RESUMEN

The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser scanning microscopy (CLSM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive three-dimensional investigation of their dynamics down to the single-cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, has radically amplified the flow of available biofilm structural data. In this contribution, we present a HCS-CLSM protocol used to analyze biofilm four-dimensional structural dynamics at high throughput. Meta-analysis of the quantitative variables extracted from HCS-CLSM will contribute to a better biological understanding of biofilm traits.

Asunto(s)

Biopelículas , Microscopía Confocal , Biopelículas/crecimiento & desarrollo , Microscopía Confocal/métodos , Microbiología de Alimentos/métodos , Imagenología Tridimensional/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos

RESUMEN

Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.

Asunto(s)

Antivirales , Fiebre Chikungunya , Virus Chikungunya , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Virus Chikungunya/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Fiebre Chikungunya/tratamiento farmacológico , Fiebre Chikungunya/virología , Humanos , Animales , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Evaluación Preclínica de Medicamentos , Replicación Viral/efectos de los fármacos , Descubrimiento de Drogas , Chlorocebus aethiops , Células Vero

RESUMEN

BACKGROUND: Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro. RESULTS: AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA). CONCLUSIONS: We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.

Asunto(s)

Antibacterianos , Inteligencia Artificial , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/enzimología , Antibacterianos/farmacología , Peptidoglicano/metabolismo , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos

RESUMEN

Droplet microfluidics has become a very powerful tool in high-throughput screening, including antibody discovery. Screens are usually carried out by physically sorting droplets hosting cells of the desired phenotype, breaking them, recovering the encapsulated cells, and sequencing the paired antibody light and heavy chain genes at the single-cell level. This series of multiple consecutive manipulation steps of rare screening hits is complex and challenging, resulting in a significant loss of clones with the desired phenotype or large fractions of cells with incomplete antibody information. Here, we present fluorescence-activated droplet sequencing, in which droplets showing the desired phenotype are selectively picoinjected with reagents for RT-PCR. Subsequently, light and heavy chain genes are natively paired, fused into a single-chain fragment variant format, and amplified before off-chip transfer and downstream nanopore sequencing. This workflow is sufficiently sensitive for obtaining different paired full-length antibody sequences from as little as five droplets, fulfilling the desired phenotype. Replacing physical sorting by specific sequencing overcomes a general bottleneck in droplet microfluidic screening and should be compatible with many more applications.

Asunto(s)

Anticuerpos , Humanos , Microfluídica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos

RESUMEN

BACKGROUND: Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato (E. granulosus), with a worldwide distribution. The current treatment strategy for CE is insufficient. Limited drug screening models severely hamper the discovery of effective anti-echinococcosis drugs. METHODS: In the present study, using high-content screening technology, we developed a novel high-throughput screening (HTS) assay by counting the ratio of propidium iodide-stained dead protoscoleces (PSCs) to the total number of PSCs. In vitro and ex vivo cyst viability assays were utilized to determine the effect of drugs on cyst viability. RESULTS: Using the newly established HTS assay, we screened approximately 12,000 clinical-stage or The Food and Drug Administration (FDA)-approved small molecules from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library, as well as the LOPAC1280 and SelleckChem libraries, as a strategic approach to facilitate the drug discovery process. Initial screening yielded 173 compounds with anti-echinococcal properties, 52 of which demonstrated dose-response efficacy against E. granulosus PSCs in vitro. Notably, two agents, omaveloxolone and niclosamide, showed complete inhibition upon further validation in cyst and microcyst viability assays in vitro after incubation for 3 days, and in an ex vivo cyst viability assay using cysts isolated from the livers of mice infected with E. granulosus, as determined by morphological assessment. CONCLUSIONS: Through the development of a novel HTS assay and by repurposing libraries, we identified omaveloxolone and niclosamide as potent inhibitors against E. granulosus. These compounds show promise as potential anti-echinococcal drugs, and our strategic approach has the potential to promote drug discovery for parasitic infections.

Asunto(s)

Reposicionamiento de Medicamentos , Equinococosis , Echinococcus granulosus , Ensayos Analíticos de Alto Rendimiento , Echinococcus granulosus/efectos de los fármacos , Animales , Ensayos Analíticos de Alto Rendimiento/métodos , Equinococosis/tratamiento farmacológico , Equinococosis/parasitología , Ratones , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos , Antihelmínticos/farmacología , Descubrimiento de Drogas , Perros

RESUMEN

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments.

Asunto(s)

Citometría de Flujo , Glioma , Citometría de Flujo/métodos , Animales , Ratas , Glioma/diagnóstico por imagen , Glioma/patología , Glioma/metabolismo , Masculino , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Imagen Óptica/métodos , Humanos , Núcleo Celular/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Colorantes Fluorescentes/química

RESUMEN

Hit screening, which involves the identification of compounds or targets capable of modulating disease-relevant processes, is an important step in drug discovery. Some assays, such as image-based high-content screenings, produce complex multivariate readouts. To fully exploit the richness of such data, advanced analytical methods that go beyond the conventional univariate approaches should be employed. In this work, we tackle the problem of hit identification in multivariate assays. As with univariate assays, a hit from a multivariate assay can be defined as a candidate that yields an assay value sufficiently far away in distance from the mean or central value of inactives. Viewed another way, a hit is an outlier from the distribution of inactives. A method was developed for identifying multivariate hit in high-dimensional data sets based on principal components and robust Mahalanobis distance (the multivariate analogue to the Z- or T-statistic). The proposed method, termed mROUT (multivariate robust outlier detection), demonstrates superior performance over other techniques in the literature in terms of maintaining Type I error, false discovery rate and true discovery rate in simulation studies. The performance of mROUT is also illustrated on a CRISPR knockout data set from in-house phenotypic screening programme.

Asunto(s)

Ensayos Analíticos de Alto Rendimiento , Análisis Multivariante , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Descubrimiento de Drogas/métodos , Algoritmos , Análisis de Componente Principal , Simulación por Computador

RESUMEN

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Asunto(s)

Bioimpresión , Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana , Humanos , Bioimpresión/métodos , Permeabilidad Capilar/fisiología , Hidrogeles/química , Impresión Tridimensional , Ensayos Analíticos de Alto Rendimiento/métodos

RESUMEN

A balanced immune system is essential to maintain adequate host defense and effective self-tolerance. While an immune system that fails to generate appropriate response will permit infections to develop, uncontrolled activation may lead to autoinflammatory or autoimmune diseases. To identify drug candidates capable of modulating immune cell functions, we screened 1200 small molecules from the Prestwick Chemical Library for their property to inhibit innate or adaptive immune responses. Our studies focused specifically on drug interactions with T cells, B cells, and polymorphonuclear leukocytes (PMNs). Candidate drugs that were validated in vitro were examined in preclinical models to determine their immunomodulatory impact in chronic inflammatory diseases, here investigated in chronic inflammatory skin diseases. Using this approach, we identified several candidate drugs that were highly effective in preclinical models of chronic inflammatory disease. For example, we found that administration of pyrvinium pamoate, an FDA-approved over-the-counter anthelmintic drug, suppressed B cell activation in vitro and halted the progression of B cell-dependent experimental pemphigoid by reducing numbers of autoantigen-specific B cell responses. In addition, in studies performed in gene-deleted mouse strains provided additional insight into the mechanisms underlying these effects, for example, the receptor-dependent actions of tamoxifen that inhibit immune-complex-mediated activation of PMNs. Collectively, our methods and findings provide a vast resource that can be used to identify drugs that may be repurposed and used to promote or inhibit cellular immune responses.

Asunto(s)

Inmunidad Adaptativa , Linfocitos B , Ensayos Analíticos de Alto Rendimiento , Inmunidad Innata , Animales , Ratones , Inmunidad Innata/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Inmunidad Adaptativa/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Reposicionamiento de Medicamentos/métodos , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Neutrófilos/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Modelos Animales de Enfermedad , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos , Ratones Noqueados

RESUMEN

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 µM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 µM, 1.87 µM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Asunto(s)

Antivirales , Virus de la Hepatitis B , Hepatocitos , Ensayos Analíticos de Alto Rendimiento , Antivirales/farmacología , Humanos , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Hepatitis B/virología , Hepatitis B/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros

RESUMEN

The level of airway constriction in thin slices of lung tissue is highly variable. Owing to the labor-intensive nature of these experiments, determining the number of airways to be analyzed in order to allocate a reliable value of constriction in one mouse is challenging. Herein, a new automated device for physiology and image analysis was used to facilitate high throughput screening of airway constriction in lung slices. Airway constriction was first quantified in slices of lungs from male BALB/c mice with and without experimental asthma that were inflated with agarose through the trachea or trans-parenchymal injections. Random sampling simulations were then conducted to determine the number of airways required per mouse to quantify maximal constriction. The constriction of 45 ± 12 airways per mouse in 32 mice were analyzed. Mean maximal constriction was 37.4 ± 32.0%. The agarose inflating technique did not affect the methacholine response. However, the methacholine constriction was affected by experimental asthma (p = 0.003), shifting the methacholine concentration-response curve to the right, indicating a decreased sensitivity. Simulations then predicted that approximately 35, 16 and 29 airways per mouse are needed to quantify the maximal constriction mean, standard deviation and coefficient of variation, respectively; these numbers varying between mice and with experimental asthma.

Asunto(s)

Asma , Pulmón , Cloruro de Metacolina , Ratones Endogámicos BALB C , Animales , Pulmón/fisiopatología , Ratones , Masculino , Cloruro de Metacolina/farmacología , Asma/fisiopatología , Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Animales de Enfermedad

RESUMEN

In major radiological events, rapid assays to detect ionizing radiation exposure are crucial for effective medical interventions. The purpose of these assays is twofold: to categorize affected individuals into groups for initial treatments, and to provide definitive dose estimates for continued care and epidemiology. However, existing high-throughput cytogenetic biodosimetry assays take about 3 days to yield results, which delays critical interventions. We have developed a multiwell-based variant of the chemical-induced G0-phase Premature Chromosome Condensation Assay that delivers same-day results. Our findings revealed that using a concentration of phosphatase inhibitor lower than recommended significantly increases the yield of cells with highly condensed chromosomes. These chromosomes exhibited increased fragmentation in a dose-dependent manner, enabling to quantify radiation damage using a custom Deep Learning algorithm. This algorithm demonstrated reasonable performance in categorizing doses into distinct treatment groups (84% and 80% accuracy for three and four iso-treatment dose bins, respectively) and showed reliability in determining the actual doses received (correlation coefficient of 0.879). This method is amendable to full automation and has the potential to address the need for same-day, high-throughput cytogenetic test for both dose categorization and dose reconstruction in large-scale radiation emergencies.

Asunto(s)

Radiometría , Humanos , Radiometría/métodos , Radiación Ionizante , Bioensayo/métodos , Relación Dosis-Respuesta en la Radiación , Ensayos Analíticos de Alto Rendimiento/métodos

RESUMEN

The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free high-throughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates real-time sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRMHR), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.

Asunto(s)

Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Descubrimiento de Drogas/métodos , Reproducibilidad de los Resultados , Humanos

RESUMEN

Assays that measure morphology, proliferation, motility, deformability, and migration are used to study the invasiveness of cancer cells. However, native invasive potential of cells may be hidden from these contextual metrics because they depend on culture conditions. We created a micropatterned chip that mimics the native environmental conditions, quantifies the invasive potential of tumor cells, and improves our understanding of the malignancy signatures. Unlike conventional assays, which rely on indirect measurements of metastatic potential, our method uses three-dimensional microchannels to measure the basal native invasiveness without chemoattractants or microfluidics. No change in cell death or proliferation is observed on our chips. Using six cancer cell lines, we show that our system is more sensitive than other motility-based assays, measures of nuclear deformability, or cell morphometrics. In addition to quantifying metastatic potential, our platform can distinguish between motility and invasiveness, help study molecular mechanisms of invasion, and screen for targeted therapeutics.

Asunto(s)

Movimiento Celular , Metástasis de la Neoplasia , Humanos , Línea Celular Tumoral , Microtecnología/métodos , Proliferación Celular , Invasividad Neoplásica , Ensayos Analíticos de Alto Rendimiento/métodos , Dispositivos Laboratorio en un Chip , Neoplasias/patología

RESUMEN

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.

Asunto(s)

Antibacterianos , Inhibidores Enzimáticos , Escherichia coli , Exorribonucleasas , Antibacterianos/farmacología , Antibacterianos/química , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Dominio Catalítico , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Bacterias/enzimología

RESUMEN

Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.

Asunto(s)

Carga Viral , Cinética , Humanos , Animales , Ensayos Analíticos de Alto Rendimiento/métodos , Virus de la Enfermedad de Newcastle/fisiología , Línea Celular

RESUMEN

Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.

Asunto(s)

Antivirales , Proteasas Similares a la Papaína de Coronavirus , Citocinas , Ensayos Analíticos de Alto Rendimiento , SARS-CoV-2 , Ubiquitinas , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Antivirales/farmacología , Antivirales/química , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Citocinas/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/química , Ubiquitinas/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Tratamiento Farmacológico de COVID-19 , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Transferencia Resonante de Energía de Fluorescencia , COVID-19/virología

RESUMEN

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Asunto(s)

Interacciones Hidrofóbicas e Hidrofílicas , Proteínas , Cromatografía por Intercambio Iónico/métodos , Concentración de Iones de Hidrógeno , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Resinas de Intercambio de Catión/química , Resinas de Intercambio Aniónico/química , Adsorción , Punto Isoeléctrico

RESUMEN

Lactate dehydrogenase A (LDHA) is highly expressed in many tumor cells and promotes the conversion of pyruvate to lactic acid in the glucose pathway, providing energy and synthetic precursors for rapid proliferation of tumor cells. Therefore, inhibition of LDHA has become a widely concerned tumor treatment strategy. However, the research and development of highly efficient and low toxic LDHA small molecule inhibitors still faces challenges. To discover potential inhibitors against LDHA, virtual screening based on molecular docking techniques was performed from Specs database of more than 260,000 compounds and Chemdiv-smart database of more than 1,000 compounds. Through molecular dynamics (MD) simulation studies, we identified 12 potential LDHA inhibitors, all of which can stably bind to human LDHA protein and form multiple interactions with its active central residues. In order to verify the inhibitory activities of these compounds, we established an enzyme activity assay system and measured their inhibitory effects on recombinant human LDHA. The results showed that Compound 6 could inhibit the catalytic effect of LDHA on pyruvate in a dose-dependent manner with an EC50 value of 14.54 ± 0.83 µM. Further in vitro experiments showed that Compound 6 could significantly inhibit the proliferation of various tumor cell lines such as pancreatic cancer cells and lung cancer cells, reduce intracellular lactic acid content and increase intracellular reactive oxygen species (ROS) level. In summary, through virtual screening and in vitro validation, we found that Compound 6 is a small molecule inhibitor for LDHA, providing a good lead compound for the research and development of LDHA related targeted anti-tumor drugs.

Asunto(s)

Antineoplásicos , Inhibidores Enzimáticos , Ensayos Analíticos de Alto Rendimiento , L-Lactato Deshidrogenasa , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular

RESUMEN

Failure in recognizing non-tuberculous mycobacteria (NTM) leads to misdiagnosis of multidrug-resistant Mycobacterium tuberculosis Complex (MTBC). There is an unmet need for diagnostic tools that can differentiate between NTMs and MTBC, and that are affordable for Low- and Middle-income Countries (LMIC). Earlier we developed a strip-based CrfA assay technology to detect the Carbapenem Resistance Factor A (CrfA) enzyme present only in MTBC. However, the strip-based CrfA assay had low turnaround time and lacked high-throughput capabilities. In this current research, we have developed a 96well-formatted CrfA assay for high-throughput detection of MTBC and differentiation with NTMs. This 96well-formatted CrfA assay displays a low turnaround time of 6-8 h with 100 % specificity and 93.75 % sensitivity on clinical samples. Based on these attributes, this 96well-formatted assay represents a valuable complementary tool to mitigate the misdiagnosis of chronic pulmonary tuberculosis with non-tuberculous mycobacteria in poorer nations.

Asunto(s)

Mycobacterium tuberculosis , Micobacterias no Tuberculosas , Sensibilidad y Especificidad , Mycobacterium tuberculosis/aislamiento & purificación , Humanos , Micobacterias no Tuberculosas/aislamiento & purificación , Micobacterias no Tuberculosas/clasificación , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Proteínas Bacterianas , Ensayos Analíticos de Alto Rendimiento/métodos