Plasmodium sporozoite egress from Anopheles midgut oocysts by targeted disruption of a stage-specific cysteine protease. Our findings show that sporozoites exit oocysts by parasite-dependent proteolysis rather than by passive oocyst rupture resulting from parasite growth. We provide genetic proof that malarial cysteine proteases are necessary for egress of invasive stages from their intracellular compartment and propose that similar cysteine protease-dependent mechanisms occur during egress from liver-stage and blood-stage schizonts.