Whole genome sequencing identifies novel mutations in malaria parasites resistant to artesunate (ATN) and to ATN + mefloquine combination.

Introduction: The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease. Methods: Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine. Results and discussion: Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.

Mutation in the 26S proteasome regulatory subunit rpn2 gene in Plasmodium falciparum confers resistance to artemisinin.

Introduction: Malaria parasites increasingly develop resistance to all drugs available in the market, hampering the goal of reducing malaria burden. Methods: Herein, we evaluated the impact of a single-nucleotide variant, E738K, present in the 26S proteasome regulatory subunit rpn2 gene, identified in Plasmodium chabaudi resistant parasites. Plasmids carrying a functional rpn2 interspecies chimeric gene with 5' recombination region from P. falciparum and 3' from P. chabaudi were constructed and transfected into Dd2 P. falciparum parasites. Results and discussion: The 738K variant parasite line presented increased parasite survival when subjected to dihydroartemisinin (DHA), as well as increased chymotrypsin-like activity and decreased accumulation of polyubiquitinated proteins. We thus conclude that the ubiquitin-proteasome pathway, including the 738K variant, play an important role in parasite response to DHA, being the first report of a mutation in a potential DHA drug target enhancing parasite survival and contributing to a significant advance in the understanding the biology of artemisinin resistance.

Antimalarial and Cytotoxic Activity of Native Plants Used in Cabo Verde Traditional Medicine.

Medicinal plants have historically been a source of drugs in multiple applications, including the treatment of malaria infections. The Cabo Verde archipelago harbors a rich diversity of native plants, most of which are used for medicinal purposes. The present study investigated the in vitro antiplasmodial activities of four native plants from Cabo Verde (i.e., Artemisia gorgonum, Lavandula rotundifolia, Sideroxylon marginatum, and Tamarix senegalensis). Traditional preparations of these medicinal plants, namely aqueous extracts (infusions) and ethanolic extracts, were tested against both chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains using the SYBR Green detection method. The in vitro cytotoxicity was evaluated in Caco-2 and PLP2 cells using a sulforhodamine B colorimetric assay. An ethanolic extract of A. gorgonum and infusions of T. senegalensis exhibited high antiplasmodial activities (EC50 < 5 µg/mL) without cytotoxicity (GI50 > 400 µg/mL). Extracts of L. rotundifolia and S. marginatum exhibited moderate activities, with EC50 values ranging from 10-30 µg/mL. The A. gorgonum ethanolic extract showed activity toward early ring stages, and parasites treated with the T. senegalensis infusions progressed to the early trophozoite stage, although did not develop further to the late trophozoite or schizont stages. Antimalarial activities and the lack of cytotoxicity of the extracts are reported in the present study and support previous claims by traditional practitioners for the use of these plants against malaria while suggesting their ethnopharmacological usefulness as future antimalarials.

Evaluation of the Polyphenolic Composition and Bioactivities of Three Native Cabo Verde Medicinal Plants.

The use of medicinal plants in a variety of health conditions remains essential for the discovery of new treatments. The present study aimed to investigate the bioactive properties of three native plants from Cabo Verde Islands, namely Artemisia gorgonum Webb, Sideroxylon marginatum (Decne. ex Webb) Cout., and Tamarix senegalensis DC., contributing to the characterization of less-known medicinal plants and their potential benefits for human health. Known compounds, such as kaempferol, quercetin, caffeyolquinic, and apigenin derivatives, among others, were detected in the plant species under study. Overall, all species demonstrated good antioxidant capacity, especially the ethanolic extracts of A. gorgonum (EC50 = 0.149 mg/mL) in TBARS assay. Moreover, the ethanolic extracts of the studied plants showed cytotoxic properties against tumor cells, and again the A. gorgonum extract proved to be the most effective in inhibiting tumor growth, mainly in the CaCO2 (GI50 = 17.3 µg/mL) and AGS (GI50 = 18.2 µg/mL) cell lines. Only the ethanolic extracts of T. senegalensis and S. marginatum demonstrated anti-inflammatory activity, albeit weak (EC50 = 35 and 43 µg/mL, respectively). The present study contributed to increased knowledge about the bioactive properties of these plants commonly used in traditional medicine, some of which was discussed for the first time, opening new perspectives for their use in a wider range of health conditions, especially in African countries, where access to modern health care is more limited.

On the contribution of the rodent model Plasmodium chabaudi for understanding the genetics of drug resistance in malaria.

Malaria is a devastating disease that still claims over half a million lives every year, mostly in sub-Saharan Africa. One of the main barriers to malaria control is the evolution and propagation of drug-resistant mutant parasites. Knowing the genes and respective mutations responsible for drug resistance facilitates the design of drugs with novel modes of action and allows predicting and monitoring drug resistance in natural parasite populations in real-time. The best way to identify these mutations is to experimentally evolve resistance to the drug in question and then comparing the genomes of the drug-resistant mutants to that of the sensitive progenitor parasites. This simple evolutive concept was the starting point for the development of a paradigm over the years, based on the use of the rodent malaria parasite Plasmodium chabaudi to unravel the genetics of drug resistance in malaria. It involves the use of a cloned parasite isolate (P. chabaudi AS) whose genome is well characterized, to artificially select resistance to given drugs through serial passages in mice under slowly increasing drug pressure. The end resulting parasites are cloned and the genetic mutations are then discovered through Linkage Group Selection, a technique conceived by Prof. Richard Carter and his group, and/or Whole Genome Sequencing. The precise role of these mutations can then be interrogated in malaria parasites of humans through allelic replacement experiments and/or genotype-phenotype association studies in natural parasite populations. Using this paradigm, all the mutations underlying resistance to the most important antimalarial drugs were identified, most of which were pioneering and later shown to also play a role in drug resistance in natural infections of human malaria parasites. This supports the use of P. chabaudi a fast-track predictive model to identify candidate genetic markers of resistance to present and future antimalarial drugs and improving our understanding of the biology of resistance.

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity.

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

Atlantic Forest Malaria: A Review of More than 20 Years of Epidemiological Investigation.

In the south and southeast regions of Brazil, cases of malaria occur outside the endemic Amazon region near the Atlantic Forest in some coastal states, where Plasmodium vivax is the recognized parasite. Characteristics of cases and vectors, especially Anopheles (Kerteszia) cruzii, raise the hypothesis of a zoonosis with simians as reservoirs. The present review aims to report on investigations of the disease over a 23-year period. Two main sources have provided epidemiological data: the behavior of Anopheles vectors and the genetic and immunological aspects of Plasmodium spp. obtained from humans, Alouatta simians, and Anopheles spp. mosquitoes. Anopheles (K.) cruzii is the most captured species in the forest canopy and is the recognized vector. The similarity between P. vivax and Plasmodium simium and that between Plasmodium malariae and Plasmodium brasilianum shared between simian and human hosts and the involvement of the same vector in the transmission to both hosts suggest interspecies transfer of the parasites. Finally, recent evidence points to the presence of Plasmodium falciparum in a silent cycle, detected only by molecular methods in asymptomatic individuals and An. (K.) cruzii. In the context of malaria elimination, it is paramount to assemble data about transmission in such non-endemic low-incidence areas.

Computational Chemogenomics Drug Repositioning Strategy Enables the Discovery of Epirubicin as a New Repurposed Hit for Plasmodium falciparum and P. vivax.

Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii Transmission-blocking activity was observed for epirubicin in vitro and in vivo Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery.

Efflux pump inhibitors as a promising adjunct therapy against drug resistant tuberculosis: a new strategy to revisit mycobacterial targets and repurpose old drugs.

INTRODUCTION: In 2018, an estimated 377,000 people developed multidrug-resistant tuberculosis (MDR-TB), urging for new effective treatments. In the last years, it has been accepted that efflux pumps play an important role in the evolution of drug resistance. Strategies are required to mitigate the consequences of the activity of efflux pumps. AREAS COVERED: Based upon the literature available in PubMed, up to February 2020, on the diversity of efflux pumps in Mycobacterium tuberculosis and their association with drug resistance, studies that identified efflux inhibitors and their effect on restoring the activity of antimicrobials subjected to efflux are reviewed. These support a new strategy for the development of anti-TB drugs, including efflux inhibitors, using in silico drug repurposing. EXPERT OPINION: The current literature highlights the contribution of efflux pumps in drug resistance in M. tuberculosis and that efflux inhibitors may help to ensure the effectiveness of anti-TB drugs. However, despite the usefulness of efflux inhibitors in in vitro studies, in most cases their application in vivo is restricted due to toxicity. In a time when new drugs are needed to fight MDR-TB and extensively drug-resistant TB, cost-effective strategies to identify safer efflux inhibitors should be implemented in drug discovery programs.

Plasmodium falciparum malarial parasites from Brazil lack artemisinin resistance-associated mutations in the kelch13 gene.

INTRODUCTION: Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.

In Silico Chemogenomics Drug Repositioning Strategies for Neglected Tropical Diseases.

Only ~1% of all drug candidates against Neglected Tropical Diseases (NTDs) have reached clinical trials in the last decades, underscoring the need for new, safe and effective treatments. In such context, drug repositioning, which allows finding novel indications for approved drugs whose pharmacokinetic and safety profiles are already known, emerging as a promising strategy for tackling NTDs. Chemogenomics is a direct descendent of the typical drug discovery process that involves the systematic screening of chemical compounds against drug targets in high-throughput screening (HTS) efforts, for the identification of lead compounds. However, different to the one-drug-one-target paradigm, chemogenomics attempts to identify all potential ligands for all possible targets and diseases. In this review, we summarize current methodological development efforts in drug repositioning that use state-of-the-art computational ligand- and structure-based chemogenomics approaches. Furthermore, we highlighted the recent progress in computational drug repositioning for some NTDs, based on curation and modeling of genomic, biological, and chemical data. Additionally, we also present in-house and other successful examples and suggest possible solutions to existing pitfalls.

Plasmodium falciparum malarial parasites from Brazil lack artemisinin resistance-associated mutations in the kelch13 gene

Abstract INTRODUCTION Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.

Is there still room to explore cyclodextrin glycosyltransferase-producers in Brazilian biodiversity?

In the present work, different Brazilian biomes aiming to identify and select cyclodextrin glycosyltransferase-producer bacteria are explored. This enzyme is responsible for converting starch to cyclodextrin, which are interesting molecules to carry other substances of economic interest applied by textile, pharmaceutical, food, and other industries. Based on the enzymatic index, 12 bacteria were selected and evaluated, considering their capacity to produce the enzyme in culture media containing different starch sources. It was observed that the highest yields were presented by the bacteria when grown in cornstarch. These bacteria were also characterized by sequencing of the 16S rRNA region and were classified as Bacillus, Paenibacillus, Gracilibacillus and Solibacillus.

QSAR-Driven Design and Discovery of Novel Compounds With Antiplasmodial and Transmission Blocking Activities.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium, affecting more than 200 million people worldwide every year and leading to about a half million deaths. Malaria parasites of humans have evolved resistance to all current antimalarial drugs, urging for the discovery of new effective compounds. Given that the inhibition of deoxyuridine triphosphatase of Plasmodium falciparum (PfdUTPase) induces wrong insertions in plasmodial DNA and consequently leading the parasite to death, this enzyme is considered an attractive antimalarial drug target. Using a combi-QSAR (quantitative structure-activity relationship) approach followed by virtual screening and in vitro experimental evaluation, we report herein the discovery of novel chemical scaffolds with in vitro potency against asexual blood stages of both P. falciparum multidrug-resistant and sensitive strains and against sporogonic development of P. berghei. We developed 2D- and 3D-QSAR models using a series of nucleosides reported in the literature as PfdUTPase inhibitors. The best models were combined in a consensus approach and used for virtual screening of the ChemBridge database, leading to the identification of five new virtual PfdUTPase inhibitors. Further in vitro testing on P. falciparum multidrug-resistant (W2) and sensitive (3D7) parasites showed that compounds LabMol-144 and LabMol-146 demonstrated fair activity against both strains and presented good selectivity versus mammalian cells. In addition, LabMol-144 showed good in vitro inhibition of P. berghei ookinete formation, demonstrating that hit-to-lead optimization based on this compound may also lead to new antimalarials with transmission blocking activity.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate.

BACKGROUND: Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. RESULTS: Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity. CONCLUSIONS: The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Outbreak of human malaria caused by Plasmodium simium in the Atlantic Forest in Rio de Janeiro: a molecular epidemiological investigation.

BACKGROUND: Malaria was eliminated from southern and southeastern Brazil over 50 years ago. However, an increasing number of autochthonous episodes attributed to Plasmodium vivax have recently been reported from the Atlantic Forest region of Rio de Janeiro state. As the P vivax-like non-human primate malaria parasite species Plasmodium simium is locally enzootic, we performed a molecular epidemiological investigation to determine whether zoonotic malaria transmission is occurring. METHODS: We examined blood samples from patients presenting with signs or symptoms suggestive of malaria as well as from local howler monkeys by microscopy and PCR. Samples were included from individuals if they had a history of travel to or resided in areas within the Rio de Janeiro Atlantic Forest, but not if they had malaria prophylaxis, blood transfusion or tissue or organ transplantation, or had travelled to known malaria endemic areas in the preceding year. Additionally, we developed a molecular assay based on sequencing of the parasite mitochondrial genome to distinguish between P vivax and P simium, and applied this assay to 33 cases from outbreaks that occurred in 2015, and 2016. FINDINGS: A total of 49 autochthonous malaria cases were reported in 2015-16. Most patients were male, with a mean age of 44 years (SD 14·6), and 82% lived in urban areas of Rio de Janeiro state and had visited the Atlantic Forest for leisure or work-related activities. 33 cases were used for mitochondrial DNA sequencing. The assay was successfully performed for 28 samples, and all were shown to be P simium, indicative of zoonotic transmission of this species to human beings in this region. Sequencing of the whole mitochondrial genome of three of these cases showed that P simium is most closely related to P vivax parasites from South America. The malaria outbreaks in this region were caused by P simium, previously considered to be a monkey-specific malaria parasite, related to but distinct from P vivax, and which has never conclusively been shown to infect people before. INTERPRETATION: This unequivocal demonstration of zoonotic transmission, 50 years after the only previous report of P simium in people, leads to the possibility that this parasite has always infected people in this region, but that it has been consistently misdiagnosed as P vivax because of an absence of molecular typing techniques. Thorough screening of local non-human primates and mosquitoes (Anopheline) is required to evaluate the extent of this newly recognised zoonotic threat to public health and malaria elimination in Brazil. FUNDING: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado de Rio de Janeiro, The Brazilian National Council for Scientific and Technological Development (CNPq), JSPS Grant-in-Aid for scientific research, Secretary for Health Surveillance of the Brazilian Ministry of Health, Global Fund, Fundaçao de amparo à pesquisa do estado de Minas Gerais (Fapemig), and PRONEX Program of the CNPq.

Chalcone Derivatives: Promising Starting Points for Drug Design.

Medicinal chemists continue to be fascinated by chalcone derivatives because of their simple chemistry, ease of hydrogen atom manipulation, straightforward synthesis, and a variety of promising biological activities. However, chalcones have still not garnered deserved attention, especially considering their high potential as chemical sources for designing and developing new effective drugs. In this review, we summarize current methodological developments towards the design and synthesis of new chalcone derivatives and state-of-the-art medicinal chemistry strategies (bioisosterism, molecular hybridization, and pro-drug design). We also highlight the applicability of computer-assisted drug design approaches to chalcones and address how this may contribute to optimizing research outputs and lead to more successful and cost-effective drug discovery endeavors. Lastly, we present successful examples of the use of chalcones and suggest possible solutions to existing limitations.

Human lagochilascariasis-A rare helminthic disease.

Lagochilascariasis is a parasitic disease caused by a helminth of the order Ascaroidea, genus Lagochilascaris that comprises 6 species, among which only Lagochilascaris minor Leiper, 1909, is implicated in the human form of the disease. It is remarkable that the majority of cases of human lagochilascariasis in the Americas have been reported in Brazil. The natural definitive hosts of this parasite seem to be wild felines and canines. Lagochilascariasis is mostly a chronic human disease that can persist for several years, in which the parasite burrows into the subcutaneous tissues of the neck, paranasal sinuses, and mastoid. L. minor exhibits remarkable ability to migrate through the tissues of its hosts, destroying even bone tissue. Fatal cases have been described in which the parasite was found in the lungs or central nervous system. Treatment is often palliative, with recurrence of lesions. This paper summarizes the main features of the disease and its etiologic agent, including prevalence, life cycle, clinical course, and treatment.

Conformation analysis of a novel fluorinated chalcone.

Chalcones are an important class of natural compounds that exhibit numerous biological activities. In this paper, we report the synthesis and characterization of new fluorinated chalcone (FCH). The molecular geometry was determined by means of single crystal X-ray diffraction, and density functional theory (DFT) at B3LYP, M06-2X functionals and MP2 method, with the 6-311++G(d,p) basis set, was applied to optimize the ground state geometry and to study the molecular conformational stability. The molecular electrostatic potential (MEP) was also investigated at the same level of theory in order to identify and quantify the possible reactive sites. The FCH crystallizes in the centrossymmetric space group [Formula: see text] with two independent conformers (α and ß) in the asymmetric unit cell. The α conformer is arranged in planar layer whereas the ß creates a layer of non-classical dimer along c axis, that differ from α in about 11° in the orientation of phenyl groups. The stabilization of the ß conformer is achieved by C-H···π arrangement. The small energy difference between the conformers (0.086 kcal mol-1) and the absence of activation energy indicates that the conversion between them can takes place at room temperature and the ß isomer is stable only in solid state. The FCH most electrophilic site occurs on the oxygen atom from the carboxyl group with absolute MEP value of about -52 kcal mol-1 whereas the MEP value calculated for F site is about -23 kcal mol-1.