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1.
Blood Adv ; 8(1): 1-13, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-37910801

RESUMO

ABSTRACT: The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. This study investigated the relationship between pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate [OV]) to stimulate phosphorylation and with 3 selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914) and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. After these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY or pS and pT via diverse mechanisms related to cell shape, thereby affecting RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.


Assuntos
Preservação de Sangue , Eritrócitos , Humanos , Preservação de Sangue/métodos , Eritrócitos/metabolismo , Membrana Eritrocítica/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo
2.
ACS Sens ; 8(7): 2533-2542, 2023 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-37335579

RESUMO

This manuscript proposes a new dual-mode cell imaging system for studying the relationships between calcium dynamics and the contractility process of cardiomyocytes derived from human-induced pluripotent stem cells. Practically, this dual-mode cell imaging system provides simultaneously both live cell calcium imaging and quantitative phase imaging based on digital holographic microscopy. Specifically, thanks to the development of a robust automated image analysis, simultaneous measurements of both intracellular calcium, a key player of excitation-contraction coupling, and the quantitative phase image-derived dry mass redistribution, reflecting the effective contractility, namely, the contraction and relaxation processes, were achieved. Practically, the relationships between calcium dynamics and the contraction-relaxation kinetics were investigated in particular through the application of two drugs─namely, isoprenaline and E-4031─known to act precisely on calcium dynamics. Specifically, this new dual-mode cell imaging system enabled us to establish that calcium regulation can be divided into two phases, an early phase influencing the occurrence of the relaxation process followed by a late phase, which although not having a significant influence on the relaxation process affects significantly the beat frequency. In combination with cutting-edge technologies allowing the generation of human stem cell-derived cardiomyocytes, this dual-mode cell monitoring approach therefore represents a very promising technique, particularly in the fields of drug discovery and personalized medicine, to identify compounds likely to act more selectively on specific steps that compose the cardiomyocyte contractility.


Assuntos
Cálcio , Células-Tronco Pluripotentes Induzidas , Humanos , Miócitos Cardíacos , Cinética , Isoproterenol/farmacologia
3.
Blood Transfus ; 21(4): 277-288, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36346887

RESUMO

BACKGROUND: The quality of red blood cells (RBCs) stored in red cell concentrates (RCCs) is influenced by processing, storage and donor characteristics, and can have a clinical impact on transfused patients. To evaluate RBC properties and their potential impact in a transfusion setting, a simple in vitro-transfusional model has been developed. MATERIALS AND METHODS: Transfusion was simulated by mixing a washed RBC pool from two male-derived RCCs stored at 4°C with a pool of 15 male-derived fresh frozen plasma (FFP) units, representing the recipient, at a hematocrit (HCT) of 30% ("control" setting) or 5% (alternative model). The mixtures were incubated at 37°C, 5% of CO2 up to 48 h. Different metabolites, hemolysis and microvesicles (MVs) were quantified at several incubation times and RBC-morphology changes and deformability after incubation. For each model, biological triplicates have been investigated with RCCs at storage days 2 and 43. RESULTS: The 5%-HCT model restored the 2,3-DPG level and maintained the ATP level. Furthermore, glucose consumption and corresponding lactate production were increased in the 5%- vs the 30%-HCT condition. Lower hemolysis was observed with 5%-HCT, but only at day 2. However, morphological analysis by digital holographic microscopy (DHM) revealed a decreased fraction of discocytes at 5% rather than at 30% of HCT at storage day 2 but at day 43, the trend was inverted. Concordantly, RBCs incubated at 5% of HCT were more deformable than at 30% at day 43 (p<0.0001). DISCUSSION: Higher metabolic activity of RBCs in the 5%-HCT condition was promoted by a higher glucose availability and limited cell-waste accumulation. The conditions of the new proposed model thus enabled rejuvenation of RBCs and maintained them in a physiological-close state in contrast to the 30%-HCT model. It may be used as a first approach to evaluate e.g., the impact of donor and recipient characteristics on RBC properties.


Assuntos
Eritrócitos , Hemólise , Humanos , Masculino , Hematócrito , Transfusão de Sangue , Preservação de Sangue , Glucose/farmacologia
5.
Sci Data ; 9(1): 610, 2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209289

RESUMO

Viruses are genetically and structurally diverse, and outnumber cells by orders of magnitude. They can cause acute and chronic infections, suppress, or exacerbate immunity, or dysregulate survival and growth of cells. To identify chemical agents with pro- or antiviral effects we conducted arrayed high-content image-based multi-cycle infection screens of 1,280 mainly FDA-approved compounds with three human viruses, rhinovirus (RV), influenza A virus (IAV), and herpes simplex virus (HSV) differing in genome organization, composition, presence of an envelope, and tropism. Based on Z'-factors assessing screening quality and Z-scores ranking individual compounds, we identified potent inhibitors and enhancers of infection: the RNA mutagen 5-Azacytidine against RV-A16; the broad-spectrum antimycotic drug Clotrimazole inhibiting IAV-WSN; the chemotherapeutic agent Raltitrexed blocking HSV-1; and Clobetasol enhancing HSV-1. Remarkably, the topical antiseptic compound Aminacrine, which is clinically used against bacterial and fungal agents, inhibited all three viruses. Our data underscore the versatility and potency of image-based, full cycle virus propagation assays in cell-based screenings for antiviral agents.


Assuntos
Anti-Infecciosos Locais , Herpes Simples , Vírus da Influenza A , Aminacrina/uso terapêutico , Anti-Infecciosos Locais/uso terapêutico , Antivirais/farmacologia , Azacitidina/uso terapêutico , Clobetasol/uso terapêutico , Clotrimazol/uso terapêutico , Herpes Simples/tratamento farmacológico , Humanos , Mutagênicos/uso terapêutico , Rhinovirus
6.
Nat Commun ; 13(1): 3823, 2022 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-35780129

RESUMO

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.


Assuntos
Ciclização , Fenômenos Biofísicos
7.
Pharmaceuticals (Basel) ; 15(6)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35745589

RESUMO

Hookworm infections cause a neglected tropical disease (NTD) affecting ~740 million people worldwide, principally those living in disadvantaged communities. Infections can cause high morbidity due to their impact on nutrient uptake and their need to feed on host blood, resulting in a loss of iron and protein, which can lead to severe anaemia and impaired cognitive development in children. Currently, only one drug, albendazole is efficient to treat hookworm infection and the scientific community fears the rise of resistant strains. As part of on-going efforts to control hookworm infections and its associated morbidities, new drugs are urgently needed. We focused on targeting the blood-feeding pathway, which is essential to the parasite survival and reproduction, using the laboratory hookworm model Nippostrongylus brasiliensis (a nematode of rodents with a similar life cycle to hookworms). We established an in vitro-drug screening assay based on a fluorescent-based measurement of parasite viability during blood-feeding to identify novel therapeutic targets. A first screen of a library of 2654 natural compounds identified four that caused decreased worm viability in a blood-feeding-dependent manner. This new screening assay has significant potential to accelerate the discovery of new drugs against hookworms.

8.
Cancers (Basel) ; 14(6)2022 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-35326726

RESUMO

Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF V600E mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRASQ61L mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.

9.
Curr Res Virol Sci ; 3: 100019, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35072124

RESUMO

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

10.
mBio ; 12(6): e0262121, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34724816

RESUMO

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.


Assuntos
Antivirais/farmacologia , Paramyxoviridae/fisiologia , Pneumovirus/fisiologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Descoberta de Drogas , Humanos , Paramyxoviridae/genética , Infecções por Paramyxoviridae/tratamento farmacológico , Infecções por Paramyxoviridae/virologia , Pneumovirus/genética , Infecções por Pneumovirus/tratamento farmacológico , Infecções por Pneumovirus/virologia
11.
Angew Chem Int Ed Engl ; 60(40): 21702-21707, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34268864

RESUMO

Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein-protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time. As a proof-of-concept, we assembled a 2700-member target-focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2-p53 protein-protein interaction and generated micromolar and sub-micromolar inhibitors. Our approach based on acoustic liquid transfer provides a general strategy for the development of macrocycle ligands.


Assuntos
Compostos Macrocíclicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Acústica , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924276

RESUMO

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape "Tirocytes". The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.


Assuntos
Eritrócitos/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Oxidantes/metabolismo , Estresse Oxidativo , Antioxidantes/farmacologia , Descoberta de Drogas , Eritrócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Resveratrol/farmacologia , Fluxo de Trabalho
13.
Viruses ; 13(1)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477492

RESUMO

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.


Assuntos
Antivirais/farmacologia , Vírus da Cinomose Canina/efeitos dos fármacos , Vírus da Cinomose Canina/fisiologia , Cinomose/virologia , Avaliação Pré-Clínica de Medicamentos , Internalização do Vírus , Animais , Antivirais/química , Sítios de Ligação , Células Cultivadas , Chlorocebus aethiops , Cinomose/tratamento farmacológico , Cinomose/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Hospedeiro-Patógeno , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Virais/metabolismo , Bibliotecas de Moléculas Pequenas , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
14.
Sci Data ; 7(1): 265, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788590

RESUMO

Human adenoviruses (HAdVs) are fatal to immuno-suppressed individuals, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HTS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny and second-round infections. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and performed robust image analyses and hit filtering. We present the entire set of the screening data including all images, image analyses and data processing pipelines. The data are made available at the Image Data Resource (IDR, idr0081). Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential.


Assuntos
Adenovírus Humanos/efeitos dos fármacos , Antivirais/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Adenovírus Humanos/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Nelfinavir/farmacologia , Replicação Viral/efeitos dos fármacos
15.
Artigo em Inglês | MEDLINE | ID: mdl-32601166

RESUMO

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-ß-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.


Assuntos
Infecções por Adenoviridae , Infecções por Adenovirus Humanos , Adenovírus Humanos , Preparações Farmacêuticas , Adenoviridae , Infecções por Adenovirus Humanos/tratamento farmacológico , Humanos , Nelfinavir/farmacologia
16.
Nat Biomed Eng ; 4(9): 863-874, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514094

RESUMO

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.


Assuntos
Técnicas de Cultura de Células/métodos , Organoides/citologia , Células-Tronco/citologia , Animais , Agregação Celular , Células Cultivadas , Dimetilpolisiloxanos/química , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Hidrogéis/química , Intestinos/citologia , Camundongos , Organogênese , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento
17.
mBio ; 11(3)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371598

RESUMO

Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in CaulobacterIMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell.


Assuntos
Antibacterianos/farmacologia , Membrana Externa Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Peptidoglicano/metabolismo , Membrana Externa Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Caulobacter crescentus/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Estresse Fisiológico
18.
Chem Sci ; 11(30): 7858-7863, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34094158

RESUMO

Macrocycles provide an attractive modality for drug development, but generating ligands for new targets is hampered by the limited availability of large macrocycle libraries. We have established a solution-phase macrocycle synthesis strategy in which three building blocks are coupled sequentially in efficient alkylation reactions that eliminate the need for product purification. We demonstrate the power of the approach by combinatorially reacting 15 bromoacetamide-activated tripeptides, 42 amines, and 6 bis-electrophile cyclization linkers to generate a 3780-compound library with minimal effort. Screening against thrombin yielded a potent and selective inhibitor (K i = 4.2 ± 0.8 nM) that efficiently blocked blood coagulation in human plasma. Structure-activity relationship and X-ray crystallography analysis revealed that two of the three building blocks acted synergistically and underscored the importance of combinatorial screening in macrocycle development. The three-component library synthesis approach is general and offers a promising avenue to generate macrocycle ligands to other targets.

19.
SLAS Discov ; 25(5): 458-470, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31779505

RESUMO

The ionotropic GABAA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABAA receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABAA receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABAA receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABAA receptor and more generally with any ligand-gated ion channels and transporters.


Assuntos
Agonistas de Receptores de GABA-A/isolamento & purificação , Antagonistas de Receptores de GABA-A/isolamento & purificação , Imagem Molecular/métodos , Receptores de GABA-A/genética , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Fenômenos Eletrofisiológicos , Agonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/química , Ensaios de Triagem em Larga Escala/métodos , Holografia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo
20.
PLoS One ; 14(1): e0211268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30695050

RESUMO

Drug combinations are extensively used to treat cancer and are often selected according to complementary mechanisms. Here, we describe a cell-based high-throughput screening assay for identification of synergistic combinations between broadly applied platinum-based chemotherapeutics and drugs from a library composed of 1280 chemically and pharmacologically diverse (mostly FDA approved) compounds. The assay was performed on chemoresistant cell lines derived from lung (A549) and pancreatic (PANC-1) carcinoma, where platinum-based combination regimens are currently applied though with limited success. The synergistic combinations identified during the screening were validated by synergy quantification using the combination index method and via high content fluorescent microscopy analysis. New promising synergistic combinations discovered using this approach include compounds currently not used as anticancer drugs, such as cisplatin or carboplatin with hycanthone and cisplatin with spironolactone in pancreatic carcinoma, and carboplatin and deferoxamine in non-small cell lung cancer. Strong synergy between cisplatin or carboplatin and topotecan in PANC-1 cells, compared to A549 cells, suggests that this combination, currently used in lung cancer treatment regimens, could be applied to pancreatic carcinoma as well. Several drugs used to treat diseases other than cancer, including pyrvinium pamoate, auranofin, terfenadine and haloprogin, showed strong cytotoxicity on their own and synergistic interactions with platinum drugs. This study demonstrates that non-obvious drug combinations that would not be selected based on complementary mechanisms can be identified via high-throughput screening.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Platina/farmacologia , Células A549 , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia de Fluorescência , Platina/uso terapêutico , Prognóstico , Neoplasias Pancreáticas
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