Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

ABSTRACTThe translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data fromMycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models ofin vivoMycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

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Zebrafish Embryo Model for Assessment of Drug Efficacy on Mycobacterial Persisters

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00801-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Susanna Commandeur ◽

Nino Iakobachvili ◽

Marion Sparrius ◽

Mariam Mohamed Nur ◽

Galina V. Mukamolova ◽

...

Keyword(s):

Drug Screening ◽

Zebrafish Embryo ◽

Neutral Lipids ◽

Drug Efficacy ◽

Infection Model ◽

Duration Of Treatment ◽

Content Type ◽

Set Up

ABSTRACT Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinum. In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo. To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo. This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters.

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Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630318803749 ◽

2018 ◽

Vol 24 (1) ◽

pp. 28-40 ◽

Cited By ~ 2

Author(s):

Kelli M. Wilson ◽

Lesley A. Mathews-Griner ◽

Tara Williamson ◽

Rajarshi Guha ◽

Lu Chen ◽

...

Keyword(s):

Cell Lines ◽

High Throughput ◽

Drug Screening ◽

Kinase Inhibitors ◽

Proteasome Inhibitors ◽

Drug Efficacy ◽

Driver Genes ◽

Human Gbm

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.

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Abstract 2787: PrimePanel provides a high throughput in vitro drug screening platform that intimately links to in vivo pharmacological analysis in PDX models.

10.1158/1538-7445.am2013-2787 ◽

2013 ◽

Author(s):

Yanxia Zhang ◽

Yuqiang Ge ◽

Yanan Liu ◽

Jing Zhao ◽

Lanhua Li ◽

...

Keyword(s):

High Throughput ◽

Drug Screening ◽

Pharmacological Analysis ◽

In Vitro Drug Screening ◽

Screening Platform ◽

Pdx Models

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A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy

Molecules ◽

10.3390/molecules23123355 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3355 ◽

Cited By ~ 14

Author(s):

Wanyoung Lim ◽

Sungsu Park

Keyword(s):

High Throughput ◽

Concentration Gradient ◽

High Throughput Screening ◽

3D Cell Culture ◽

Drug Efficacy ◽

Cancer Drug ◽

Spheroid Culture ◽

Gradient Generator ◽

Micro Channels

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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A versatile automated high-throughput drug screening platform for zebrafish embryos

10.1101/2020.12.16.423108 ◽

2020 ◽

Author(s):

Alexandra Lubin ◽

Jason Otterstrom ◽

Yvette Hoade ◽

Ivana Bjedov ◽

Eleanor Stead ◽

...

Keyword(s):

High Throughput ◽

Drug Screening ◽

Imaging System ◽

Cell Types ◽

Wide Range ◽

Stem And Progenitor Cells ◽

Multiple Cell ◽

Flexible Imaging ◽

Automated Screening ◽

Screening Platform

AbstractZebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed a easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan®Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft®Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high content screening in zebrafish.

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Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00363-20 ◽

2020 ◽

Vol 64 (11) ◽

Cited By ~ 2

Author(s):

Matt D. Johansen ◽

Wassim Daher ◽

Françoise Roquet-Banères ◽

Clément Raynaud ◽

Matthéo Alcaraz ◽

...

Keyword(s):

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Zebrafish Model ◽

Content Type ◽

Attractive Option ◽

Conventional Antibiotics ◽

Susceptibility Profiles ◽

Combination Regimens

ABSTRACT Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.

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