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1.
Front Immunol ; 13: 962220, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36110843

RESUMO

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.


Assuntos
Leucócitos Mononucleares , Transcrição Reversa , Biomarcadores , Citocinas , Epitopos , Ensaios de Triagem em Larga Escala , Imunoadsorventes
2.
Curr Protoc ; 2(9): e542, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36102902

RESUMO

The potential neurotoxicity from an increasing number of drugs and untested environmental chemicals creates a need to develop reliable and efficient in vitro methods for identifying chemicals that may adversely affect the nervous system. An important process in neurodevelopment is neurite outgrowth, which can be affected by developmental neurotoxicity. Currently, neurite outgrowth assays rely mainly on staining, which requires multiple sample processing steps, particularly washing steps, that may introduce variation and limit throughput. Here, we describe a neurite outgrowth assay that uses induced pluripotent stem cell (iPSC)-derived human cortical glutamatergic neurons and/or spinal motor neurons labeled with green fluorescent protein (GFP) to test compounds in a high-content and high-throughput format. This method enables live and time-lapse imaging of GFP-labeled neurons using an assay plate that is continuously imaged at multiple times after chemical treatment. In this article, we describe how to thaw frozen GFP-labeled neurons, culture them, treat them with a compound of interest, and analyze neurite outgrowth using a high-content imaging platform. In this assay, GFP-labeled iPSC-derived human neurons represent a promising tool for identifying and prioritizing compounds with potential developmental neurotoxicity for further hazard characterization. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Thawing and seeding of iPSC-derived neurons Basic Protocol 2: Compound plate preparation and treatment of neurons Basic Protocol 3: High-content imaging and analysis.


Assuntos
Células-Tronco Pluripotentes Induzidas , Síndromes Neurotóxicas , Proteínas de Fluorescência Verde/genética , Ensaios de Triagem em Larga Escala , Humanos , Crescimento Neuronal , Neurônios
3.
Methods Cell Biol ; 172: 135-143, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064220

RESUMO

The radiochemotherapy- or chemotherapy-induced stimulation of immunogenic cell death (ICD) affecting malignant cells ignites antitumor immune responses that are clinically relevant as they allow to achieve durable responses beyond treatment discontinuation. The mechanistic exploration of ICD and the discovery of agents and interventions that are endowed with the capacity to elicit ICD is of the utmost importance. Here, we describe an assay for the assessment of type I interferon (IFN) production, which is one of the salient features of ICD. Biosensor cells that express GFP under the control of the IFN-inducible MX dynamin like GTPase 1 (MX1) gene promoter are employed, and the fluorescent signal is assessed by automated microscopy. The described workflow is automation-friendly, rendering it compatible with high-throughput screening (HTS) for drug discovery.


Assuntos
Interferon Tipo I , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Morte Celular Imunogênica
4.
Curr Protoc ; 2(9): e529, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36066205

RESUMO

Tumor spheroid models are widely used for drug screening as in vitro models of the tumor microenvironment. There are various ways in which tumor spheroid models can be prepared, including the self-assembly of cells using low-adherent plates, micro-patterned plates, or hanging-drop plates. Recently, drug high-throughput screening (HTS) approaches have incorporated the use of these culture systems. These HTS culture systems, however, require complicated equipment, such as robot arms, detectors, and software for handling solutions and data processing. Here, we describe protocols that allow tumor spheroids to be tested with different concentrations of a drug in a parallel fashion using a microfluidic device that generates a gradient of anti-cancer drugs. This microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) enables the formation of 50 tumor spheroids and the testing of drugs at five different concentrations. First, we provide a protocol for the fabrication of the µFSCD-CGG, which has both a culture array in which tumor cells are injected and aggregate to form spheroids and a concentration gradient generator for drug testing. Second, we provide a protocol for tumor spheroid formation and HTS of anti-cancer drugs using the device. Finally, we provide a protocol for assessing the response of tumor spheroids at different drug concentrations. To address the needs of the pharmaceutical industry, this protocol can be used for various cell types, including stem cells, and the number of tumor spheroids and drug concentration ranges that can be tested in the µFSCD-CGG can be increased. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of a microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) Basic Protocol 2: Seeding cells and formation of spheroids in the µFSCD-CGG Basic Protocol 3: Drug treatment and assessment of cell viability in the µFSCD-CGG.


Assuntos
Antineoplásicos , Dispositivos Lab-On-A-Chip , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Esferoides Celulares
5.
Anal Chim Acta ; 1227: 340322, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36089324

RESUMO

Microalgae are a group of photoautotrophic microorganisms which could use carbon dioxide for autosynthesis. They have been envisioned as one of the most prospective feedstock for renewable oil. However, great endeavors will still be needed to increase their economic feasibility. The screening of competitive species and suitable culture conditions are such issues. To greatly accelerate these rather laborious steps and also improve their experimental lump-sum-manner, we developed a microfluidic droplet-based 2 × 103 resolution "identification card", which allowed high throughput real-time monitoring of individual algae among population. A novel fluid-blocking-based droplet generating and trapping performance were integrated in the platform which made it excellent in operational simplicity, rapidity and stability and full of the potentials in single-cell-isolation/screening. The developed platform was successfully used to screen three unicellular algae, namely, Isochrysis zhanjiangensis, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis. In situ bioassays of the lipid accumulation and cell proliferation at single cell level for interspecies comparison were possible. Furthermore, lipid-producing inhomogeneity was demonstrated among cells in the same specie and batch. Nitrogen stress condition can be identified that induce positive-skewed frequency distribution of lipid content, even among individual inhomogeneous cells over the typically used culture condition.


Assuntos
Clorófitas , Microalgas , Ensaios de Triagem em Larga Escala , Lipídeos , Microfluídica , Estudos Prospectivos
6.
Sci Rep ; 12(1): 14879, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050506

RESUMO

We performed a high-throughput phenotypic whole cell screen of Mycobacterium tuberculosis against a diverse chemical library of approximately 100,000 compounds from the AbbVie corporate collection and identified 24 chemotypes with anti-tubercular activity. We selected two series for further exploration and conducted structure-activity relationship studies with new analogs for the 4-phenyl piperidines (4PP) and phenylcyclobutane carboxamides (PCB). Strains with mutations in MmpL3 demonstrated resistance to both compound series. We isolated resistant mutants for the two series and found mutations in MmpL3. These data suggest that MmpL3 is the target, or mechanism of resistance for both series.


Assuntos
Mycobacterium tuberculosis , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Ensaios de Triagem em Larga Escala , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismo
7.
STAR Protoc ; 3(3): 101602, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-35959496

RESUMO

We present a high-content screening (HCS) protocol for quantifying mitochondrial activity in live neural cells from human induced pluripotent stem cells (iPSCs). The assessment is based on mitochondrial membrane potential, which is influenced by the efficiency of mitochondrial bioenergetics. We describe how to perform the analysis using both an HCS platform and the open-source software CellProfiler. The protocol can identify the mitochondrial fitness of human neurons and may be used to carry out high-throughput compound screenings in patient-derived neural cells. For complete details on the use and execution of this protocol, please refer to Lorenz et al. (2017) and Zink et al. (2020).


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/fisiologia , Neurônios , Células-Tronco Pluripotentes/metabolismo
8.
STAR Protoc ; 3(3): 101641, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36035796

RESUMO

Drug repositioning represents a cost- and time-efficient strategy for drug development. Here, we present a workflow of in silico screening of ACE2 enzymatic activators to treat COVID-19-induced metabolic complications. By using structure-based virtual screening and signature-based off-target effect identification via the Connectivity Map database, we provide a ranked list of the repositioning candidates as potential ACE2 enzymatic activators to ameliorate COVID-19-induced metabolic complications. The workflow can also be applied to other diseases with ACE2 as a potential target. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).


Assuntos
COVID-19 , Enzima de Conversão de Angiotensina 2 , Ativadores de Enzimas , Ensaios de Triagem em Larga Escala , Humanos , SARS-CoV-2
9.
Methods Mol Biol ; 2542: 115-126, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36008660

RESUMO

Traditional small molecule antifungal discovery efforts often utilize high-throughput (HTP) biochemical or whole-cell phenotypic screens to identify novel candidates. However, both methods have limitations which hinder the rapid identification of physiologically active compounds that act via a defined mechanism of action. The method described herein is an efficient, sensitive, and HTP compatible approach that utilizes the principles of competitive fitness to rapidly identify small molecules that functionally interact with a specific target protein within whole cells.


Assuntos
Antifúngicos , Proteínas , Antifúngicos/farmacologia , Fenômenos Biofísicos , Ensaios de Triagem em Larga Escala/métodos
10.
Biomolecules ; 12(8)2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-36008948

RESUMO

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an innate immune pattern recognition receptor responsible for the recognition of bacterial peptidoglycan fragments. Given its central role in the formation of innate and adaptive immune responses, NOD2 represents a valuable target for modulation with agonists and antagonists. A major challenge in the discovery of novel small-molecule NOD2 modulators is the lack of a co-crystallized complex with a ligand, which has limited previous progress to ligand-based design approaches and high-throughput screening campaigns. To that end, a hybrid docking and pharmacophore modeling approach was used to identify key interactions between NOD2 ligands and residues in the putative ligand-binding site. Following docking of previously reported NOD2 ligands to a homology model of human NOD2, a structure-based pharmacophore model was created and used to virtually screen a library of commercially available compounds. Two compounds, 1 and 3, identified as hits by the pharmacophore model, exhibited NOD2 antagonist activity and are the first small-molecule NOD2 modulators identified by virtual screening to date. The newly identified NOD2 antagonist scaffolds represent valuable starting points for further optimization.


Assuntos
Ensaios de Triagem em Larga Escala , Simulação de Dinâmica Molecular , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Proteína Adaptadora de Sinalização NOD2
11.
Anal Chem ; 94(33): 11659-11669, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35942642

RESUMO

The "design-build-test-learn" (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.


Assuntos
Gentamicinas , Ensaios de Triagem em Larga Escala , Reatores Biológicos , Computadores , Aprendizado de Máquina
12.
Molecules ; 27(16)2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-36014542

RESUMO

The 3D cell migration assay was developed for the evaluation of drugs that inhibit cell migration using high throughput methods. Wound-healing assays have commonly been used for cell migration assays. However, these assays have limitations in mimicking the in vivo microenvironment of the tumor and measuring cell viability for evaluation of cell migration inhibition without cell toxicity. As an attempt to manage these limitations, cells were encapsulated with Matrigel on the surface of the pillar, and an analysis of the morphology of cells attached to the pillar through Matrigel was performed for the measurement of cell migration. The micropillar/microwell chips contained 532 pillars and wells, which measure the migration and viability of cells by analyzing the roundness and size of the cells, respectively. Cells seeded in Matrigel have a spherical form. Over time, cells migrate through the Matrigel and attach to the surface of the pillar. Cells that have migrated and adhered have a diffused shape that is different from the initial spherical shape. Based on our analysis of the roundness of the cells, we were able to distinguish between the diffuse and spherical shapes. Cells in Matrigel on the pillar that were treated with migration-inhibiting drugs did not move to the surface of the pillar and remained in spherical forms. During the conduct of experiments, 70 drugs were tested in single chips and migration-inhibiting drugs without cell toxicity were identified. Conventional migration assays were performed using transwell for verification of the four main migration-inhibiting drugs found on the chip.


Assuntos
Técnicas de Cultura de Células , Ensaios de Triagem em Larga Escala , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Ensaios de Migração Celular , Movimento Celular , Sobrevivência Celular
13.
Proc Natl Acad Sci U S A ; 119(35): e2122004119, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994666

RESUMO

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).


Assuntos
Códon sem Sentido , Doenças Genéticas Inatas , Guanidinas , Quinazolinas , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Gentamicinas/farmacologia , Guanidinas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacologia
14.
J Chem Inf Model ; 62(16): 3854-3862, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35938299

RESUMO

High-throughput virtual screening is an indispensable technique utilized in the discovery of small molecules. In cases where the library of molecules is exceedingly large, the cost of an exhaustive virtual screen may be prohibitive. Model-guided optimization has been employed to lower these costs through dramatic increases in sample efficiency compared to random selection. However, these techniques introduce new costs to the workflow through the surrogate model training and inference steps. In this study, we propose an extension to the framework of model-guided optimization that mitigates inference costs using a technique we refer to as design space pruning (DSP), which irreversibly removes poor-performing candidates from consideration. We study the application of DSP to a variety of optimization tasks and observe significant reductions in overhead costs while exhibiting similar performance to the baseline optimization. DSP represents an attractive extension of model-guided optimization that can limit overhead costs in optimization settings where these costs are non-negligible relative to objective costs, such as docking.


Assuntos
Ensaios de Triagem em Larga Escala , Fluxo de Trabalho
15.
Eur J Med Chem ; 241: 114643, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-35961069

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a highly fatal disease that lacks appropriate treatments and highly effective drugs. Many reported indicated that the TGF-ß1/Smad3 signaling pathway played a pivotal role in development of IPF. In this case, it was hypothesized that discovery novel compounds to block the TGF-ß1/Smad3 signaling pathway might be useful for treatment of IPF. Therefore, a high-throughput screening system based on stably transfected CAGA-NIH3T3 cells was established for discovering lead compounds which could validly suppress the TGF-ß1/Smad3 signal path. In this study, a series of novel Pleuromutilin derivatives were prepared and quickly evaluated by high-throughput assay. The lead compound 32 was discovered to be able to remarkably suppress the TGF-ß1/Smad3 pathway in vitro. Further biological evaluation revealed that compound 32 could remarkably decrease the myofibroblast stimulation and extracellular matrix (ECM) deposition. More importantly, compound 32 could remarkably mitigate bleomycin (BLM)-triggered lung fibrosis in mice models. Additionally, the lead compound possess excellent pharmacokinetics properties, good oral availability and low toxicity. In general, our study has demonstrated the potency of a novel Pleuromutilin derivative (compound 32), which might be a prospective candidate for developing anti-IPF medicines by suppress the TGF-ß1/Smad3 signal pathway.


Assuntos
Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Animais , Bleomicina/metabolismo , Bleomicina/farmacologia , Bleomicina/uso terapêutico , Diterpenos , Fibroblastos , Ensaios de Triagem em Larga Escala , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Compostos Policíclicos , Fator de Crescimento Transformador beta1/metabolismo
16.
Methods Mol Biol ; 2538: 131-144, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35951298

RESUMO

Escherichia coli remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for E. coli yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in E. coli to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Amiloide/genética , Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala , Humanos
17.
Anal Chem ; 94(34): 11838-11847, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35977405

RESUMO

A higher correlation of epidermal growth factor receptor (EGFR)-targeting drugs has been reported with the use of the cell proliferation receptor-enhanced three-dimensional high-throughput screening model (CPRE 3D-HTS model) compared with two-dimensional (2D) cell-based HTS. A greater expression of differential human EGFR 2 (HER2) protein between HER2-positive and HER2-negative cell lines was observed in breast cancer (BC) cell lines cultured using the CPRE 3D-HTS model compared with 2D-cultured cells. When using 2D-cultured cells, properties such as the expression of the cell proliferation receptor are lost as the cells attach to the bottom of the well plate. In an effort to solve this problem, the CPRE 3D-HTS model expressing high cell proliferation receptors was optimized by the selection of alginate as the extracellular matrix. Results from the use of the CPRE 3D-HTS model showed higher drug resistance with increased expression of drug resistance-related proteins. Of particular interest, a higher correlation of HER2-targeted drugs was observed with the use of the CPRE 3D-HTS model. In order to validate this higher correlation of target drugs observed in the CPRE 3D-HTS model, the results of Western blot analysis and high content imaging analysis were analyzed, which confirmed that 3D-cultured BC cell lines showed a greater difference in the expression of HER2-positive and HER2-negative BC cell lines than 2D-cultured cells. Thus, the use of CPRE 3D-HTS using a 384-pillar plate resulted in increased accuracy when screening HER2-targeted drugs in BC, and it is a very useful platform for analyzing the efficacy of targeted drugs by enhancing the expression of HER2.


Assuntos
Antineoplásicos , Neoplasias da Mama , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Detecção Precoce de Câncer , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Receptor ErbB-2/metabolismo
18.
Metab Eng ; 73: 144-157, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35921946

RESUMO

Metabolic engineering seeks to rewire the metabolic network of cells for the efficient production of value-added compounds from renewable substrates. However, it remains challenging to evaluate and identify strains with the desired phenotype from the vast rational or random mutagenesis library. One effective approach to resolve this bottleneck is to design an efficient high-throughput screening (HTS) method to rapidly detect and analyze target candidates. L-cysteine is an important sulfur-containing amino acid and has been widely used in agriculture, pharmaceuticals, cosmetics, and food additive industries. However, HTS methods that enable monitoring of L-cysteine levels and screening of the enzyme variants and strains to confer superior L-cysteine biosynthesis remain unavailable, greatly limiting the development of efficient microbial cell factories for L-cysteine production at the industrial scale. Here, we took advantage of the L-cysteine-responsive transcriptional regulator CcdR to develop a genetically encoded biosensor for engineering and screening the L-cysteine overproducer. The in vivo L-cysteine-responsive assays and in vitro electrophoretic mobility shift assay (EMSA) and DNase I footprint analysis indicated that CcdR is a transcriptional activator that specifically interacts with L-cysteine and binds to its regulatory region to induce the expression of target genes. To improve the response performance of the L-cysteine biosensor, multilevel optimization strategies were performed, including regulator engineering by semi-rational design and systematic optimization of the genetic elements by modulating the promoter and RBS combination. As a result, the dynamic range and sensitivity of the biosensor were significantly improved. Using this the excellent L-cysteine biosensor, a HTS platform was established by coupling with fluorescence-activated cell sorting (FACS) and was successfully applied to achieve direct evolution of the key enzyme in the L-cysteine biosynthetic pathway to increase its catalytic performance and to screen the high L-cysteine-producing strains from the random mutagenesis library. These results presented a paradigm of design and optimization of biosensors to dynamically detect metabolite concentrations and provided a promising tool enabling HTS and metabolic regulation to construct L-cysteine hyperproducing strains to satisfy industrial demand.


Assuntos
Técnicas Biossensoriais , Cisteína , Técnicas Biossensoriais/métodos , Cisteína/genética , Ensaios de Triagem em Larga Escala , Engenharia Metabólica/métodos , Fatores de Transcrição/genética
19.
Toxicol In Vitro ; 84: 105451, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35921976

RESUMO

In response to the need to minimize the use of experimental animals, new approach methodologies (NAMs) using advanced technology have emerged in the 21st century. ToxCast/Tox21 aims to evaluate the adverse effects of chemicals quickly and efficiently using a high-throughput screening and to transform the paradigm of toxicity assessment into mechanism-based toxicity prediction. The ToxCast/Tox21 database, which contains extensive data from over 1400 assays with numerous biological targets and activity data for over 9000 chemicals, can be used for various purposes in the field of chemical prioritization and toxicity prediction. In this study, an overview of the database was explored to aid mechanism-based chemical prioritization and toxicity prediction. Implications for the utilization of the ToxCast/Tox21 database in chemical prioritization and toxicity prediction were derived. The research trends in ToxCast/Tox21 assay data were reviewed in the context of toxicity mechanism identification, chemical priority, environmental monitoring, assay development, and toxicity prediction. Finally, the potential applications and limitations of using ToxCast/Tox21 assay data in chemical risk assessment were discussed. The analysis of the toxicity mechanism-based assays of ToxCast/Tox21 will help in chemical prioritization and regulatory applications without the use of laboratory animals.


Assuntos
Bioensaio , Ensaios de Triagem em Larga Escala , Animais , Bases de Dados Factuais , Monitoramento Ambiental , Ensaios de Triagem em Larga Escala/métodos , Medição de Risco
20.
J Med Chem ; 65(16): 11322-11339, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-35943172

RESUMO

High-throughput screening provides one of the most common ways of finding hit compounds. Lead-like libraries, in particular, provide hits with compatible functional groups and vectors for structural elaboration and physical properties suitable for optimization. Library synthesis approaches can lead to a lack of chemical diversity because they employ parallel derivatization of common building blocks using single reaction types. We address this problem through a "build-couple-transform" paradigm for the generation of lead-like libraries with scaffold diversity. Nineteen transformations of a 4-oxo-2-butenamide scaffold template were optimized, including 1,4-cyclizations, 3,4-cyclizations, reductions, and 1,4-additions. A pool-transformation approach efficiently explored the scope of these transformations for nine different building blocks and synthesized a >170-member library with enhanced chemical space coverage and favorable drug-like properties. Screening revealed hits against CDK2. This work establishes the build-couple-transform concept for the synthesis of lead-like libraries and provides a differentiated approach to libraries with significantly enhanced scaffold diversity.


Assuntos
Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Bibliotecas de Moléculas Pequenas/química
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