Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

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.

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

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.

Chemical Genomic Profiling Unveils the in Vitro and in Vivo Antiplasmodial Mechanism of Açaí (Euterpe oleracea Mart.) Polyphenols.

Malaria remains a major detrimental parasitic disease in the developing world, with more than 200 million cases annually. Widespread drug-resistant parasite strains push for the development of novel antimalarial drugs. Plant-derived natural products are key sources of antimalarial molecules. Euterpe oleracea Martius ("açaí") originates from Brazil and has anti-inflammatory and antineoplasic properties. Here, we evaluated the antimalarial efficacy of three phenolic fractions of açaí; total phenolics (1), nonanthocyanin phenolics (2), and total anthocyanins (3). In vitro, fraction 2 moderately inhibited parasite growth in chloroquine-sensitive (HB3) and multiresistant (Dd2) Plasmodium falciparum strains, while none of the fractions was toxic to noncancer cells. Despite the limited activity in vitro, the oral treatment with 20 mg/kg of fraction 1 reduced parasitemia by 89.4% in Plasmodium chabaudi-infected mice and prolonged survival. Contrasting in vitro and in vivo activities of 1 suggest key antiplasmodial roles for polyphenol metabolites rather than the fraction itself. Finally, we performed haploinsufficiency chemical genomic profiling (HIP) utilizing heterozygous Saccharomyces cerevisiae deletion mutants to identify molecular mechanisms of açaí fractions. HIP results indicate proteostasis as the main cellular pathway affected by fraction 2. These results open avenues to develop açaí polyphenols as potential new antimalarial candidates.

Clinical and epidemiological aspects of feline leishmaniasis in Brazil / Aspectos clínicos e epidemiológicos da leishmaniose felina no Brasil

Tegumentary and visceral leishmaniasis are severe and unfortunately common parasitic diseases in Brazil. Among domestic animals, dogs are considered the main urban reservoir of the protozoan parasites, however, there is evidence that infected cats can also contribute towards the disease pool. The number of cats diagnosed with leishmaniasis has greatly increased in the last few years, highlighting the importance of thorough investigations on the role of the cat in the epidemiological cycle of the disease and in public health related issues. The main clinical manifestations of leishmaniasis suffered by cats, even when infected with Leishmania chagasi, a viscerotropic species, are skin abnormalities, which can be confounded with multiple other diseases. Indirect ELISA should be used as a screening test in epidemiological investigations for being a sensitive technique, followed by more specific laboratory tests. The standardization and validation of rapid, economical and reproducible diagnostic methods, to be employed in epidemiological surveillance, are still required.(AU)

Leishmaniose tegumentar e visceral são doenças parasitárias graves e, infelizmente, comuns no Brasil. Entre os animais domésticos, o cão é considerado o principal reservatório urbano do parasito protozoário; no entanto, há indícios de que gatos infectados também possam contribuir para essas doenças. O número de gatos com diagnóstico de leishmaniose aumentou muito nos últimos anos, destacando a importância de investigações aprofundadas sobre o papel desse hospedeiro no ciclo epidemiológico da doença e em Saúde Pública. As principais manifestações clínicas da leishmaniose felina são anormalidades na pele, o que pode ser confundida com várias outras doenças, mesmo em casos de infecção por Leishmania chagasi, uma espécie viscerotrópica. ELISA indireto poderia ser usado como teste de triagem em investigações epidemiológicas, por ser um método sensível, seguido de exames laboratoriais mais específicos. A padronização e validação de métodos de diagnóstico rápidos, econômicos e reprodutíveis a serem empregados na vigilância epidemiológica ainda são necessárias.(AU)

Aspectos clínicos e epidemiológicos da leishmaniose felina no Brasil / Clinical and epidemiological aspects of feline leishmaniasis in Brazil

Leishmaniose tegumentar e visceral são doenças parasitárias graves e, infelizmente, comuns no Brasil. Entre os animais domésticos, o cão é considerado o principal reservatório urbano do parasito protozoário; no entanto, há indícios de que gatos infectados também possam contribuir para essas doenças. O número de gatos com diagnóstico de leishmaniose aumentou muito nos últimos anos, destacando a importância de investigações aprofundadas sobre o papel desse hospedeiro no ciclo epidemiológico da doença e em Saúde Pública. As principais manifestações clínicas da leishmaniose felina são anormalidades na pele, o que pode ser confundida com várias outras doenças, mesmo em casos de infecção por Leishmania chagasi, uma espécie viscerotrópica. ELISA indireto poderia ser usado como teste de triagem em investigações epidemiológicas, por ser um método sensível, seguido de exames laboratoriais mais específicos. A padronização e validação de métodos de diagnóstico rápidos, econômicos e reprodutíveis a serem empregados na vigilância epidemiológica ainda são necessárias.

Tegumentary and visceral leishmaniasis are severe and unfortunately common parasitic diseases in Brazil. Among domestic animals, dogs are considered the main urban reservoir of the protozoan parasites, however, there is evidence that infected cats can also contribute towards the disease pool. The number of cats diagnosed with leishmaniasis has greatly increased in the last few years, highlighting the importance of thorough investigations on the role of the cat in the epidemiological cycle of the disease and in public health related issues. The main clinical manifestations of leishmaniasis suffered by cats, even when infected with Leishmania chagasi, a viscerotropic species, are skin abnormalities, which can be confounded with multiple other diseases. Indirect ELISA should be used as a screening test in epidemiological investigations for being a sensitive technique, followed by more specific laboratory tests. The standardization and validation of rapid, economical and reproducible diagnostic methods, to be employed in epidemiological surveillance, are still required.  

Clinical and epidemiological aspects of feline leishmaniasis in Brazil / Aspectos clínicos e epidemiológicos da leishmaniose felina no Brasil

Tegumentary and visceral leishmaniasis are severe and unfortunately common parasitic diseases in Brazil. Among domestic animals, dogs are considered the main urban reservoir of the protozoan parasites, however, there is evidence that infected cats can also contribute towards the disease pool. The number of cats diagnosed with leishmaniasis has greatly increased in the last few years, highlighting the importance of thorough investigations on the role of the cat in the epidemiological cycle of the disease and in public health related issues. The main clinical manifestations of leishmaniasis suffered by cats, even when infected with Leishmania chagasi, a viscerotropic species, are skin abnormalities, which can be confounded with multiple other diseases. Indirect ELISA should be used as a screening test in epidemiological investigations for being a sensitive technique, followed by more specific laboratory tests. The standardization and validation of rapid, economical and reproducible diagnostic methods, to be employed in epidemiological surveillance, are still required.

Leishmaniose tegumentar e visceral são doenças parasitárias graves e, infelizmente, comuns no Brasil. Entre os animais domésticos, o cão é considerado o principal reservatório urbano do parasito protozoário; no entanto, há indícios de que gatos infectados também possam contribuir para essas doenças. O número de gatos com diagnóstico de leishmaniose aumentou muito nos últimos anos, destacando a importância de investigações aprofundadas sobre o papel desse hospedeiro no ciclo epidemiológico da doença e em Saúde Pública. As principais manifestações clínicas da leishmaniose felina são anormalidades na pele, o que pode ser confundida com várias outras doenças, mesmo em casos de infecção por Leishmania chagasi, uma espécie viscerotrópica. ELISA indireto poderia ser usado como teste de triagem em investigações epidemiológicas, por ser um método sensível, seguido de exames laboratoriais mais específicos. A padronização e validação de métodos de diagnóstico rápidos, econômicos e reprodutíveis a serem empregados na vigilância epidemiológica ainda são necessárias.

Aspectos clínicos e epidemiológicos da leishmaniose felina no Brasil / Clinical and epidemiological aspects of feline leishmaniasis in Brazil

Leishmaniose tegumentar e visceral são doenças parasitárias graves e, infelizmente, comuns no Brasil. Entre os animais domésticos, o cão é considerado o principal reservatório urbano do parasito protozoário; no entanto, há indícios de que gatos infectados também possam contribuir para essas doenças. O número de gatos com diagnóstico de leishmaniose aumentou muito nos últimos anos, destacando a importância de investigações aprofundadas sobre o papel desse hospedeiro no ciclo epidemiológico da doença e em Saúde Pública. As principais manifestações clínicas da leishmaniose felina são anormalidades na pele, o que pode ser confundida com várias outras doenças, mesmo em casos de infecção por Leishmania chagasi, uma espécie viscerotrópica. ELISA indireto poderia ser usado como teste de triagem em investigações epidemiológicas, por ser um método sensível, seguido de exames laboratoriais mais específicos. A padronização e validação de métodos de diagnóstico rápidos, econômicos e reprodutíveis a serem empregados na vigilância epidemiológica ainda são necessárias.

Tegumentary and visceral leishmaniasis are severe and unfortunately common parasitic diseases in Brazil. Among domestic animals, dogs are considered the main urban reservoir of the protozoan parasites, however, there is evidence that infected cats can also contribute towards the disease pool. The number of cats diagnosed with leishmaniasis has greatly increased in the last few years, highlighting the importance of thorough investigations on the role of the cat in the epidemiological cycle of the disease and in public health related issues. The main clinical manifestations of leishmaniasis suffered by cats, even when infected with Leishmania chagasi, a viscerotropic species, are skin abnormalities, which can be confounded with multiple other diseases. Indirect ELISA should be used as a screening test in epidemiological investigations for being a sensitive technique, followed by more specific laboratory tests. The standardization and validation of rapid, economical and reproducible diagnostic methods, to be employed in epidemiological surveillance, are still required.