Multicenter study of the natural history and therapeutic responses of patients with chikungunya, focusing on acute and chronic musculoskeletal manifestations - a study protocol from the clinical and applied research in Chikungunya (REPLICK network).

BACKGROUND: Chikungunya is associated with high morbidity and the natural history of symptomatic infection has been divided into three phases (acute, post-acute, and chronic) according to the duration of musculoskeletal symptoms. Although this classification has been designed to help guide therapeutic decisions, it does not encompass the complexity of the clinical expression of the disease and does not assist in the evaluation of the prognosis of severity nor chronic disease. Thus, the current challenge is to identify and diagnose musculoskeletal disorders and to provide the optimal treatment in order to prevent perpetuation or progression to a potentially destructive disease course. METHODS: The study is the first product of the Clinical and Applied Research Network in Chikungunya (REPLICK). This is a prospective, outpatient department-based, multicenter cohort study in Brazil. Four work packages were defined: i. Clinical research; ii) Translational Science - comprising immunology and virology streams; iii) Epidemiology and Economics; iv) Therapeutic Response and clinical trials design. Scheduled appointments on days 21 (D21) ± 7 after enrollment, D90 ± 15, D120 ± 30, D180 ± 30; D360 ± 30; D720 ± 60, and D1080 ± 60 days. On these visits a panel of blood tests are collected in addition to the clinical report forms to obtain data on socio-demographic, medical history, physical examination and questionnaires devoted to the evaluation of musculoskeletal manifestations and overall health are performed. Participants are asked to consent for their specimens to be maintained in a biobank. Aliquots of blood, serum, saliva, PAXgene, and when clinically indicated to be examined, synovial fluid, are stored at -80° C. The study protocol was submitted and approved to the National IRB and local IRB at each study site. DISCUSSION: Standardized and harmonized patient cohorts are needed to provide better estimates of chronic arthralgia development, the clinical spectra of acute and chronic disease and investigation of associated risk factors. This study is the largest evaluation of the long-term sequelae of individuals infected with CHIKV in the Brazilian population focusing on musculoskeletal manifestations, mental health, quality of life, and chronic pain. This information will both define disease burden and costs associated with CHIKV infection, and better inform therapeutic guidelines.

Methylprednisolone as Adjunctive Therapy for Patients Hospitalized With Coronavirus Disease 2019 (COVID-19; Metcovid): A Randomized, Double-blind, Phase IIb, Placebo-controlled Trial.

BACKGROUND: Steroid use for coronavirus disease 2019 (COVID-19) is based on the possible role of these drugs in mitigating the inflammatory response, mainly in the lungs, triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the efficacy of methylprednisolone (MP) among hospitalized patients with suspected COVID-19. METHODS: A parallel, double-blind, placebo-controlled, randomized, Phase IIb clinical trial was performed with hospitalized patients aged ≥18 years with clinical, epidemiological, and/or radiological suspected COVID-19 at a tertiary care facility in Manaus, Brazil. Patients were randomly allocated (1:1 ratio) to receive either intravenous MP (0.5 mg/kg) or placebo (saline solution) twice daily for 5 days. A modified intention-to-treat (mITT) analysis was conducted. The primary outcome was 28-day mortality. RESULTS: From 18 April to 16 June 2020, 647 patients were screened, 416 were randomized, and 393 were analyzed as mITT, with 194 individuals assigned to MP and 199 to placebo. SARS-CoV-2 infection was confirmed by reverse transcriptase polymerase chain reaction in 81.3%. The mortality rates at Day 28 were not different between groups. A subgroup analysis showed that patients over 60 years old in the MP group had a lower mortality rate at Day 28. Patients in the MP arm tended to need more insulin therapy, and no difference was seen in virus clearance in respiratory secretion until Day 7. CONCLUSIONS: The findings of this study suggest that a short course of MP in hospitalized patients with COVID-19 did not reduce mortality in the overall population. CLINICAL TRIALS REGISTRATION: NCT04343729.

Covid-19 Automated Diagnosis and Risk Assessment through Metabolomics and Machine Learning.

COVID-19 is still placing a heavy health and financial burden worldwide. Impairment in patient screening and risk management plays a fundamental role on how governments and authorities are directing resources, planning reopening, as well as sanitary countermeasures, especially in regions where poverty is a major component in the equation. An efficient diagnostic method must be highly accurate, while having a cost-effective profile. We combined a machine learning-based algorithm with mass spectrometry to create an expeditious platform that discriminate COVID-19 in plasma samples within minutes, while also providing tools for risk assessment, to assist healthcare professionals in patient management and decision-making. A cross-sectional study enrolled 815 patients (442 COVID-19, 350 controls and 23 COVID-19 suspicious) from three Brazilian epicenters from April to July 2020. We were able to elect and identify 19 molecules related to the disease's pathophysiology and several discriminating features to patient's health-related outcomes. The method applied for COVID-19 diagnosis showed specificity >96% and sensitivity >83%, and specificity >80% and sensitivity >85% during risk assessment, both from blinded data. Our method introduced a new approach for COVID-19 screening, providing the indirect detection of infection through metabolites and contextualizing the findings with the disease's pathophysiology. The pairwise analysis of biomarkers brought robustness to the model developed using machine learning algorithms, transforming this screening approach in a tool with great potential for real-world application.

Machine Learning Models Identify Inhibitors of SARS-CoV-2.

With the rapidly evolving SARS-CoV-2 variants of concern, there is an urgent need for the discovery of further treatments for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need, and numerous compounds have already been selected for in vitro testing by several groups. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein, we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA-approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, lumefantrine, an antimalarial was selected for testing and showed limited antiviral activity in cell-based assays while demonstrating binding (Kd 259 nM) to the spike protein using microscale thermophoresis. Several other compounds which we prioritized have since been tested by others and were also found to be active in vitro. This combined machine learning and in vitro testing approach can be expanded to virtually screen available molecules with predicted activity against SARS-CoV-2 reference WIV04 strain and circulating variants of concern. In the process of this work, we have created multiple iterations of machine learning models that can be used as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 with over 500 compounds is now freely available at www.assaycentral.org.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

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.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria.

BACKGROUND: Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE: To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS: Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS: Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1ß and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS: Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Administration of endothelial progenitor cells accelerates the resolution of arterial thrombus in mice.

BACKGROUND: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl3 lesion and in vessel re-endothelization in the mouse carotid artery. METHODS: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation. RESULTS: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro. CONCLUSIONS: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process.

Acute hemolytic vascular inflammatory processes are prevented by nitric oxide replacement or a single dose of hydroxyurea.

Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

MyD88 signaling is directly involved in the development of murine placental malaria.

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.

A decade of malaria during pregnancy in Brazil: what has been done concerning prevention and management.

In Brazil, malaria remains a disease of major epidemiological importance because of the high number of cases in the Amazonian Region. Plasmodium spp infections during pregnancy are a significant public health problem with substantial risks for the pregnant woman, the foetus and the newborn child. In Brazil, the control of malaria during pregnancy is primarily achieved by prompt and effective treatment of the acute episodes. Thus, to assure rapid diagnosis and treatment for pregnant women with malaria, one of the recommended strategy for low transmission areas by World Health Organization and as part of a strategy by the Ministry of Health, the National Malaria Control Program has focused on integrative measures with woman and reproductive health. Here, we discuss the approach for the prevention and management of malaria during pregnancy in Brazil over the last 10 years (2003-2012) using morbidity data from Malaria Health Information System. Improving the efficiency and quality of healthcare and education and the consolidation of prevention programmes will be challenges in the control of malaria during pregnancy in the next decade.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon.

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Amazonian plant natural products: perspectives for discovery of new antimalarial drug leads.

Plasmodium falciparum and P. vivax malaria parasites are now resistant, or showing signs of resistance, to most drugs used in therapy. Novel chemical entities that exhibit new mechanisms of antiplasmodial action are needed. New antimalarials that block transmission of Plasmodium spp. from humans to Anopheles mosquito vectors are key to malaria eradication efforts. Although P. vivax causes a considerable number of malaria cases, its importance has for long been neglected. Vivax malaria can cause severe manifestations and death; hence there is a need for P. vivax-directed research. Plants used in traditional medicine, namely Artemisia annua and Cinchona spp. are the sources of the antimalarial natural products artemisinin and quinine, respectively. Based on these compounds, semi-synthetic artemisinin-derivatives and synthetic quinoline antimalarials have been developed and are the most important drugs in the current therapeutic arsenal for combating malaria. In the Amazon region, where P. vivax predominates, there is a local tradition of using plant-derived preparations to treat malaria. Here, we review the current P. falciparum and P. vivax drug-sensitivity assays, focusing on challenges and perspectives of drug discovery for P. vivax, including tests against hypnozoites. We also present the latest findings of our group and others on the antiplasmodial and antimalarial chemical components from Amazonian plants that may be potential drug leads against malaria.

Association of Ang/Tie2 pathway mediators with endothelial barrier integrity and disease severity in COVID-19.

Endothelial barrier (EB) disruption contributes to acute lung injury in COVID-19, and levels of both VEGF-A and Ang-2, which are mediators of EB integrity, have been associated with COVID-19 severity. Here we explored the participation of additional mediators of barrier integrity in this process, as well as the potential of serum from COVID-19 patients to induce EB disruption in cell monolayers. In a cohort from a clinical trial consisting of thirty patients with COVID-19 that required hospital admission due to hypoxia we demonstrate that i) levels of soluble Tie2 were increase, and of soluble VE-cadherin were decreased when compared to healthy individuals; ii) sera from these patients induce barrier disruption in monolayers of endothelial cells; and iii) that the magnitude of this effect is proportional to disease severity and to circulating levels of VEGF-A and Ang-2. Our study confirms and extends previous findings on the pathogenesis of acute lung injury in COVID-19, reinforcing the concept that EB is a relevant component of this disease. Our results pave the way for future studies that can refine our understanding of the pathogenesis of acute lung injury in viral respiratory disorders, and contribute to the identification of new biomarkers and therapeutic targets for these conditions.

Update and elucidation of Plasmodium kinomes: Prioritization of kinases as potential drug targets for malaria.

Malaria is a tropical disease caused by Plasmodium spp. and transmitted by the bite of infected Anopheles mosquitoes. Protein kinases (PKs) play key roles in the life cycle of the etiological agent of malaria, turning these proteins attractive targets for antimalarial drug discovery campaigns. As part of an effort to understand parasite signaling functions, we report the results of a bioinformatics pipeline analysis of PKs of eight Plasmodium species. To date, no P. malariae and P. ovale kinome assemble has been conducted. We classified, curated and annotated predicted kinases to update P. falciparum, P. vivax, P. yoelii, P. berghei, P. chabaudi, and P. knowlesi kinomes published to date, as well as report for the first time the kinomes of P. malariae and P. ovale. Overall, from 76 to 97 PKs were identified among all Plasmodium spp. kinomes. Most of the kinases were assigned to seven of nine major kinase groups: AGC, CAMK, CMGC, CK1, STE, TKL, OTHER; and the Plasmodium-specific group FIKK. About 30% of kinases have been deeply classified into group, family and subfamily levels and only about 10% remained unclassified. Furthermore, updating and comparing the kinomes of P. vivax and P. falciparum allowed for the prioritization and selection of kinases as potential drug targets that could be explored for discovering new drugs against malaria. This integrated approach resulted in the selection of 37 protein kinases as potential targets and the identification of investigational compounds with moderate in vitro activity against asexual P. falciparum (3D7 and Dd2 strains) stages that could serve as starting points for the search of potent antimalarial leads in the future.

Computational and Experimental Approaches Identify Beta-Blockers as Potential SARS-CoV-2 Spike Inhibitors.

Finding antivirals for SARS-CoV-2 is still a major challenge, and many computational and experimental approaches have been employed to find a solution to this problem. While the global vaccination campaigns are the primary driver of controlling the current pandemic, orally bioavailable small-molecule drugs and biologics are critical to overcome this global issue. Improved therapeutics and prophylactics are required to treat people with circulating and emerging new variants, addressing severe infection, and people with underlying or immunocompromised conditions. The SARS-CoV-2 envelope spike is a challenging target for viral entry inhibitors. Pindolol presented a good docking score in a previous virtual screening using computational docking calculations after screening a Food and Drug Administration (FDA)-approved drug library of 2400 molecules as potential candidates to block the SARS-CoV-2 spike protein interaction with the angiotensin-converting enzyme 2 (ACE-2). Here, we expanded the computational evaluation to identify five beta-blockers against SARS-CoV-2 using several techniques, such as microscale thermophoresis, NanoDSF, and in vitro assays in different cell lines. These data identified carvedilol with a K d of 364 ± 22 nM for the SARS-CoV-2 spike and in vitro activity (EC50 of 7.57 µM, CC50 of 18.07 µM) against SARS-CoV-2 in Calu-3 cells. We have shown how we can apply multiple computational and experimental approaches to find molecules that can be further optimized to improve anti-SARS-CoV-2 activity.

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein.

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases.

The discovery and development of a new drug is a complex, time consuming and costly process that typically takes over 10 years and costs around 1 billion dollars from bench to market. This scenario makes the discovery of novel drugs targeting neglected tropical diseases (NTDs), which afflict in particular people in low-income countries, prohibitive. Despite the intensive use of High-Throughput Screening (HTS) in the past decades, the speed with which new drugs come to the market has remained constant, generating doubts about the efficacy of this approach. Here we review a few of the yeast-based high-throughput approaches that can work synergistically with parasite-based, in vitro, or in silico methods to identify and optimize novel antiparasitic compounds. These yeast-based methods range from HTP screens to identify novel hits against promising parasite kinase targets to the identification of potential antiparasitic kinase inhibitors extracted from databases of yeast chemical genetic screens.

QSAR-Based Virtual Screening of Natural Products Database for Identification of Potent Antimalarial Hits.

With about 400,000 annual deaths worldwide, malaria remains a public health burden in tropical and subtropical areas, especially in low-income countries. Selection of drug-resistant Plasmodium strains has driven the need to explore novel antimalarial compounds with diverse modes of action. In this context, biodiversity has been widely exploited as a resourceful channel of biologically active compounds, as exemplified by antimalarial drugs such as quinine and artemisinin, derived from natural products. Thus, combining a natural product library and quantitative structure-activity relationship (QSAR)-based virtual screening, we have prioritized genuine and derivative natural compounds with potential antimalarial activity prior to in vitro testing. Experimental validation against cultured chloroquine-sensitive and multi-drug-resistant P. falciparum strains confirmed the potent and selective activity of two sesquiterpene lactones (LDT-597 and LDT-598) identified in silico. Quantitative structure-property relationship (QSPR) models predicted absorption, distribution, metabolism, and excretion (ADME) and physiologically based pharmacokinetic (PBPK) parameters for the most promising compound, showing that it presents good physiologically based pharmacokinetic properties both in rats and humans. Altogether, the in vitro parasite growth inhibition results obtained from in silico screened compounds encourage the use of virtual screening campaigns for identification of promising natural compound-based antimalarial molecules.