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1.
Sci Rep ; 14(1): 3875, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365924

RESUMO

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.


Assuntos
Antineoplásicos , Histonas , Histonas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli ADP Ribosilação , Ensaios de Triagem em Larga Escala , Poli(ADP-Ribose) Polimerase-1/metabolismo , Reparo do DNA , Dano ao DNA
2.
Brief Bioinform ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38324621

RESUMO

Single-cell clustered regularly interspaced short palindromic repeats-sequencing (scCRISPR-seq) is an emerging high-throughput CRISPR screening technology where the true cellular response to perturbation is coupled with infected proportion bias of guide RNAs (gRNAs) across different cell clusters. The mixing of these effects introduces noise into scCRISPR-seq data analysis and thus obstacles to relevant studies. We developed scDecouple to decouple true cellular response of perturbation from the influence of infected proportion bias. scDecouple first models the distribution of gene expression profiles in perturbed cells and then iteratively finds the maximum likelihood of cell cluster proportions as well as the cellular response for each gRNA. We demonstrated its performance in a series of simulation experiments. By applying scDecouple to real scCRISPR-seq data, we found that scDecouple enhances the identification of biologically perturbation-related genes. scDecouple can benefit scCRISPR-seq data analysis, especially in the case of heterogeneous samples or complex gRNA libraries.


Assuntos
Ensaios de Triagem em Larga Escala , RNA Guia de Sistemas CRISPR-Cas
3.
Sci Rep ; 14(1): 3312, 2024 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-38332234

RESUMO

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.


Assuntos
Heparinoides , Junções Íntimas , Humanos , Idoso , Claudina-1/metabolismo , Junções Íntimas/metabolismo , Heparinoides/metabolismo , Ensaios de Triagem em Larga Escala , Queratinócitos/metabolismo , Claudina-4/metabolismo
4.
Int Ophthalmol ; 44(1): 63, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38347388

RESUMO

PURPOSE: Pterygium is a hyaline degenerative disease of the conjunctiva characterized by the progression of fibrovascular connective tissue from the bulbar conjunctiva to the cornea. The mechanism of pterygium formation is still not fully understood. Transient receptor potential (TRP) channels are a group of ion channels with distinct characteristics. Recent indications suggest TRP channels may play a significant regulatory role in pterygium development, but previous studies have mainly focused on in silico analysis. Accordingly, in the present study, we aimed to decipher the expression signatures and role of TRP channels in pterygium development. METHODS: The study encompassed a cohort of 45 patients matched for age and gender distribution, comprising 30 individuals with primary pterygium (PP) and 15 individuals with recurrent pterygium (RP). The control group consisted of unaffected conjunctival tissue obtained from the same set of patients. High-throughput screening of differentially expressed TRP channels in pterygium tissues was achieved with the help of Fluidigm 96.96 Dynamic Array Expression Chip and reactions were held in BioMark™ HD System Real-Time PCR platform. RESULTS: Statistically significant increases were found in the expression of 21 genes, mainly TRPA1 (p = 0.021), TRPC2 (p = 0.001), and TRPM8 (p = 0.003), in patients with PP, and in TRPC5 (p = 0.05), TRPM2 (p = 0.029), TRPM4 (p = 0.03), TRPM6 (p = 0.045), TRPM8 (p = 0.038), TRPV1 (p = 0.01) and TRPV4 (p = 0.025) genes in RP tissues. CONCLUSION: Collectively, TRP channel proteins appear to play pivotal roles in both the development and progression of pterygium, making them promising candidates for future therapeutic interventions in patients afflicted by this condition.


Assuntos
Túnica Conjuntiva/anormalidades , Pterígio , Canais de Potencial de Receptor Transitório , Humanos , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Pterígio/diagnóstico , Ensaios de Triagem em Larga Escala , Túnica Conjuntiva/metabolismo
5.
J Vis Exp ; (203)2024 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-38314817

RESUMO

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.


Assuntos
Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , Espécies Reativas de Oxigênio/metabolismo , Etídio
6.
Methods Cell Biol ; 182: 265-284, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38359982

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent and recombination-based mechanism used by approximately 15% of human cancers to maintain telomere length and to sustain proliferation. ALT-positive cells display unique features that could be exploited for tailored cancer therapies. A key limitation for the development of ALT-specific treatments is the lack of an assay to detect ALT-positive cells that is easy to perform and that can be scaled up. One of the most broadly used assays for ALT detection, CCA (C-circle assay), does not provide single-cell information and it is not amenable to High-Throughput Screening (HTS). To overcome these limitations, we developed Native-FISH (N-FISH) as an alternative method to visualize ALT-specific single-stranded telomeric DNA. N-FISH produces single-cell data, can be applied to fixed tissues, does not require DNA isolation or amplification steps, and it can be miniaturized in a 384-well format. This protocol details the steps to perform N-FISH protocol both in a low- and high-throughput format to analyze ALT. While low-throughput N-FISH is useful to assay the ALT state of cell lines, we expect that the miniaturized N-FISH assay coupled with high-throughput imaging will be useful in functional genomics and chemical screens to identify novel cellular factors that regulate ALT and potential ALT therapeutic targets for cancer therapies directed against ALT-positive tumors, respectively.


Assuntos
Ensaios de Triagem em Larga Escala , Neoplasias , Humanos , Animais , DNA , Telômero/genética , Peixes/genética
7.
J Med Virol ; 96(1): e29382, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38235833

RESUMO

Japanese encephalitis (JE) caused by JE virus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at-risk populations. This underscores the urgent need for prophylaxis and potent anti-JEV drugs. In this context, a high-content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high-throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA-dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti-JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Animais , Camundongos , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Antivirais/farmacologia , Antivirais/uso terapêutico , Replicação Viral
8.
Int J Mol Sci ; 25(1)2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38203828

RESUMO

High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.


Assuntos
Endonucleases , Testes Genéticos , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Fenótipo
9.
Biosens Bioelectron ; 248: 115972, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38171222

RESUMO

Enzymes, as biocatalysts, play a cumulatively important role in environmental purification and industrial production of chemicals and pharmaceuticals. However, natural enzymes are limited by their physiological properties in practice, which need to be modified driven by requirements. Screening and isolating certain enzyme variants or ideal industrial strains with high yielding of target product enzymes is one of the main directions of enzyme engineering research. Droplet-based high-throughput screening (DHTS) technology employs massive monodisperse emulsion droplets as microreactors to achieve single strain encapsulation, as well as continuous monitoring for the inside mutant library. It can effectively sort out strains or enzymes with desired characteristics, offering a throughput of 108 events per hour. Much of the early literature focused on screening various engineered strains or designing signalling sorting strategies based on DHTS technology. However, the field of enzyme engineering lacks a comprehensive overview of advanced methods for microfluidic droplets and their cutting-edge developments in generation and manipulation. This review emphasizes the advanced strategies and frontiers of microfluidic droplet generation and manipulation facilitating enzyme engineering development. We also introduce design for various screening signals that cooperate with DHTS and devote to enzyme engineering.


Assuntos
Técnicas Biossensoriais , Ensaios de Triagem em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos
10.
Food Res Int ; 177: 113902, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38225144

RESUMO

A microtiter plate (MTP) method was developed to screen 1064 unique microorganisms-substrate fermentations for production of 68 target aroma compounds. Based on the number of hits identified by GC-MS, 50 fermentations were repeated at 50-mL scale in flasks. Comparison of GC-MS data showed that scaling up from MTP to flask did not generally result in large differences between the volatile profiles, even with a wide variety of substrates (juice, food slurry and food side-streams) and microorganisms (yeast, bacteria and fungi) used. From the screening results, Lactobacillus plantarum fermentation of chilli pepper was further studied as a high amount of phenols, especially guaiacol and 4-ethylphenol, was produced after fermentation. From HPLC-MS and sensory analysis, capsaicin was shown to be a probable precursor for these phenols and a potential mechanism was proposed. The protocol described herein to screen aroma compounds from fermentation of agri-food products and side streams can support development of clean label flavourful food ingredients.


Assuntos
Ensaios de Triagem em Larga Escala , Odorantes , Fermentação , Ensaios de Triagem em Larga Escala/métodos , Fenóis , Saccharomyces cerevisiae
11.
Neurotherapeutics ; 21(1): e00291, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38241154

RESUMO

Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.


Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Ensaios de Triagem em Larga Escala
12.
J Chromatogr A ; 1716: 464632, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38219623

RESUMO

Recombinant adeno-associated virus (AAV) has emerged as one of the most promising systems for therapeutic gene delivery and has demonstrated clinical success in a wide range of genetic disorders. However, manufacturing of high-quality AAV in large amounts still remains a challenge. A significant difficulty for downstream processing is the need to remove empty capsids that are generated in all currently utilized expression systems and that represent product-related impurities that adversely affect safety and efficacy of AAV vectors. Empty and full capsids exhibit only subtle differences in surface charge and size, making chromatography-based separations highly challenging. Here, we present a rapid methodology for the systematic process development of the crucial AAV full/empty capsid separation on ion-exchange media based on high-throughput screening and mechanistic modeling. Two of the most commonly employed serotypes, AAV8 and AAV9, are used as case studies. First, high-throughput studies in filter-plate format are performed that allow the rapid and comprehensive study of binding and elution behavior of AAV on different resins, using different buffer systems, pH, salt conditions, and solution additives. Small amounts of separated empty and full AAV capsids are generated by iodixanol gradient centrifugation that allow studying the binding and elution behavior of the two vector species separately in miniaturized format. Process conditions that result in maximum differences in elution behavior between empty and full capsids are then transferred to benchtop chromatography systems that are used to generate calibration data for the estimation of steric mass-action isotherm and mass transport parameters for process simulation. The resulting column models are employed for in-silico process development that serves to enhance understanding of separation constraints and to identify optimized conditions for the removal of empty particles. Finally, optimized separation conditions are verified experimentally. The methodology presented in this work provides a systematic framework that affords mechanistic understanding of the crucial empty/full capsid separation and accelerates the development of a scalable AAV downstream process.


Assuntos
Capsídeo , Dependovirus , Capsídeo/química , Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Ensaios de Triagem em Larga Escala , Vetores Genéticos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/análise
13.
J Chem Inf Model ; 64(3): 638-652, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38294781

RESUMO

A simple approach was developed to computationally construct a polymer dataset by combining simplified molecular-input line-entry system (SMILES) strings of a targeted polymer backbone and a variety of molecular fragments. This method was used to create 14 polymer datasets by combining seven polymer backbones and molecules from two large molecular datasets (MOSES and QM9). Polymer backbones that were studied include four polydimethylsiloxane (PDMS) based backbones, poly(ethylene oxide) (PEO), poly(allyl glycidyl ether) (PAGE), and polyphosphazene (PPZ). The generated polymer datasets can be used for various cheminformatics tasks, including high-throughput screening for gas permeability and selectivity. This study utilized machine learning (ML) models to screen the polymers for CO2/CH4 and CO2/N2 gas separation using membranes. Several polymers of interest were identified. The results highlight that employing an ML model fitted to polymer selectivities leads to higher accuracy in predicting polymer selectivity compared to using the ratio of predicted permeabilities.


Assuntos
Dióxido de Carbono , Polímeros , Polietilenoglicóis , Quimioinformática , Ensaios de Triagem em Larga Escala
14.
Phys Chem Chem Phys ; 26(6): 5377-5386, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38269624

RESUMO

Due to the crucial regulatory mechanism of cyclin-dependent kinase 9 (CDK9) in mRNA transcription, the development of kinase inhibitors targeting CDK9 holds promise as a potential treatment strategy for cancer. A structure-based virtual screening approach has been employed for the discovery of potential novel CDK9 inhibitors. First, compounds with kinase inhibitor characteristics were identified from the ZINC15 database via virtual high-throughput screening. Next, the predicted binding modes were optimized by molecular dynamics simulations, followed by precise estimation of binding affinities using absolute binding free energy calculations based on the free energy perturbation scheme. The binding mode of molecule 006 underwent an inward-to-outward flipping, and the new binding mode exhibited binding affinity comparable to the small molecule T6Q in the crystal structure (PDB ID: 4BCF), highlighting the essential role of molecular dynamics simulation in capturing a plausible binding pose bridging docking and absolute binding free energy calculations. Finally, structural modifications based on these findings further enhanced the binding affinity with CDK9. The results revealed that enhancing the molecule's rigidity through ring formation, while maintaining the major interactions, reduced the entropy loss during the binding process and, thus, enhanced binding affinities.


Assuntos
Quinase 9 Dependente de Ciclina , Ensaios de Triagem em Larga Escala , Ligação Proteica , Entropia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular
15.
J Med Chem ; 67(3): 2118-2128, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38270627

RESUMO

We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry, automated purification, in situ quantification, and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays. In using microscale chemistry and thus consuming minimal chemical matter, the platform is not only efficient but also follows green chemistry principles. By reorienting existing high-throughput assay technology, the platform can generate a full package of relevant data on each set of compounds in every learning cycle. The multiparameter exploration of chemical and property space is hereby driven by active learning models. The enhanced compound optimization process is generating knowledge for drug discovery projects in a time frame never before possible.


Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala
16.
Proc Natl Acad Sci U S A ; 121(5): e2318718121, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38252820

RESUMO

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 µM), Apicidin (10 µM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.


Assuntos
Aterosclerose , Células Endoteliais , Morfolinas , Pironas , Humanos , Animais , Camundongos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Aterosclerose/tratamento farmacológico , Hemodinâmica , Inflamação
17.
J Immunol ; 212(2): 235-243, 2024 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-38166249

RESUMO

Abs are versatile molecules with the potential to achieve exceptional binding to target Ags, while also possessing biophysical properties suitable for therapeutic drug development. Protein display and directed evolution systems have transformed synthetic Ab discovery, engineering, and optimization, vastly expanding the number of Ab clones able to be experimentally screened for binding. Moreover, the burgeoning integration of high-throughput screening, deep sequencing, and machine learning has further augmented in vitro Ab optimization, promising to accelerate the design process and massively expand the Ab sequence space interrogated. In this Brief Review, we discuss the experimental and computational tools employed in synthetic Ab engineering and optimization. We also explore the therapeutic challenges posed by developing Abs for infectious diseases, and the prospects for leveraging machine learning-guided protein engineering to prospectively design Abs resistant to viral escape.


Assuntos
Anticorpos , Engenharia de Proteínas , Anticorpos/genética , Aprendizado de Máquina , Proteínas , Ensaios de Triagem em Larga Escala
18.
Nat Commun ; 15(1): 42, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168091

RESUMO

To curb viral epidemics and pandemics, antiviral drugs are needed with activity against entire genera or families of viruses. Here, we develop a cell-based multiplex antiviral assay for high-throughput screening against multiple viruses at once, as demonstrated by using three distantly related orthoflaviviruses: dengue, Japanese encephalitis and yellow fever virus. Each virus is tagged with a distinct fluorescent protein, enabling individual monitoring in cell culture through high-content imaging. Specific antisera and small-molecule inhibitors are employed to validate that multiplexing approach yields comparable inhibition profiles to single-virus infection assays. To facilitate downstream analysis, a kernel is developed to deconvolute and reduce the multidimensional quantitative data to three cartesian coordinates. The methodology is applicable to viruses from different families as exemplified by co-infections with chikungunya, parainfluenza and Bunyamwera viruses. The multiplex approach is expected to facilitate the discovery of broader-spectrum antivirals, as shown in a pilot screen of approximately 1200 drug-like small-molecules.


Assuntos
Viroses , Vírus , Humanos , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Técnicas de Cultura de Células , Replicação Viral
19.
Appl Microbiol Biotechnol ; 108(1): 66, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38194139

RESUMO

Biocatalysis is one of the greatest tools for implementing the 12 principles of Green chemistry. Biocatalysts are bio-based, highly efficient and selective, operate at moderate conditions, and can be reused multiple times. However, the wider application of biocatalysts is plagued by a plethora of drawbacks, such as poor stability at operating conditions, inadequate efficiency of catalytic systems, a small number of commercially available biocatalysts, and a lack of substrates or methods for their discovery and development. In this work, we address the lack of suitable substrates for high-throughput screening of laccase by synthesising and investigating a newly developed phenazine-type substrate - Ferbamine. Investigation of Ferbamine pH and thermal stability indicated that its long-term stability in an aqueous medium is superior to that of commercially available substrates and does not require organic solvents. Ferbamine displayed convincing performance in detecting laccase activity on Ferbamine-agar plates in commercial laccase products and the collection of extracts from wild terrestrial fungi (42 species, 65 extracts), of which 26 species have not been described to have laccase activity prior to this work. Incubation of microorganisms on Ferbamine-agar plates showed its compatibility with live colonies. Ferbamine proved to be an easy-to-use substrate, which could be a great addition to the toolbox of methods for the functional analysis of laccases.


Assuntos
Ensaios de Triagem em Larga Escala , Lacase , Ágar , Biocatálise , Fenazinas
20.
Sci Total Environ ; 914: 169783, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38184261

RESUMO

Ecotoxicology has long relied on assessing the hazard potential of chemicals through traditional in vivo testing methods to understand the possible risk exposure could pose to ecological taxa. In the past decade, the development of non-animal new approach methods (NAMs) for assessing chemical hazard and risk has quickly grown. These methods are often cheaper and faster than traditional toxicity testing, and thus are amenable to high-throughput toxicity testing (HTT), resulting in large datasets. The ToxCast/Tox21 HTT programs have produced in vitro data for thousands of chemicals covering a large space of biological activity. The relevance of these data to in vivo mammalian toxicity has been much explored. Interest has also grown in using these data to evaluate the risk of environmental exposures to taxa of ecological importance such as fish, aquatic invertebrates, etc.; particularly for the purpose of estimating the risk of exposure from real-world complex mixtures. Understanding the relationship and relative sensitivity of NAMs versus standardized ecotoxicological whole organism models is a key component of performing reliable read-across from mammalian in vitro data to ecotoxicological in vivo data. In this work, we explore the relationship between in vivo ecotoxicity data from several publicly available databases and the ToxCast/Tox21 data. We also performed several case studies in which we compare how using different ecotoxicity datasets, whether traditional or ToxCast-based, affects risk conclusions based on exposure to complex mixtures derived from existing large-scale chemical monitoring data. Generally, predictive value of ToxCast data for traditional in vivo endpoints (EPs) was poor (r ≤ 0.3). Risk conclusions, including identification of different chemical risk drivers and prioritized monitoring sites, were different when using HTT data vs. traditional in vivo data.


Assuntos
Ecotoxicologia , Ensaios de Triagem em Larga Escala , Animais , Ensaios de Triagem em Larga Escala/métodos , Exposição Ambiental , Testes de Toxicidade , Misturas Complexas , Medição de Risco , Mamíferos
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