Investigation of an Allosteric Deoxyhypusine Synthase Inhibitor in P. falciparum.

The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites' genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC50 value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC50 values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors.

Recombinant Active Peptides and their Therapeutic Functions.

Recombinant active peptides are utilized as diagnostic and biotherapeutics in various maladies and as bacterial growth inhibitors in the food industry. This consequently stimulated the need for recombinant peptides' production, which resulted in about 19 approved biotech peptides of 1- 100 amino acids commercially available. While most peptides have been produced by chemical synthesis, the production of lengthy and complicated peptides comprising natural amino acids has been problematic with low quantity. Recombinant peptide production has become very vital, costeffective, simple, environmentally friendly with satisfactory yields. Several reviews have focused on discussing expression systems, advantages, disadvantages, and alternatives strategies. Additionally, the information on the antimicrobial activities and other functions of multiple recombinant peptides is challenging to access and is scattered in literature apart from the food and drug administration (FDA) approved ones. From the reports that come to our knowledge, there is no existing review that offers substantial information on recombinant active peptides developed by researchers and their functions. This review provides an overview of some successfully produced recombinant active peptides of ≤100 amino acids by focusing on their antibacterial, antifungal, antiviral, anticancer, antioxidant, antimalarial, and immune-modulatory functions. It also elucidates their modes of expression that could be adopted and applied in future investigations. We expect that the knowledge available in this review would help researchers involved in recombinant active peptide development for therapeutic uses and other applications.

Identification of mRNA 5' cap-associated proteins in the human malaria parasite Plasmodium falciparum.

Eukaryotic messenger RNA is translated via a 5' cap-dependent initiation mechanism. Experimental evidence for proteins involved with translation initiation among eukaryotic parasites is lacking, including Plasmodium falciparum, the human malaria parasite. Native P. falciparum proteins from asexual stage parasites were enriched using a 5' cap affinity matrix. Proteomic analysis of enriched protein eluates revealed proteins putatively associated with the 5' cap. The canonical 5' cap-binding protein eIF4E (PF3D7_0315100) was the most reproducibly enriched protein. The eIF4A and eIF4G proteins hypothesized to form the eIF4F initiation complex with eIF4E were also detected as 5' cap enriched, albeit with low reproducibility. Surprisingly, enolase (ENO) was the second most enriched protein after eIF4E. Recombinant ENO protein did not demonstrate 5' cap activity, suggesting an indirect association of the native ENO with the 5' cap.

A cell-based ribozyme reporter system employing a chromosomally-integrated 5' exonuclease gene.

BACKGROUND: Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. RESULTS: We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5' exonuclease is induced from a chromosomally-integrated gene in the same cell. CONCLUSIONS: The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

Validation of Plasmodium falciparum deoxyhypusine synthase as an antimalarial target.

BACKGROUND: Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. METHODS: Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. RESULTS: Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N 1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. DISCUSSION: Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.

Tools for attenuation of gene expression in malaria parasites.

An understanding of the biology of Plasmodium parasites, which are the causative agents of the disease malaria, requires study of gene function. Various reverse genetic tools have been described for determining gene function. These tools can be broadly grouped as trans- and cis-acting. Trans-acting tools control gene functions through synthetic nucleic acid probe molecules matching the sequence of the gene of interest. Once delivered to the parasite, the probe engages with the mRNA of the target gene and attenuates its function. Cis-acting tools control gene function through elements introduced into the gene of interest by DNA transfection. The expression of the modified gene can be controlled using external agents, typically small molecule ligands. In this review, we discuss the strengths and weaknesses of these tools to guide researchers in selecting the appropriate tool for studies of gene function, and for guiding future refinements of these tools.

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites.

Molecular characterization of Plasmodium falciparum Bruno/CELF RNA binding proteins.

The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory mechanisms in different stages of its life cycle. Despite the importance of post-transcriptional regulation, key elements of these processes, namely RNA binding proteins (RBPs), are poorly characterized. In this study, the RNA binding properties of P. falciparum proteins were characterized including two putative members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate synthase (PfDHFR-TS), and adenosine deaminase (PfAda). RNA binding activity was tested using UV-crosslinking and electrophoretic mobility shift assays. PfCELF1 and PfDHFR-TS demonstrated RNA binding activity, whereas PfAda and PfCELF2 were RBP-negative. Intracellular protein localization of RBPs was studied using GFP-tagged transgenic parasite lines. PfCELF1 protein may shuttle between nucleus and cytoplasm, as shown by a predominantly nuclear PfCELF1 cell population and another predominantly cytoplasmic. In contrast, PfDHFR-TS protein is predominantly cytoplasmic. PfCELF1 may thus have several roles, including pre-mRNA processing. The mRNA targets of these P. falciparum proteins were investigated by ribonomics using DNA microarrays. A sequence motif similar to that recognized by CELF proteins in other species is common in the introns of target mRNAs identified for PfCELF1, suggesting that nuclear-localized PfCELF1 may regulate pre-mRNA splicing in P. falciparum, as has been found for CELF proteins in other species. In contrast, none or very few mRNA targets were found for the other proteins, suggesting that they do not have biologically relevant roles as RBPs in the asexual stages of P. falciparum.

Pleiotropic control of secondary metabolism and morphological development by KsbC, a butyrolactone autoregulator receptor homologue in Kitasatospora setae.

The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel ß-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel ß-carboline alkaloids, indicating that these genes were biosynthetic genes for ß-carboline alkaloid and thus are the first such genes to be discovered in bacteria.

Kitasetaline, a novel ß-carboline alkaloid from Kitasatospora setae NBRC 14216T.

With the genetically modified Kitasatospora setae NBRC 14216(T) strain, a new ß-carboline alkaloid, kitasetaline (1), was produced on solid medium. The structure was elucidated on the basis of physicochemical evidence. This is the first report of this type of alkaloid found in the genus Kitasatospora.

Isolation and characterization of bamA genes, homologues of the gamma-butyrolactone autoregulator-receptor gene in Amycolatopsis mediterranei, a rifamycin producer.

Four genes (bamA1, bamA2, bamA3 and bamA4) encoding homologues of the gamma-butyrolactone autoregulator receptor of Streptomyces were found and cloned from Amycolatopsis mediterranei, a typical non-Streptomyces actinomycetes and a producer of rifamycin, one of the major anti-tuberculosis drugs in clinical treatment. Transcriptional analysis demonstrated that bamA1 and bamA2 are transcribed in a growth-dependent manner, while bamA3 and bamA4 are constitutively transcribed during growth. Binding assays using (3)H-labeled autoregulator analogues as ligands confirmed that all of the recombinant BamA proteins expressed in Escherichia coli have clear binding activity toward several types of Streptomyces autoregulators. The ligand specificity of the recombinant BamA1 protein was identical to that of the crude cell-free lysates of A. mediterranei reported in our previous work. These results suggest that A. mediterranei, which is phylogenetically situated in a distal clade from the genus Streptomyces as non-Streptomyces actinomycetes, has an autoregulator-mediated signaling system.