Antiplasmodial Properties and Cytotoxicity of Endophytic Fungi from Symphonia globulifera (Clusiaceae)

Ateba JET, Toghueo RMK, Awantu AF, et al. Antiplasmodial Properties and Cytotoxicity of Endophytic Fungi from Symphonia globulifera (Clusiaceae). JOURNAL OF FUNGI. 2018;4(2): UNSP 70.There is continuing need for new and improved drugs to tackle malaria, which remains a major public health problem, especially in tropical and subtropical regions of the world. Natural products represent credible sources of new antiplasmodial agents for antimalarial drug development. Endophytes that widely colonize healthy tissues of plants have been shown to synthesize a great variety of secondary metabolites that might possess antiplasmodial benefits. The present study was carried out to evaluate the antiplasmodial potential of extracts from endophytic fungi isolated from Symphonia globulifera against a chloroquine-resistant strain of Plasmodium falciparum (PfINDO). Sixty-one fungal isolates with infection frequency of 67.77% were obtained from the bark of S. globulifera. Twelve selected isolates were classified into six different genera including Fusarium, Paecilomyces, Penicillium, Aspergillus, Mucor, and Bipolaris. Extracts from the 12 isolates were tested against PfINDO, and nine showed good activity (IC50 < 10 mu g.mL(-1)) with three fungi including Paecilomyces lilacinus (IC50 = 0.44 mu g.mL(-1)), Penicillium janthinellum (IC50 = 0.2 mu g.mL(-1)), and Paecilomyces sp. (IC50 = 0.55 mu g.mL(-1)) showing the highest promise. These three isolates were found to be less cytotoxic against the HEK293T cell line with selectivity indices ranging from 24.52 to 70.56. Results from this study indicate that endophytic fungi from Symphonia globulifera are promising sources of hit compounds that might be further investigated as novel drugs against malaria. The chemical investigation of active extracts is ongoing

Anti-plasmodial action of <it>de novo</it>-designed, cationic, lysine-branched, amphipathic, helical peptides

<p>Abstract</p> <p>Background</p> <p>A lack of vaccine and rampant drug resistance demands new anti-malarials.</p> <p>Methods</p> <p><it>In vitro</it> blood stage anti-plasmodial properties of several <it>de novo</it>-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against <it>Plasmodium falciparum</it> using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis.</p> <p>Results</p> <p>Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH₂) (X = F,ΔF) (Fm_, ΔFm IC₅₀ >100 μM)}, the lysine-branched,dimeric versions showed far greater potency {IC₅₀ (μM) Fd 1.5 , ΔFd 1.39}. The more helical and proteolytically stable ΔFd was studied for mechanistic details. ΔFq, a K-K₂ dendrimer of ΔFm and (ΔFm)₂ a linear dimer of ΔFm showed IC₅₀ (μM) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (ΔFd), >20 (ΔFm)₂ and 10 (ΔFq). FITC-ΔFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ΔFd binds DNA. Trophozoites and schizonts incubated with ΔFd (2.5 μM) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent.</p> <p>Conclusion</p> <p>Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of ΔFd against malaria.</p

Specificity of tyrosine protein kinases of the structurally related receptors for insulin and insulin-like growth factor I: Tyr-containing synthetic polymers as specific inhibitors or substrates

The receptors for insulin and insulin-like growth factor (IGF) I are structurally similar transmembrane proteins. Ligand binding to the extracellular domain of the receptor stimulates its cytoplasmic tyrosine protein kinase which phosphorylates its own β subunit as well as exogenous substrates. It is believed, from several lines of evidence, that tyrosine-specific protein kinases are mediating some or all of the actions of insulin (or IGF-I). In order to gain insights into the substrate specificity of the structurally related insulin and IGF-I receptor kinases, we have studied the action of highly purified receptors isolated from human placental membranes. Present studies using selected tyrosine-containing polymers have revealed: (i) Polymers such as (Y,A,E)n and (Y-A-E)n inhibit β subunit autophosphorylation and exogenous substrate phosphorylation by autophosphorylated receptors, (ii) Insulin receptor kinase is at least 10 times more sensitive to these inhibitors than IGF-I receptor kinase. (iii) (Y-A-E)n is ~8 times more potent an inhibitor than (Y,A,E)n toward both receptors, (iv) While (E4Y1)n and (E6,A3,Y1)n are good substrates for both receptor kinases, the ratio of phosphate incorporation into the former to the latter is characteristically high (~4) for the IGF-I receptor and low (~1) for the insulin receptor. These results imply that the substrate specificity and enzymatic action of these two receptor kinases are distinct

Synergy with Rifampin and Kanamycin Enhances Potency, Kill Kinetics, and Selectivity of De Novo-Designed Antimicrobial Peptides▿

By choosing membranes as targets of action, antibacterial peptides offer the promise of providing antibiotics to which bacteria would not become resistant. However, there is a need to increase their potency against bacteria along with achieving a reduction in toxicity to host cells. Here, we report that three de novo-designed antibacterial peptides (ΔFm, ΔFmscr, and Ud) with poor to moderate antibacterial potencies and kill kinetics improved significantly in all of these aspects when synergized with rifampin and kanamycin against Escherichia coli. (ΔFm and ΔFmscr [a scrambled-sequence version of ΔFm] are isomeric, monomeric decapeptides containing the nonproteinogenic amino acid α,β-didehydrophenylalanine [ΔF] in their sequences. Ud is a lysine-branched dimeric peptide containing the helicogenic amino acid α-aminoisobutyric acid [Aib].) In synergy with rifampin, the MIC of ΔFmscr showed a 34-fold decrease (67.9 μg/ml alone, compared to 2 μg/ml in combination). A 20-fold improvement in the minimum bactericidal concentration of Ud was observed when the peptide was used in combination with rifampin (369.9 μg/ml alone, compared to 18.5 μg/ml in combination). Synergy with kanamycin resulted in an enhancement in kill kinetics for ΔFmscr (no killing until 60 min for ΔFmscr alone, versus 50% and 90% killing within 20 min and 60 min, respectively, in combination with kanamycin). Combination of the dendrimeric peptide ΔFq (a K-K2 dendrimer for which the sequence of ΔFm constitutes each of the four branches) (MIC, 21.3 μg/ml) with kanamycin (MIC, 2.1 μg/ml) not only lowered the MIC of each by 4-fold but also improved the therapeutic potential of this highly hemolytic (37% hemolysis alone, compared to 4% hemolysis in combination) and cytotoxic (70% toxicity at 10× MIC alone, versus 30% toxicity in combination) peptide. Thus, synergy between peptide and nonpeptide antibiotics has the potential to enhance the potency and target selectivity of antibacterial peptides, providing regimens which are more potent, faster acting, and safer for clinical use

De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions

Design of helical super secondary structural motifs is expected to provide important scaffolds to incorporate functional sites, thus allowing the engineering of novel miniproteins with function. An α,β-dehydrophenylalanine containing 21-residue apolar peptide was designed to mimic the helical hairpin motif by using a simple geometrical design strategy. The synthetic peptide folds into the desired structure as assessed crystallographically at 1.0-Å resolution. The two helices of the helical-hairpin motif, connected by a flexible (Gly)(4) linker, are docked to each other by the concerted influence of weak interactions. The folding of the peptide without binary patterning of amino acids, disulfide bonds, or metal ions is a remarkable observation. The results demonstrate that preferred interactions among the hydrophobic residues selectively discriminate their putative partners in space, leading to the unique folding of the peptide, also a hallmark of the unique folding of hydrophobic core in globular proteins. We demonstrate here the engineering of molecules by using weak interactions pointing to their possible further exploitation in the de novo design of protein super secondary structural elements

Antimalarial and antiplasmodial activity of husk extract and fractions of Zea mays

Context: Zea mays L. (Poacae) husk decoctions are traditionally used in the treatment of malaria by various tribes in Nigeria. Objective: To assess the antimalarial and antiplasmodial potentials of the husk extract and fractions on malaria parasites using in vivo and in vitro models. Materials and methods: The ethanol husk extract and fractions (187–748 mg/kg, p.o.) of Zea mays were investigated for antimalarial activity against Plasmodium berghei using rodent (mice) malaria models and in vitro activity against chloroquine sensitive (Pf 3D7) and resistant (Pf INDO) strains of Plasmodium falciparum using the SRBR green assay method. Median lethal dose and cytotoxic activities against HeLa and HEKS cells were also carried out. The GCMS analysis of the most active fraction was carried out. Results: The husk extract (187–748 mg/kg, p.o.) with LD50 of 1874.83 mg/kg was found to exert significant (p 100 μg/mL against both HeLa and HEKS cell lines. Discussion and conclusion: These results suggest that the husk extract/fractions of Zea mays possesses antimalarial and antiplasmodial activities and these justify its use in ethnomedicine to treat malaria infections

Bioactivity guided fractionation of leaves extract of Nyctanthes arbor tristis (Harshringar) against P falciparum.

BACKGROUND: Nyctanthes arbor-tristis (Harshringar, Night Jasmine) has been traditionally used in Ayurveda, Unani and other systems of medicine in India. The juice of its leaves has been used by various tribal populations of India in treatment of fevers resembling malaria. AIM OF THE STUDY: This work reports the antiplasmodial activity guided fractionation of Harshringar leaves extract. METHODOLOGY: Crude ethanolic Harshringar leaves extract and its RPHPLC purified fractions were studied for antiplasmodial potency against 3D7 (CQ sensitive) and Dd2 (CQ resistant) strains of P.falciparum and subsequently subjected to bioassay guided fractionation using reverse phase chromatography to pursue the isolation of active fractions. PRINCIPAL FINDINGS: Harshringar crude leaves extract and some of its RPHPLC purified fractions exhibited promising antiplasmodial potency against 3D7 and Dd2 strains of P.falciparum. CONCLUSIONS: The present study has provided scientific validity to the traditional use of leaves extract of Harshringar against malaria leading to the conclusion that this plant holds promise with respect to antimalarial phytotherapy. This is the first scientific report of antiplasmodial activity of RPHPLC fractions of Harshringar leaves extract against P.falciparum strains

Isolation, characterization and UPLC-DAD based quantification of antiplasmodial isoquinoline alkaloids from Cissampelos pareira L.

C. pareira L. is a centuries-old traditional medicinal plant utilized to treat various diseases like asthma, diarrhea, fever, heart disorders, snakebite, vomiting, malaria, pneumonia, dog bite, inflammation and abdominal pain. Globally, based on traditional knowledge, different parts of this plant are being used individually or in combination in various forms to manage malaria. However, the scientific investigation for validating the most effective part of this plant against malaria parasite has not been done. Therefore, current study aimed to evaluate in vitro antiplasmodial activity of extracts/fractions (whole plant) and decoctions from different parts (roots, stem, leaves and whole plant) of C. pareira against different strains of Plasmodium falciparum followed by antiplasmodial activity guided isolation and quantification of isoquinoline alkaloids in extracts/fractions and decoctions. All extracts/fractions/decoctions and molecules isolated from active fractions were investigated for antiplasmodial activity. Results showed that the chloroform fraction of whole plant was the most promising with IC50 (µg/mL) of 0.79 (Pf3D7) and 2.26 (PfINDO) followed by root decoction having IC50 (µg/mL) 10.22 (Pf3D7) and 7.7 (PfINDO). Among three isolated molecules, two bisbenzylisoquinoline alkaloids namely curine (2) [IC50 (µM) 1.46 (Pf3D7) and 0.51 (PfINDO)], and O,O-dimethylcurine (1) [IC50 (µM) 0.92 (Pf3D7) and 2.6 (PfINDO)], were found to be the most potent against P. falciparum strains. The antiplasmodial activity of chloroform fraction was further validated by the developed UPLC-DAD method, which showed the highest quantities of curine (2) (~107 mg/g) and O,O-dimethylcurine (1) (~15 mg/g) in this fraction. This study showed that the root decoction was more effective than decoctions of each of the other parts of the plant and whole plant hydroalcoholic extract. Further, for the first time, this study validates the traditional use of C. pareira whole plant to manage malaria, providing further opportunity to explore the tremendous structural and chemical diversity of isoquinoline alkaloids for antimalarial drug development

Dendrimeric Template of <i>Plasmodium falciparum</i> Histidine Rich Protein II Repeat Motifs Bearing Asp→Asn Mutation Exhibits Heme Binding and β-Hematin Formation

<div><p><i>Plasmodium falciparum</i> (<i>Pf</i>) employs a crucial <i>Pf</i>HRPII catalyzed reaction that converts toxic heme into hemozoin. Understanding heme polymerization mechanism is the first step for rational design of new drugs, targeting this pathway. Heme binding and hemozoin formation have been ascribed to <i>Pf</i>HRPII aspartate carboxylate-heme metal ionic interactions. To investigate, if this ionic interaction is indeed pivotal, we examined the comparative heme binding and β-hematin forming abilities of a wild type dendrimeric peptide BNT1 {harboring the native sequence motif of <i>Pf</i>HRPII (AHHAHHAADA)} versus a mutant dendrimeric peptide BNTM {in which ionic Aspartate residues have been replaced by the neutral Asparaginyl residues (AHHAHHAANA)}. UV and IR data reported here reveal that at pH 5, both BNT1 and BNTM exhibit comparable heme binding as well as β-hematin forming abilities, thus questioning the role of <i>Pf</i>HRPII aspartate carboxylate-heme metal ionic interactions in heme binding and β-hematin formation. Based on our data and information in the literature we suggest the possible role of weak dispersive interactions like N-H···π and lone-pair···π in heme binding and hemozoin formation.</p></div