Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen

Abstract

The 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 from Mycobacterium 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 of in vivo Mycobacterium 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.

FIG 1

Scheme of drug efficacy methods using antibacterial activity and uptake assays in the zebrafish model integrated in conventional disease-screening pipeline. Different models are used in our workflow to determine the efficacy of antitubercular compounds (from left to right). (a) Initially, the in vitro efficacy of the tested compounds is determined by their MIC against Mycobacterium marinum and Mycobacterium tuberculosis cultures using fluorimetric and colorimetric readouts from the resazurin assay. (b to d) Biological validation subsequently is performed in in vivo models. (b) First, compound efficacy is screened in M. marinum-infected zebrafish larvae. Embryos are robotically injected, and following compound treatment, the percentage of inhibition is determined using the fluorescence readout of the COPAS system. (c) To unravel whether certain compounds fail to be active in zebrafish larvae due to the lack of antibacterial activity or poor uptake, our microneedle sampling method combined with mass spectrometry is used to assess uptake levels from samples of the yolk. (d) As a gold standard in antitubercular drug development, compound efficacy is established by determining the rate of the CFU reduction in the lungs of M. tuberculosis-infected rodents. After setting (arbitrary) cutoffs in all models, compounds could be categorized into positive or negative groups. By the comparison of these groups along the pipeline, our improved zebrafish platform may give a more predictive value for human efficacy of drugs. Cycle times are indicated for each model.

FIG 2

Rifampin and moxifloxacin quantities measured by the whole-zebrafish-larva lysis method (a) and microneedle sampling method from the yolk (b). Three-day-old zebrafish larvae were exposed to a combination of rifampin (RIF; gray bars) and moxifloxacin (MOX; white bars) dosed at a 150 μM concentration for 0, 17, and 40 h (n = 10). Note that as a control, at the zero time point (T = 0 h) the zebrafish larvae were exposed to the compounds (at the same dose) for only a few seconds. This short exposure was followed by three consecutive washing steps with 50% methanol. In the readout, after lysis of the whole larvae the presence of compounds is detected even at T = 0 h (a); however, via our microneedle method the compounds are undetectable in the yolk at T = 0 h (b). All data are expressed as the means ± standard errors of the means.

FIG 3

Correlation between the uptake level of preclinical antitubercular compounds and their efficacy in M. marinum zebrafish infection model. (a) Uptake levels of 15 preclinical antitubercular GSK compounds were measured from samples taken from the yolk of 5-day-old zebrafish larvae after 40 h of exposure at 10 μM concentration (black bars) (n = 10). The efficacy of the compounds was assessed by monitoring fluorescent bacterial burden in the total body (red bars) or tail region (orange bars) of larvae at 5 days p.i. using the COPAS system after 40 h of treatment (n = 200). Efficacy is expressed as a percentage of inhibition of mycobacterial proliferation relative to the level for DMSO-treated control groups. The bar graphs depict the correlation between the uptake and the relative inhibition, both in the total body and the tail region. Significance in inhibition is indicated with asterisks (***, P < 0.001). (b and c) Correlation between the efficacy in the total body (b) or in the tail (c) and the uptake of the GSK compounds. The efficacy of several compounds shown in panels b and c is below detection limits, as shown in panel a.

FIG 4

Correlation between uptake levels and compound hydrophobicity. chromlogD is a representation of hydrophobicity; therefore, it represents the solubility/lipophilicity of a compound. To examine the relationship between chromlogD of the compounds and their uptake, chromlogD is plotted against uptake levels measured in 5-day-old zebrafish larvae after 40 h of exposure.

FIG 5

Correlation between uptake, in vitro MIC, and in vivo zebrafish antibacterial activities of compounds. The measured uptake data in zebrafish larvae were normalized with the in vitro MIC of compounds against M. marinum [log(uptake/MIC)]. This ratio is plotted against the observed percentage of inhibition in the zebrafish M. marinum infection assay. The size of the circles indicates their MIC values, meaning smaller circles correspond to lower MIC values with higher potency. Green color represents compounds significantly reducing bacterial burden in the zebrafish infection model, considered active compounds, while red color indicates inactive compounds.