Inhibition of malaria parasite blood stages by tyrocidines, membrane-active cyclic peptide antibiotics from Bacillus brevis

Tyrothricin, a complex mixture of antibiotic peptides from Bacillus brevis, was reported in 1944 to have antimalarial activity rivalling that of quinine in chickens infected with Plasmodium gallinaceum. We have isolated the major components of tyrothricin, cyclic decapeptides collectively known as the tyrocidines, and tested them against the human malaria parasite Plasmodium falciparum using standard in vitro assays. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their observed 50% parasite inhibitory concentrations (IC₅₀) spanned three orders of magnitude (0.58 to 360 nM). Activity correlated strictly with increased apparent hydrophobicity and reduced total side-chain surface area and the presence of ornithine and phenylalanine in key positions. In contrast, mammalian cell toxicity and haemolytic activities of the respective peptides were considerably less variable (2.6 to 28 μM). Gramicidin S, a structurally analogous antimicrobial peptide, was less active (IC₅₀ = 1.3 μM) and selective than the tyrocidines. It exerted its parasite inhibition by rapid and selective lysis of infected erythrocytes as judged by fluorescence and light microscopy. The tyrocidines, however, did not cause an overt lysis of infected erythrocytes, but an inhibition of parasite development and life-cycle progression.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants expressing human beta-defensin-2 are more resistant to fungal attack: functional homology between plant and human defensins

Human beta-defensin-2 (hBD-2) is a small antimicrobial peptide with potent activity against different Gram-negative bacteria and fungal/yeast species. Since human beta-defensins and plant defensins share structural homology, we set out to analyse whether there also exists a functional homology between these defensins of different eukaryotic kingdoms. To this end, we constructed a plant transformation vector harbouring the hBD-2 coding sequence, which we transformed to Arabidopsis thaliana plants, giving rise to A. thaliana plants indeed expressing hBD-2. Furthermore, we could demonstrate that this heterologously produced hBD-2 possesses antifungal activity in vitro. Finally, we could show that hBD-2 expressing A. thaliana plants are more resistant against the broad-spectrum fungal pathogen Botrytis cinerea as compared to untransformed A. thaliana plants, and that this resistance is correlated with the level of active hBD-2 produced in these transgenic plants. Hence, we demonstrated a functional homology, next to the already known structural homology, between defensins originating from different eukaryotic kingdoms. To our knowledge, this is the first time that this is specifically demonstrated for plant and mammalian defensins.     

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three-dimensional solution structure by (1)H two-dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts,fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.     

Mitochondrial localization of the threonine peptidase PfHslV, a ClpQ ortholog in Plasmodium falciparum

Plasmodium falciparum belongs to a group of eukaryotes expressing an ortholog of the prokaryotic T1-threonine peptidase, heat shock locus V (HslV). Bacterial HslV is a particularly well studied protease, due to its structural and biochemical similarity to the eukaryotic proteasome. Plasmodium falciparum HslV (PfHslV) is expressed in schizonts and merozoites of the asexual blood stage. Strong sequence conservation between plasmodial species, absence of HslV homologs in the human genome, and availability of specific inhibitors led us to explore its function and potential use as a drug target. In a first step, we investigated localization of PfHslV, using a bioinformatics approach and a transgenic P. falciparum line expressing a PfHslV-enhanced yellow fluorescent protein (EYFP) fusion protein from the endogenous pfhslV locus. PfHslV-EYFP was found in the mitochondrial matrix under fluorescence and immunoelectron microscopy. Endogenous, non-modified PfHslV was present in purified mitochondria and interference with mitochondrial membrane potential by drug treatment led to impairment of PfHslV processing. Import of heterologous EYFP into the plasmodial mitochondrion is mediated by the N-terminal 37 amino acids of PfHslV. PfHslV’s targeting sequence is also functional in human cells, demonstrating strong conservation of mitochondrial targeting in eukaryotes. In conclusion, our data shows that PfHslV is located to the plasmodial mitochondrion and presumably has vital function within this organelle which makes it an attractive target for interventions.     

Antiplasmodial benzophenones from the trunk latex of Moronobea coccinea (Clusiaceae)

In an effort to find antimalarial drugs, a systematic in vitro evaluation on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) was undertaken on sixty plant extracts collected in French Guiana. The methanol extract obtained from the latex of Moronobea coccinea exhibited a strong antiplasmodial activity (95% at 10 μg/ml). The phytochemical investigation of this extract led to the isolation of eleven polycyclic polyprenylated acylphloroglucinols (PPAPs), from which eight showed potent antiplasmodial activity with IC50 ranged from 3.3 μM to 37.2 μM.     

Evaluation of substituted phenalenone analogues as antiplasmodial agents

A set of derivatives encompassing structural modifications on the privileged phenalenone scaffold were assessed for their antiplasmodial activities against a strain of chloroquine sensitive Plasmodium falciparum F32. Two compounds exhibited considerable effects against the malaria parasite (IC₅₀ ? 1 μg/mL), one of which maintained the same level of activity in a chloroquine-resistant strain. This is the first record of antiplasmodial activity on this type of scaffold, providing a new structural motif as a new lead for antimalarial activity.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification

Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a K_d value of [¹²⁵I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-β-d-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [¹²⁵I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1.     

Two novel defensin-encoding genes of the Chagas disease vector Triatoma brasiliensis (Reduviidae, Triatominae): Gene expression and peptide-structure modeling

Defensins are cysteine-rich peptides involved in the innate immunity of insects and many other organisms. In the present study, two novel defensin-encoding cDNAs and the respective genomic DNAs (def3 and def4) of Triatoma brasiliensis were identified and their tissue-specific and temporal expression was characterized. Both of the deduced mature peptides consisted of 43 amino acid residues and were highly similar to previously identified triatomine defensins (81.4-100%). Semi-quantitative RT-PCR data showed that def3 was constitutively expressed in the fat body and was induced in salivary glands and the small intestine at 5 and 3 days after feeding (daf), respectively. The def4 mRNA level was highly up-regulated in the stomach and fat-body tissues at 5 and 3 daf, respectively. The three-dimensional structures of these defensins were predicted using a homology modeling approach with Def-AAA, the defensin from Anopheles gambiae, as template (62-74% identity). A map of the electrostatic potential of these models revealed that, despite their similar folding patterns, mature Def2 and Def4 have a more cationic structure than is the case for Def1 and Def3. Such differences may orient the antimicrobial profile of these defensins against distinct targets in different organs of the insect.     

Ampelozyziphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria in the Amazon Region, hampers the development of Plasmodium berghei sporozoites

Most medicinal plants used against malaria in endemic areas aim to treat the acute symptoms of the disease such as high temperature fevers with periodicity and chills. In some endemic areas of the Brazilian Amazon region one medicinal plant seems to be an exception: Ampelozyziphus amazonicus, locally named “Indian beer” or “Saracura-mira”, used to prevent the disease when taken daily as a cold suspension of powdered dried roots. In previous work we found no activity of the plant extracts against malaria blood parasites in experimentally infected animals (mice and chickens) or in cultures of Plasmodium falciparum. However, in infections induced by sporozoites, chickens treated with plant extracts were partially protected against Plasmodium gallinaceum and showed reduced numbers of exoerythrocytic forms in the brain. We now present stronger evidence that the ethanolic extract of “Indian beer” roots hampers in vitro and in vivo development of Plasmodium berghei sporozoites, a rodent malaria parasite. Some mice treated with high doses of the plant extract did not become infected after sporozoite inoculation, whereas others had a delayed prepatent period and lower parasitemia. Our data validates the use of “Indian beer” as a remedy for malaria prophylaxis in the Amazon, where the plant exists and the disease represents an important problem which is difficult to control. Studies aiming to identify the active compounds responsible for the herein described causal prophylactic activity are needed and may lead to a new antimalarial prophylactic.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Opioid δ1 receptor antagonist 7-benzylidenenaltrexone as an effective resistance reverser for chloroquine-resistant Plasmodium chabaudi

We evaluated antimalarial and/or chloroquine-resistance reversing effects of five opioid receptor antagonists. Although none of the evaluated compounds showed antimalarial effects, some of them, especially the δ₁ receptor antagonist, 7-benzylidenenaltrexone (BNTX) exhibited potent chloroquine-resistance reversing effects in Plasmodium chabaudi.     

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds

Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC₅₀ ≤ 1 μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3′-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.     

Expression of a biologically-active conotoxin PrIIIE in Escherichia coli

Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5 mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure-function studies.     

First report on exploring structural requirements of alpha and beta thymidine analogs for PfTMPK inhibitory activity using in silico studies

With the emergence of multi-drug resistance of the currently available antimalarial drugs including the “magic bullet” artemisinin derivatives in the market, there is an urgent need for discovery and development of new potent antimalarial molecules. The present work deals with quantitative structure–activity relationship (QSAR) modeling, pharmacophore mapping and docking studies of a series of 35 thymidine analogs as inhibitors of Plasmodium falciparum thymidylate kinase (PfTMPK), an enzyme that catalyzes phosphorylation of thymidine monophosphate (TMP) to thymidine diphosphate (TDP). The models were validated both internally and externally and significant statistical results were obtained, indicating the robustness and reliability of the developed models. The docking study was performed using the LigandFit option of receptor–ligand interactions protocol section available in Discovery Studio 2.1 where lower RMSD values (0.6931 Å) between the co-crystallized ligand and re-docked ligand assured that the ligand was bound in the same binding pocket. The QSAR, pharmacophore mapping and docking studies provide an understanding of important structural requirements or essential molecular properties, or features of molecules, and important binding interactions, and provide an important guidance for the chemist to synthesis of new molecules with improved PfTMPK inhibitory activity profile. This work revealed the importance of –NH-fragment, electrophilicity of the molecules and the number of oxygen atom towards the PfTMPK inhibitory activity of the molecules. To the best of our knowledge, this work presents the first QSAR and pharmacophore report for thymidine analogs which may serve as an efficient tool for the design and synthesis of potent molecules as PfTMPK inhibitors to address the increasing threat of multi-drug resistance against P. falciparum.