[1-7-NαC]-Crocaorb A1 and A2, orbitides from the latex of Croton campanulatus.

Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 µM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 µM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds.

Optimization of Plasmodium vivax infection of colonized Amazonian Anopheles darlingi.

Obtaining Plasmodium vivax sporozoites is essential for in vitro culture of liver stage parasites, not only to understand fundamental aspects of parasite biology, but also for drug and vaccine development. A major impediment to establish high-throughput in vitro P. vivax liver stage assays for drug development is obtaining sufficient numbers of sporozoites. To do so, female anopheline mosquitoes have to be fed on blood from P. vivax-infected patients through an artificial membrane-feeding system, which in turns requires a well-established Anopheles colony. In this study we established conditions to provide a robust supply of P. vivax sporozoites. Adding a combination of serum replacement and antibiotics to the membrane-feeding protocol was found to best improve sporozoite production. A simple centrifugation method appears to be a possible tool for rapidly obtaining purified sporozoites with a minimal loss of yield. However, this method needs to be better defined since sporozoite viability and hepatocyte infection were not evaluated.

Transmission-blocking activity of antimalarials for Plasmodium vivax malaria in Anopheles darlingi.

Malaria is caused by parasite of the genus Plasmodium and is still one of the most important infectious diseases in the world. Several biological characteristics of Plasmodium vivax contribute to the resilience of this species, including early gametocyte production, both of which lead to efficient malaria transmission to mosquitoes. This study evaluated the impact of currently used drugs on the transmission of P. vivax. Participants received one of the following treatments for malaria: i) chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 7 days]; ii) Chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with one-dose of Tafenoquine [300 mg on day 1]; and iii) Artesunate and Mefloquine [100 mg and 200 mg on day 1, 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 14 days]. Patient blood was collected before treatment and 4 h, 24 h, 48 h and 72 h after treatment. The blood was used to perform a direct membrane feeding assay (DMFA) using Anopheles darlingi mosquitoes. The results showed 100% inhibition of the mosquito infection after 4 h using ASMQ+PQ, after 24 h for the combination of CQ+PQ and 48 h using CQ+TQ. The density of gametocytes declined over time in all treatment groups, although the decline was more rapid in the ASMQ+PQ group. In conclusion, it was possible to demonstrate the transmission-blocking efficacy of the malaria vivax treatment and that ASMQ+PQ acts faster than the two other treatments.

Population-based genomic study of Plasmodium vivax malaria in seven Brazilian states and across South America.

Background: Brazil is a unique and understudied setting for malaria, with complex foci of transmission associated with human and environmental conditions. An understanding of the population genomic diversity of P. vivax parasites across Brazil can support malaria control strategies. Methods: Through whole genome sequencing of P. vivax isolates across 7 Brazilian states, we use population genomic approaches to compare genetic diversity within country (n = 123), continent (6 countries, n = 315) and globally (26 countries, n = 885). Findings: We confirm that South American isolates are distinct, have more ancestral populations than the other global regions, with differentiating mutations in genes under selective pressure linked to antimalarial drugs (pvmdr1, pvdhfr-ts) and mosquito vectors (pvcrmp3, pvP45/48, pvP47). We demonstrate Brazil as a distinct parasite population, with signals of selection including ABC transporter (PvABCI3) and PHIST exported proteins. Interpretation: Brazil has a complex population structure, with evidence of P. simium infections and Amazonian parasites separating into multiple clusters. Overall, our work provides the first Brazil-wide analysis of P. vivax population structure and identifies important mutations, which can inform future research and control measures. Funding: AI is funded by an MRC LiD PhD studentship. TGC is funded by the Medical Research Council (Grant no. MR/M01360X/1, MR/N010469/1, MR/R025576/1, MR/R020973/1 and MR/X005895/1). SC is funded by Medical Research Council UK grants (MR/M01360X/1, MR/R025576/1, MR/R020973/1 and MR/X005895/1) and Bloomsbury SET (ref. CCF17-7779). FN is funded by The Shloklo Malaria Research Unit - part of the Mahidol Oxford Research Unit, supported by the Wellcome Trust (Grant no. 220211). ARSB is funded by São Paulo Research Foundation - FAPESP (Grant no. 2002/09546-1). RLDM is funded by Brazilian National Council for Scientific and Technological Development - CNPq (Grant no. 302353/2003-8 and 471605/2011-5); CRFM is funded by FAPESP (Grant no. 2020/06747-4) and CNPq (Grant no. 302917/2019-5 and 408636/2018-1); JGD is funded by FAPESP fellowships (2016/13465-0 and 2019/12068-5) and CNPq (Grant no. 409216/2018-6).

New Strategies for Novel Drugs: Antimicrobial Peptides Containing Ferrocene with Improved Antifungal and Antiplasmodial Biological Activity.

BACKGROUND: Fungal and parasitic diseases are global health problems, and the available treatments are becoming ineffective, mainly due to the emergence of resistant strains of pathogens. Furthermore, the drugs currently in use exhibit high toxicity and side effects. The scarcity of efficient treatments for fungal and parasitic diseases has motivated the search for new drug candidates, including antimicrobial peptides. The chemokine class RP1 peptide shows inhibitory activity against bacteria, viruses, cancer cells and parasites. In addition, the organometallic compound ferrocene showed antiparasitic activity. OBJECTIVE: Study aimed to assess the effect of conjugation of the RP1 peptide with ferrocene in terms of its structure, biological activity against fungi and parasites and toxicity. METHODS: Peptides and conjugates were synthesized using solid phase peptide synthesis (SPPS). The Fc-RP1 peptide showed antifungal and antimalarial activities with low toxicity in the U87 and HepG2 cell lines. RESULTS: The mechanism of action of these peptides, analyzed by flow cytometry in the fungus Cryptococcus neoformans, was through membrane permeabilization, with an emphasis on the Fc-RP1 peptide that presented the highest rate of PI-positive cell marking. CONCLUSION: In conclusion, ferrocene conjugated to antimicrobial peptide RP1 is an attractive biomolecule for drug discovery against fungal and parasitic diseases.

New Isoquinoline Alkaloids from Paraphaeosphaeria sporulosa F03, a Fungal Endophyte Isolated from Paepalanthus planifolius.

As part of our continuing efforts to discover new bioactive compounds from endophytic fungal sources, we have investigated the extract of the Paraphaeosphaeria sporulosa F03 strain. The study led to the isolation of four new 3-methyl-isoquinoline alkaloids (1:  - 4: ) and four known polyketides (5:  - 8: ). The structures of compounds 1:  - 4: were elucidated by 1D and 2D NMR experiments and HRMS analysis. The absolute configuration of 4: was determined by comparison of its experimental electronic circular dichroism spectrum with calculated data. Compounds 1:  - 4: exhibited antifungal activity with minimal inhibitory concentration values ranging from 6.25 - 50 µg/mL against six Candida species but they did not present any cytotoxic activity against the human tumor cell lines A549 (lung), MCF-7 (breast), and HepG2 (hepatocellular). In addition, compound 4: exhibited antiplasmodial activity in the low micromolar range (IC50 = 4 µM).

A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Plasmodium vivax Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Plasmodium berghei Parasite.

Infections with Plasmodium vivax are predominant in the Americas, representing 75% of malaria cases. Previously perceived as benign, malaria vivax is, in fact, a highly debilitating and economically important disease. Considering the high complexity of the malaria parasite life cycle, it has been hypothesized that an effective vaccine formulation against Plasmodium should contain multiple antigens expressed in different parasite stages. Based on that, we analyzed a recombinant P. vivax vaccine formulation mixing the apical membrane antigen 1 ectodomain (PvAMA-1) and a full-length circumsporozoite protein (PvCSP-AllFL) previously studied by our group, which elicits a potent antibody response in mice. Genetically distinct strains of mice (C57BL/6 and BALB/c) were immunized with the proteins, alone or in combination, in the presence of poly(I:C) adjuvant, a TLR3 agonist. In C57BL/6, high-antibody titers were induced against PvAMA-1 and the three PvCSP variants (VK210, VK247, and P. vivax-like). Meanwhile, mixing PvAMA-1 with PvCSP-AllFL had no impact on total IgG antibody titers, which were long-lasting. Moreover, antibodies from immunized mice recognized VK210 sporozoites and blood-stage parasites by immunofluorescence assay. However, in the BALB/c model, the antibody response against PvCSP-AllFL was relatively low. PvAMA-1-specific CD3+CD4+ and CD3+CD8+ T-cell responses were observed in C57BL/6 mice, and the cellular response was impaired by PvCSP-AllFL combination. More relevant, the multistage vaccine formulation provided partial protection in mice challenged with a transgenic Plasmodium berghei sporozoite expressing the homologous PvCSP protein.

Primaquine-thiazolidinones block malaria transmission and development of the liver exoerythrocytic forms.

BACKGROUND: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, arenealdehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block malaria transmission to mosquitoes. RESULTS: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evaluated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day delay in the malaria pre-patent period. CONCLUSIONS: The compound 4m was the most promising, blocking malaria transmissions and reducing the number of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also caused a 3-day delay in the malaria pre-patent period.

Chemical Composition, Antiprotozoal and Cytotoxic Activities of Indole Alkaloids and Benzofuran Neolignan of Aristolochia cordigera.

This is a comparative study on the intraspecific chemical variability of Aristolochia cordigera species, collected in two different regions of Brazil, Biome Cerrado (semiarid) and Biome Amazônia (coastal). The use of GC-MS and statistical methods led to the identification of 56 compounds. A higher percentage of palmitone and germacrene-D in the hexanes extracts of the leaves of plants from these respective biomes was observed. Phytochemical studies on the extracts led to the isolation and identification of 19 known compounds, including lignans, neolignans, aristolochic acids, indole-ß-carboline, and indole alkaloids. In addition, two new indole alkaloids, 3,4-dihydro-hyrtiosulawesine and 6-O-(ß-glucopyranosyl)hyrtiosulawesine, were isolated and a new neolignan, cis-eupomatenoid-7, was obtained in a mixture with its known isomer eupomatenoid-7. Their structures were determined by spectroscopic methods, mainly by 1D- and 2D-NMR. The occurrence of indole alkaloids is being described for the first time in the Aristolochiaceae family. Moreover, the in vitro susceptibility of intracellular amastigote and promastigote forms of Leishmania amazonensis to the alkaloids and eupomatenoid-7 were evaluated. This neolignan exhibited low activity against promastigotes (IC50 = 46 µM), while the alkaloids did not show inhibitory activity. The new alkaloid 6-O-(ß-glucopyranosyl)hyrtiosulawesine exhibited activity in the low micromolar range against Plasmodium falciparum, with an IC50 value of 5 µM and a selectivity index higher than 50.

BbMP-1, a new metalloproteinase isolated from Bothrops brazili snake venom with in vitro antiplasmodial properties.

This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bß chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC50 of 3.2 ± 2.0 µg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity.

Ethnopharmacological evaluation of medicinal plants used against malaria by quilombola communities from Oriximiná, Brazil.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is the most important parasitic disease in the world, including in the Amazon region, due to its high incidence. In addition, malaria is difficult to control because of the geographical characteristics of the endemic Amazon region. The quilombola communities of Oriximina, located in remote rainforest areas, have extensive experience with medicinal plants due to their close contact with and dependence on local biodiversity as a therapeutic resource. To search for active bioproducts against malaria, based on in vitro tests using blood culture-derived parasites and plants selected by an ethno-directed approach in traditional quilombola communities of Oriximiná, in the Amazon region of Brazil. MATERIALS AND METHODS: Ethnobotanical data were collected from 35 informants in the quilombola communities of Oriximiná, Brazil, by a free-listing method for the survey of species locally indicated to be effective against malaria and related symptoms. Data were analyzed by salience index (S) and major use agreement. The activity of extracts from 11 plants, selected based on their Salience values (four plants with S>1; seven plants with S<0.1), was measured in vitro in cultures of W2 clone Plasmodium falciparum parasites resistant to chloroquine. RESULTS: Thirty-five ethnospecies comprising 40 different plants belonging to 23 botanical families and 37 genera were listed as antimalarials by the ethno-directed approach. Among these, 11 species selected based on their S values were assayed against P. falciparum. The most active plant extracts, with an IC50 as low as 1.6µg/mL, were obtained from Aspidosperma rigidum (Apocynaceae), Bertholletia excelsa (Lecythidaceae) and Simaba cedron (Simaroubaceae), all of which displayed an S value>1. CONCLUSION: A strong correlation between the consensus of the informants from quilombola communities living in a malaria endemic area and the salience index indicating antiplasmodial activity was observed, where the ethnospecies mostly cited to be effective against malaria produced the most active plant extracts in vitro. It was also evident from the data that these groups approached the treatment of malaria with an holistic view, making use of purgative, depurative, emetic and adaptogen plants.

Plasmodium vivax and Plasmodium falciparum ex vivo susceptibility to anti-malarials and gene characterization in Rondônia, West Amazon, Brazil.

BACKGROUND: Chloroquine (CQ), a cost effective antimalarial drug with a relatively good safety profile and therapeutic index, is no longer used by itself to treat patients with Plasmodium falciparum due to CQ-resistant strains. P. vivax, representing over 90% of malaria cases in Brazil, despite reported resistance, is treated with CQ as well as with primaquine to block malaria transmission and avoid late P. vivax malaria relapses. Resistance to CQ and other antimalarial drugs influences malaria control, thus monitoring resistance phenotype by parasite genotyping is helpful in endemic areas. METHODS: A total of 47 P. vivax and nine P. falciparum fresh isolates were genetically characterized and tested for CQ, mefloquine (MQ) and artesunate (ART) susceptibility in vitro. The genes mdr1 and pfcrt, likely related to CQ resistance, were analyzed in all isolates. Drug susceptibility was determined using short-term parasite cultures of ring stages for 48 to 72 hour and thick blood smears counts. Each parasite isolate was tested with the antimalarials to measure the geometric mean of 50% inhibitory concentration. RESULTS: The low numbers of P. falciparum isolates reflect the species prevalence in Brazil; most displayed low sensitivity to CQ (IC50 70 nM). However, CQ resistance was rare among P. vivax isolates (IC50 of 32 nM). The majority of P. vivax and P. falciparum isolates were sensitive to ART and MQ. One hundred percent of P. falciparum isolates carried non-synonymous mutations in the pfmdr1 gene in codons 184, 1042 and 1246, 84% in codons 1034 and none in codon 86, a well-known resistance mutation. For the pfcrt gene, mutations were observed in codons 72 and 76 in all P. falciparum isolates. One P. falciparum isolate from Angola, Africa, showing sensitivity to the antimalarials, presented no mutations. In P. vivax, mutations of pvmdr1 and the multidrug resistance gene 1 marker at codon F976 were absent. CONCLUSION: All P. falciparum Brazilian isolates showed CQ resistance and presented non-synonymous mutations in pfmdr1 and pfcrt. CQ resistant genotypes were not present among P. vivax isolates and the IC50 values were low in all samples of the Brazilian West Amazon.

New approaches in antimalarial drug discovery and development: a review.

Malaria remains a major world health problem following the emergence and spread of Plasmodium falciparum that is resistant to the majority of antimalarial drugs. This problem has since been aggravated by a decreased sensitivity of Plasmodium vivax to chloroquine. This review discusses strategies for evaluating the antimalarial activity of new compounds in vitro and in animal models ranging from conventional tests to the latest high-throughput screening technologies. Antimalarial discovery approaches include the following: the discovery of antimalarials from natural sources, chemical modifications of existing antimalarials, the development of hybrid compounds, testing of commercially available drugs that have been approved for human use for other diseases and molecular modelling using virtual screening technology and docking. Using these approaches, thousands of new drugs with known molecular specificity and active against P. falciparum have been selected. The inhibition of haemozoin formation in vitro, an indirect test that does not require P. falciparum cultures, has been described and this test is believed to improve antimalarial drug discovery. Clinical trials conducted with new funds from international agencies and the participation of several industries committed to the eradication of malaria should accelerate the discovery of drugs that are as effective as artemisinin derivatives, thus providing new hope for the control of malaria.

Synthesis and antimalarial activity of thioetherhydroxyethylsulfonamides, potential aspartyl protease inhibitors, Part 3.

A series of novel thioetherhydroxyethylsulfonamide derivatives has been synthesized from the coupling of intermediates 3-amino-4-phenyl-1-thioetherazine-butan-2-oles 6,7 with arenesulfonyl chlorides in good yields. Characterizations of products were achieved by NMR techniques and specifically for compound 8e by X-ray crystallography. Preliminary results of antimalarial activity in vitro against the Plasmodium falciparum W2 clone (chloroquine resistant and mefloquine sensitive) showed moderate activity for hydroxyethylsulfonamide 8f. In addition, none of the compounds tested showed cytotoxicity at high concentration tested against HepG2 and BGM cell lines.