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.
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.
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.
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.
NCT04343729.
Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes ...(encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells.
Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein.
We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs.
Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein ...function.
We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1).
STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs.
We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants.
This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.
Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently ...undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0 μM). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low μM range (IC50 2.8 and 5.3 μM, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50 > 200 μM), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.
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•Plasmodium falciparum DHODH is a validated target for malaria treatment.•New PfDHODH inhibitors are designed and synthetized using hydroxyazole scaffolds.•X-ray crystallography determined the binding mode inside the PfDHODH binding site.•Selectively over the human isoform, the hydroxypyrazole 7e inhibit PfDHODH (IC50 = 2.8 μM).•3 best compounds were tested against Pf-infected erythrocytes (EC50 between 26 and 40 μM).
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
, 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
CK2α inhibitors, we utilized an
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 (
), 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
inhibits yeast Cka1, which has a hinge region with high similarity to
CK2α. This finding is in agreement with our
results suggesting that
inhibits
CK2α via hinge region interaction.
Abstract
The malaria parasite uses actin-based mechanisms throughout its lifecycle to control a range of biological processes including intracellular trafficking, gene regulation, parasite motility ...and invasion. In this work we assign functions to the
Plasmodium falciparum
formins 1 and 2 (FRM1 and FRM2) proteins in asexual and sexual blood stage development. We show that FRM1 is essential for merozoite invasion and FRM2 is required for efficient cell division. We also observed divergent functions for FRM1 and FRM2 in gametocyte development. Conditional deletion of FRM1 leads to a delay in gametocyte stage progression. We show that FRM2 controls the actin and microtubule cytoskeletons in developing gametocytes, with premature removal of the protein resulting in a loss of transmissible stage V gametocytes. Lastly, we show that targeting formin proteins with the small molecule inhibitor of formin homology domain 2 (SMIFH2) leads to a multistage block in asexual and sexual stage parasite development.
Malaria is a tropical infectious disease that affects over 219 million people worldwide. Due to the constant emergence of parasitic resistance to the current antimalarial drugs, the discovery of new ...antimalarial drugs is a global health priority. Multi-target drug discovery is a promising and innovative strategy for drug discovery and it is currently regarded as one of the best strategies to face drug resistance. Aiming to identify new multi-target antimalarial drug candidates, we developed an integrative computational approach to select multi-kinase inhibitors for
calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4) and protein kinase 6 (PK6). For this purpose, we developed and validated shape-based and machine learning models to prioritize compounds for experimental evaluation. Then, we applied the best models for virtual screening of a large commercial database of drug-like molecules. Ten computational hits were experimentally evaluated against asexual blood stages of both sensitive and multi-drug resistant
strains. Among them, LabMol-171, LabMol-172, and LabMol-181 showed potent antiplasmodial activity at nanomolar concentrations (EC
≤ 700 nM) and selectivity indices >15 folds. In addition, LabMol-171 and LabMol-181 showed good
inhibition of
ookinete formation and therefore represent promising transmission-blocking scaffolds. Finally, docking studies with protein kinases CDPK1, CDPK4, and PK6 showed structural insights for further hit-to-lead optimization studies.
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.
Computer-aided drug design approaches were applied to identify chalcones with antiplasmodial activity.
The virtual screening was performed as follows: structural standardization of in-house database ...of chalcones; identification of potential
protein targets for the chalcones; homology modeling of the predicted
targets; molecular docking studies; and
experimental validation.
Using these models, we prioritized 16 chalcones with potential antiplasmodial activity, for further experimental evaluation. Among them, LabMol-86 and LabMol-87 showed potent
antiplasmodial activity against
, while LabMol-63 and LabMol-73 were potent inhibitors of
progression into mosquito stages.
Our results encourage the exploration of chalcones in hit-to-lead optimization studies for tackling malaria.
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 (“açaı́”) originates from Brazil and has anti-inflammatory and antineoplasic properties. Here, we evaluated the antimalarial efficacy of three phenolic fractions of açaı́; 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 açaı́ fractions. HIP results indicate proteostasis as the main cellular pathway affected by fraction 2. These results open avenues to develop açaı́ polyphenols as potential new antimalarial candidates.
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