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

Funding Information: The authors are incredibly grateful to Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq, CAPES, Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG), and to the Swedish Research Council (grants 2016–05627 and 2021–03667). K.C.P.T. thanks FAPESP (grants 2018/05926–2, and 2019/17062–5). FAPESP funded T.A.T. (2019/27626-3), J.V.B.B. (2019/21854-4), K.B.M. and R.M.C. (2014/50897-0), V.M.A. (2022/00743-2), G.C.C. (2015/20774-6), and F.T.M.C. (grants 2017/18611–7 and 2018/07007–4). L.C.S.A. was funded by CNPq (162117/2018– 3). E.B. was supported by FAPESP (2015/03553-6 and 2018/07007–4) and CAPES (88887.304810/2018–00). J.V.B.B. and J.T.M.F. were supported by CAPES (Finance Code 001). C.H.A. and M.M. thanks FAPEG (grants 20171026700006 and 202010267000272). C.H.A. thanks the “L'Oréal-UNESCO-ABC Para Mulheres na Ciência” and “L’Oréal-UNESCO International Rising Talents” for the awards and fellowships received, which partially funded this work. C.H.A. and F.T.M.C. are CNPq research fellows. We are thankful to Liam B. King for his critical review of the report. Publisher Copyright: © 2023 American Society for Microbiology. All Rights Reserved.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.publishersversionpublishe

A spatially resolved single-cell lung atlas integrated with clinical and blood signatures distinguishes COVID-19 disease trajectories

COVID-19 is characterized by a broad range of symptoms and disease trajectories. Understanding the correlation between clinical biomarkers and lung pathology during acute COVID-19 is necessary to understand its diverse pathogenesis and inform more effective treatments. Here, we present an integrated analysis of longitudinal clinical parameters, peripheral blood markers, and lung pathology in 142 Brazilian patients hospitalized with COVID-19. We identified core clinical and peripheral blood signatures differentiating disease progression between patients who recovered from severe disease compared with those who succumbed to the disease. Signatures were heterogeneous among fatal cases yet clustered into two patient groups: "early death" (&lt;15 days until death) and "late death" (&gt;15 days). Progression to early death was characterized systemically and in lung histopathological samples by rapid endothelial and myeloid activation and the presence of thrombi associated with SARS-CoV-2+ macrophages. In contrast, progression to late death was associated with fibrosis, apoptosis, and SARS-CoV-2+ epithelial cells in postmortem lung tissue. In late death cases, cytotoxicity, interferon, and T helper 17 (TH17) signatures were only detectable in the peripheral blood after 2 weeks of hospitalization. Progression to recovery was associated with higher lymphocyte counts, TH2 responses, and anti-inflammatory-mediated responses. By integrating antemortem longitudinal blood signatures and spatial single-cell lung signatures from postmortem lung samples, we defined clinical parameters that could be used to help predict COVID-19 outcomes.</p

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.publishersversionpublishe

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine, and Pyronaridine: In Vitro Activity against SARS-CoV-2 and Potential Mechanisms

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with Kd values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic

Chemical genomic profiling unveils the in vitro and in vivo antiplasmodial mechanism of açaı́ (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 ("acai") originates from Brazil and has anti-inflammatory and antineoplasic properties. Here, we evaluated the antimalarial efficacy of three phenolic fractions of acai; 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 acai fractions. HIP results indicate proteostasis as the main cellular pathway affected by fraction 2. These results open avenues to develop acai polyphenols as potential new antimalarial candidates4131562815635CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação002/20142017/18611-7; 2015/03553-6; 2018/07007-4; 2017/50400-6; 2017/01986-8; 2018/05328-8his work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—Grants 2017/18611-7 and 2015/03553-6), FCT-FAPESP (Grant 2018/07007-4), Texas A&M University TAMU FAPESP Grant (2017/50400-6), Texas A&M AgriLife Vector Disease Program Funding, College Station, TX, Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-TAMU 002/2014), and the Swedish Research Council (Grant 2016-05627). L.T.F. and T.A.T. were supported by a CAPES fellowship. L.D.A. was sponsored by a FAPESP fellowship (2017/01986-8). G.S.P. was sponsored by a FAPESP fellowship (2018/05328-8). F.T.M.C. is a CNPq researcher fellow level 1C