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1.
Cells ; 12(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36672225

RESUMO

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Gases em Plasma , Humanos , Cloroquina/farmacologia , Células A549 , Gases em Plasma/farmacologia , Apoptose , Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo
2.
J Interferon Cytokine Res ; 43(1): 35-42, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36651846

RESUMO

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.


Assuntos
Infecções por Coronavirus , Coronavirus Humano OC43 , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Interferon beta/farmacologia , Interferon beta/uso terapêutico , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Interferons/metabolismo
3.
Braz. j. biol ; 83: e247422, 2023. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1285631

RESUMO

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.


Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.


Assuntos
Plasmodium falciparum/genética , Antimaláricos/farmacologia , Paquistão , Proteínas de Membrana Transportadoras/genética , Resistência a Medicamentos/genética , Proteínas de Protozoários/genética , Cloroquina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Alelos
4.
Int Immunopharmacol ; 113(Pt A): 109403, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36461598

RESUMO

Growing evidence describes the host immune response mechanism involved in malaria. Despite the spread of drug resistance, chloroquine (CQ) remains the main antimalarial drug in most countries in Latin America and Asia. Studies have indicated an immunomodulatory activity of CQ, however, the potential implications for CQ on immunological memory recognizing the malaria parasite are still being elucidated. Our study suggests that CQ treatment significantly delayed the initiation of parasitemia during infection of mice with the rodent malaria parasite, Plasmodium chabaudi (P.c.). Additionally, there was a decrease in T follicular helper cells (Tfh), CD4+ effector memory T cells, memory B cells (MBC), IgG2a memoryB cells, along with IgG2a plasma cells; while antibody production was not affected atthe observation time points. After PD-1 blockade and CQ treatment, no reductions in the numbers of CD4+ effector memory T cells, MBC, and IgG2a memoryB cells were observed compared with the P.c. group. Therefore, CQ might regulate immunological memory via the PD-1/PD-L1 signaling pathway. Compared with antibody secretion, the inhibition of CQ on immune memory cells was a more sensitive indicator.


Assuntos
Malária , Plasmodium chabaudi , Animais , Camundongos , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Receptor de Morte Celular Programada 1 , Antígeno B7-H1 , Malária/tratamento farmacológico , Imunoglobulina G
5.
PLoS One ; 17(12): e0274763, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36454967

RESUMO

Chloroquine often causes serious eye and vision problems, which are mainly mediated by lysosomotropic alteration. In this study, we investigated whether the ginsenoside protopanaxadiol relieves chloroquine-induced retinopathy by restoring lysosomotropic abnormalities in human adult retinal pigment epithelial-19 cells. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological alterations in autophagosomes of adult retinal pigment epithelial-19 cells was detected using confocal microscopy. Apoptosis was examined using flow cytometry, whereas cellular reactive oxygen species levels were determined by measuring the fluorescence intensity of 5-(and-6)-carboxy-2'-7'-dichlorohydrofluorescein diacetate. Lysosomal function was assessed by measuring lysosomal pH and enzyme activity. Immunoprecipitation and western blotting analyses were performed. Adult retinal pigment epithelial-19 cells accumulated autophagosomes with fusion defects in lysosomes and reactive oxygen species formation following exposure to chloroquine. This effect trapped Beclin-1 and B-cell lymphoma 2 interfering with autophagy initiation and autophagosome development. Protopanaxadiol alleviated chloroquine-induced toxicity by modulating the interaction between Beclin-1 and Bcl-2, which was mediated by the AMP-activated protein kinase-mammalian target of rapamycin signal axis. Furthermore, autophagy and apoptosis were simultaneously controlled by protopanaxadiol via upregulation of autophagy flux and decreased reactive oxygen species formation and apoptotic protein expression. These findings suggest that protopanaxadiol is a promising treatment strategy for chloroquine-mediated retinopathy.


Assuntos
Ginsenosídeos , Doenças Retinianas , Adulto , Humanos , Ginsenosídeos/farmacologia , Cloroquina/farmacologia , Proteína Beclina-1 , Espécies Reativas de Oxigênio , Autofagia , Apoptose , Pigmentos da Retina
6.
Front Cell Infect Microbiol ; 12: 1063407, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36530422

RESUMO

Introduction: The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: 'delayed death' by inhibiting the bacterium-like ribosomes of the apicoplast, and 'quick-killing' that kills rapidly across the entire blood stage development. Methods: Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans). Results: Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (<12 hrs treatment) and were >5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment. Discussion: The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.


Assuntos
Antimaláricos , Malária Falciparum , Malária , Parasitos , Animais , Humanos , Antimaláricos/farmacologia , Azitromicina/farmacologia , Plasmodium falciparum , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Malária/tratamento farmacológico , Malária/parasitologia
7.
Front Cell Infect Microbiol ; 12: 1047269, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36530433

RESUMO

In the fight against malaria, the key is early treatment with antimalarial chemotherapy, such as artemisinin-based combination treatments (ACTs). However, Plasmodium has acquired multidrug resistance, including the emergence of P. falciparum strains with resistance to ACT. The development of novel antimalarial molecules, that are capable of interfering in the asexual and sexual blood stages, is important to slow down the transmission in endemic areas. In this work, we studied the ability of the mettalo copper-cinchonine complex to interfere in the sexual and asexual stages of Plasmodium. The tested compound in the in vitro assay was a cinchonine derivative, named CinCu (Bis[Cinchoninium Tetrachlorocuprate(II)]trihydrate). Its biological functions were assessed by antiplasmodial activity in vitro against chloroquine-resistant P. falciparum W2 strain. The mice model of P. berghei ANKA infection was used to analyze the antimalarial activity of CinCu and chloroquine and their acute toxicity. The oocyst formation-blocking assay was performed by experimental infection of Anopheles aquasalis with P. vivax infected blood, which was treated with different concentrations of CinCu, cinchonine, and primaquine. We found that CinCu was able to suppress as high as 81.58% of parasitemia in vitro, being considered a molecule with high antiplasmodial activity and low toxicity. The in vivo analysis showed that CinCu suppressed parasitemia at 34% up to 87.19%, being a partially active molecule against the blood-stage forms of P. berghei ANKA, without inducing severe clinical signs in the treated groups. The transmission-blocking assay revealed that both cinchonine and primaquine were able to reduce the infection intensity of P. vivax in A. aquasalis, leading to a decrease in the number of oocysts recovered from the mosquitoes' midgut. Regarding the effect of CinCu, the copper-complex was not able to induce inhibition of P. vivax infection; however, it was able to induce an important reduction in the intensity of oocyst formation by about 2.4 times. It is plausible that the metallo-compound also be able to interfere with the differentiation of parasite stages and/or ookinete-secreted chitinase into the peritrophic matrix of mosquitoes, promoting a reduction in the number of oocysts formed. Taken together, the results suggest that this compound is promising as a prototype for the development of new antimalarial drugs. Furthermore, our study can draw a new pathway for repositioning already-known antimalarial drugs by editing their chemical structure to improve the antimalarial activity against the asexual and sexual stages of the parasite.


Assuntos
Antimaláricos , Malária Falciparum , Malária Vivax , Plasmodium , Camundongos , Animais , Antimaláricos/farmacologia , Primaquina/farmacologia , Primaquina/uso terapêutico , Oocistos , Parasitemia/parasitologia , Cobre/farmacologia , Malária Falciparum/parasitologia , Cloroquina/farmacologia , Plasmodium falciparum
8.
Malar J ; 21(1): 394, 2022 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-36566182

RESUMO

BACKGROUND: Despite significant progress in eliminating malaria from the state of Odisha, India, the disease is still considered endemic. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been introduced since 2010 as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and artesunate (ART) in P. falciparum parasites circulating in the state. METHODS: A total of 239 isolates of P. falciparum mono infection were collected during July 2018-November 2020 from the four different geographical regions of the state. Genomic DNA was extracted from 200 µL of venous blood and amplified using nested polymerase chain reaction. Mutations on gene associated with CQ (Pfcrt and Pfmdr1) were assessed by PCR amplification and restriction fragment length polymorphism, artemisinin (Pfk13) gene by DNA sequencing and SP (Pfdhfr and Pfdhps) genes by allele-specific polymerase chain reaction (AsPCR). RESULTS: The point mutation in Pfcrt (K76T) was detected 2.1%, in Pfmdr1 (N86Y) 3.4%, and no mutations were found in Pfkelch13 propeller domain. Prevalence of Pfdhfr, Pfdhps and Pfhdfr-Pfdhps (two locus) gene mutations were 50.43%, 47.05% and 49.79% respectively. The single, double, triple and quadruple point mutations in Pfdhfr gene was 11.2%, 8.2%, 17.2% and 3.4% while, in Pfdhps gene was 10.9%,19.5%, 9.5% and 2.7% respectively. Of the total 13 haplotypes found in Pfdhfr, 8 were detected for the first time in the state and of the total 26 haplotypes found in Pfdhps, 7 were detected for the fisrt time in the state. The linked quintuple mutation Pfdhfr (N51I-C59R-S108N)-Pfdhps (A437G-K540E) responsible for clinical failure (RIII level of resistance) of SP resistance and A16V-S108T mutation in Pfdhfr responsible for cycloguanil was absent. CONCLUSION: The study has demonstrated a low prevalence of CQ resistance alleles in the study area. Despite the absence of the Pfkelch13 mutations, high prevalence of Pfdhfr and Pfdhps point mutations undermine the efficacy of SP partner drug, thereby threatening the P. falciparum malaria treatment policy. Therefore, continuous molecular and in vivo monitoring of ACT efficacy is warranted in Odisha.


Assuntos
Antimaláricos , Resistência a Medicamentos , Malária Falciparum , Plasmodium falciparum , Proteínas de Protozoários , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artesunato/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Combinação de Medicamentos , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/uso terapêutico , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Sulfadoxina/farmacologia , Sulfadoxina/uso terapêutico , Índia/epidemiologia
9.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499465

RESUMO

4-O-methylascochlorin (MAC) is a 4-fourth carbon-substituted derivative of ascochlorin, a compound extracted from a phytopathogenic fungus Ascochyta viciae. MAC induces apoptosis and autophagy in various cancer cells, but the effects of MAC on apoptosis and autophagy in cervical cancer cells, as well as how the interaction between apoptosis and autophagy mediates the cellular anticancer effects are not known. Here, we investigated that MAC induced apoptotic cell death of cervical cancer cells without regulating the cell cycle and promoted autophagy by inhibiting the phosphorylation of serine-threonine kinase B (Akt), mammalian target of rapamycin (mTOR), and 70-kDa ribosomal protein S6 kinase (p70S6K). Additional investigations suggested that Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP-3), but not Hypoxia-inducible factor 1 alpha (HIF-1α), is a key regulator of MAC-induced apoptosis and autophagy. BNIP-3 siRNA suppressed MAC-induced increases in cleaved- poly (ADP-ribose) polymerase (PARP) and LC3II expression. The pan-caspase inhibitor Z-VAD-FMK suppressed MAC-induced cell death and enhanced MAC-induced autophagy. The autophagy inhibitor chloroquine (CQ) enhanced MAC-mediated cell death by increasing BNIP-3 expression. These results indicate that MAC induces apoptosis to promote cell death and stimulates autophagy to promote cell survival by increasing BNIP-3 expression. This study also showed that co-treatment of cells with MAC and CQ further enhanced the death of cervical cancer cells.


Assuntos
Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Autofagia , Apoptose , Cloroquina/farmacologia
10.
Elife ; 112022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36476338

RESUMO

Mice are the most commonly used model animals for itch research and for development of anti-itch drugs. Most laboratories manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID (Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine. The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that could replace manual quantification for mouse scratching behavior in different itch models and for drug screening.


Assuntos
Aprendizado Profundo , Camundongos , Animais , Prurido/induzido quimicamente , Comportamento Animal , Injeções , Cloroquina/farmacologia , Modelos Animais de Doenças
11.
BMC Med ; 20(1): 448, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36397090

RESUMO

BACKGROUND: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials. METHODS: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined. RESULTS: The PPQ median IC50 interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC50s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC50s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC50 and Pfcrt K76T allele (P=0.0026). CONCLUSIONS: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.


Assuntos
Antimaláricos , Artemisininas , Parasitos , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum/genética , Quênia/epidemiologia , Proteínas de Protozoários/genética , Combinação Arteméter e Lumefantrina , Artemeter , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Lumefantrina , Genômica
12.
Mol Pharm ; 19(12): 4631-4643, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36346968

RESUMO

Hydroxychloroquine (HCQ) has been the subject of multiple recent preclinical and clinical studies for its beneficial use in the combination treatments of different types of cancers. Polymeric HCQ (PCQ), a macromolecular multivalent version of HCQ, has been shown to be effective in various cancer models both in vitro and in vivo as an inhibitor of cancer cell migration and experimental lung metastasis. Here, we present detailed in vitro studies that show that low concentrations of PCQ can efficiently inhibit cancer cell migration and colony formation orders of magnitude more effectively compared to HCQ. After intraperitoneal administration of PCQ in vivo, high levels of tumor accumulation and penetration are observed, combined with strong antimetastatic activity in an orthotopic pancreatic cancer model. These studies support the idea that PCQ may be effectively used at low doses as an adjuvant in the therapy of pancreatic cancer. In conjunction with previously published literature, these studies further undergird the potential of PCQ as an anticancer agent.


Assuntos
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Hidroxicloroquina/uso terapêutico , Hidroxicloroquina/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Antineoplásicos/uso terapêutico , Polímeros/uso terapêutico
13.
Cell Commun Signal ; 20(1): 189, 2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36434621

RESUMO

BACKGROUND: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. RESULTS: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. CONCLUSIONS: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion. Video abstract.


Assuntos
Autofagossomos , Autofagia , Autofagossomos/metabolismo , Lisossomos/metabolismo , Cloroquina/farmacologia , Cloroquina/metabolismo , Colesterol/metabolismo
14.
Molecules ; 27(22)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36431900

RESUMO

Due to the urgent need of innovation in the antimalarial therapeutic arsenal, a series of thirty-seven ring-substituted N-arylcinnamanilides prepared by microwave-assisted synthesis were subjected to primary screening against the chloroquine-sensitive strain of P. falciparum 3D7/MRA-102. The lipophilicity of all compounds was experimentally determined as the logarithm of the capacity factor k, and these data were subsequently used in the discussion of structure-activity relationships. Among the screened compounds, fourteen derivatives exhibited IC50 from 0.58 to 31 µM, whereas (2E)-N-(4-bromo-2-chlorophenyl)-3-phenylprop-2-enamide (24) was the most effective agent (IC50 = 0.58 µM). In addition, (2E)-N-[2,6-dibromo-4-(trifluoromethyl)- phenyl]-3-phenylprop-2-enamide (36), (2E)-N-[4-nitro-3-(trifluoromethyl)phenyl]-3-phenylprop- 2-enamide (18), (2E)-N-(2-bromo-5-fluorophenyl)-3-phenylprop-2-enamide (23), and (2E)-3-phenyl-N-(3,4,5-trichlorophenyl)prop-2-enamide (33) demonstrated efficacy in the IC50 range from 2.0 to 4.3 µM, comparable to the clinically used standard chloroquine. The results of a cell viability screening performed using THP1-Blue™ NF-κB cells showed that none of these highly active compounds displayed any significant cytotoxic effect up to 20 µM, which makes them promising Plasmodium selective substances for further investigations.


Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Humanos , Antimaláricos/farmacologia , Cloroquina/farmacologia , Relação Estrutura-Atividade
15.
PLoS Pathog ; 18(10): e1010926, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36306287

RESUMO

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.


Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Parasitos , Animais , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Cloroquina/farmacologia , Cloroquina/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/metabolismo , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Malária Falciparum/parasitologia , Antimaláricos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Artemisininas/farmacologia , Mutação , Hemoglobinas/metabolismo , Heme/metabolismo
16.
Front Cell Infect Microbiol ; 12: 952993, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36310859

RESUMO

Cerebral malaria (CM) is one of the most severe forms of malaria and is a neuropathology that can lead to death. Monocytes have been shown to accumulate in the brain microvasculature at the onset of neurological symptoms during CM. Monocytes have a remarkable ability to adapt their function to their microenvironment from pro-inflammatory to resolving activities. This study aimed to describe the behavior of monocyte subpopulations during infection and its resolution. C57BL/6 mice were infected with the Plasmodium berghei ANKA strain and treated or not with chloroquine (CQ) on the first day of the onset of neurological symptoms (day 6) for 4 days and followed until day 12 to mimic neuroinflammation and its resolution during experimental CM. Ly6C monocyte subpopulations were identified by flow cytometry of cells from the spleen, peripheral blood, and brain and then quantified and characterized at different time points. In the brain, the Ly6Cint and Ly6Clow monocytes were associated with neuroinflammation, while Ly6Chi and Ly6Cint were mobilized from the peripheral blood to the brain for resolution. During neuroinflammation, CD36 and CD163 were both involved via splenic monocytes, whereas our results suggest that the low CD36 expression in the brain during the neuroinflammation phase was due to degradation. The resolution phase was characterized by increased expressions of CD36 and CD163 in blood Ly6Clow monocytes, a higher expression of CD36 in the microglia, and restored high expression levels of CD163 in Ly6Chi monocytes localized in the brain. Thus, our results suggest that increasing the expressions of CD36 and CD163 specifically in the brain during the neuroinflammatory phase contributes to its resolution.


Assuntos
Malária Cerebral , Monócitos , Animais , Camundongos , Monócitos/metabolismo , Malária Cerebral/tratamento farmacológico , Malária Cerebral/patologia , Camundongos Endogâmicos C57BL , Cloroquina/farmacologia , Encéfalo/patologia , Antígenos CD36/metabolismo
17.
Front Cell Infect Microbiol ; 12: 1015957, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36310880

RESUMO

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016-2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.


Assuntos
Antimaláricos , Malária Falciparum , Humanos , Antimaláricos/farmacologia , Plasmodium falciparum/genética , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , Malária Falciparum/parasitologia , Gana , Artemeter/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Lumefantrina/farmacologia , Lumefantrina/uso terapêutico , Proteínas de Protozoários/genética
18.
Antimicrob Agents Chemother ; 66(11): e0028422, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314800

RESUMO

Drug combinations and drug repurposing have emerged as promising strategies to develop novel treatments for infectious diseases, including Chagas disease. In this study, we aimed to investigate whether the repurposed drugs chloroquine (CQ) and colchicine (COL), known to inhibit Trypanosoma cruzi infection in host cells, could boost the anti-T. cruzi effect of the trypanocidal drug benznidazole (BZN), increasing its therapeutic efficacy while reducing the dose needed to eradicate the parasite. The combination of BZN and COL exhibited cytotoxicity to infected cells and low antiparasitic activity. Conversely, a combination of BZN and CQ significantly reduced T. cruzi infection in vitro, with no apparent cytotoxicity. This effect seemed to be consistent across different cell lines and against both the partially BZN-resistant Y and the highly BZN-resistant Colombiana strains. In vivo experiments in an acute murine model showed that the BZN+CQ combination was eight times more effective in reducing T. cruzi infection in the acute phase than BZN monotherapy. In summary, our results demonstrate that the concomitant administration of CQ and BZN potentiates the trypanocidal activity of BZN, leading to a reduction in the dose needed to achieve an effective response. In a translational context, it could represent a higher efficacy of treatment while also mitigating the adverse effects of high doses of BZN. Our study also reinforces the relevance of drug combination and repurposing approaches in the field of Chagas disease drug discovery.


Assuntos
Doença de Chagas , Nitroimidazóis , Tripanossomicidas , Trypanosoma cruzi , Camundongos , Animais , Reposicionamento de Medicamentos , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico
19.
BMC Biol ; 20(1): 197, 2022 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-36271358

RESUMO

BACKGROUND: By 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen. RESULTS: Attempts to exacerbate the P. falciparum proteome's propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen's viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid ß peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent. CONCLUSIONS: Inhibiting protein aggregation in Plasmodium significantly reduces the parasite's viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.


Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Humanos , Antimaláricos/farmacologia , Plasmodium falciparum , Agregados Proteicos , Peptídeos beta-Amiloides , Proteoma , Resistência a Medicamentos , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Malária Falciparum/parasitologia , Cloroquina/química , Cloroquina/farmacologia , Cloroquina/uso terapêutico
20.
Nat Commun ; 13(1): 6163, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36257944

RESUMO

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.


Assuntos
Antimaláricos , Malária Falciparum , Humanos , Plasmodium falciparum , Mefloquina/farmacologia , Mefloquina/uso terapêutico , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Proteínas de Protozoários/genética , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Cloroquina/farmacologia
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