Dataset Information

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

ABSTRACT: Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the ?-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical ?-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

SUBMITTER: Neuckermans J

PROVIDER: S-EPMC6775094 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Neuckermans Jessie J Mertens Alan A De Win Dinja D Schwaneberg Ulrich U De Kock Joery J

Scientific reports 20191002 1

Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human ...[more]

PMID: 31578365

Similar Datasets

Project description:Quantitative high throughput screening (qHTS) assays use cells or tissues to screen thousands of compounds in a short period of time. Data generated from qHTS assays are then evaluated using nonlinear regression models, such as the Hill model, and decisions regarding toxicity are made using the estimates of the parameters of the model. For any given compound, the variability in the observed response may either be constant across dose groups (homoscedasticity) or vary with dose (heteroscedasticity). Since thousands of compounds are simultaneously evaluated in a qHTS assay, it is not practically feasible for an investigator to perform residual analysis to determine the variance structure before performing statistical inferences on each compound. Since it is well-known that the variance structure plays an important role in the analysis of linear and nonlinear regression models it is therefore important to have practically useful and easy to interpret methodology which is robust to the variance structure. Furthermore, given the number of chemicals that are investigated in the qHTS assay, outliers and influential observations are not uncommon. In this article we describe preliminary test estimation (PTE) based methodology which is robust to the variance structure as well as any potential outliers and influential observations. Performance of the proposed methodology is evaluated in terms of false discovery rate (FDR) and power using a simulation study mimicking a real qHTS data. Of the two methods currently in use, our simulations studies suggest that one is extremely conservative with very small power in comparison to the proposed PTE based method whereas the other method is very liberal. In contrast, the proposed PTE based methodology achieves a better control of FDR while maintaining good power. The proposed methodology is illustrated using a data set obtained from the National Toxicology Program (NTP). Additional information, simulation results, data and computer code are available online as supplementary materials.

Project description:Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD's quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD's ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.

Dataset Information

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Publications

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets