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Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

ABSTRACT: It is well established that chemical-peptide conjugation represents the molecular initiating event (MIE) in skin sensitization. This MIE has been successfully exploited in the development of in chemico peptide reactivity assays, with the Direct Peptide Reactivity Assay (DPRA) being validated as a screening tool for skin sensitization hazard as well as an OECD test guideline. This test relies on the use of a high-performance liquid chromatography/ultraviolet detection method to quantify chemical-peptide conjugation through measurement of the depletion of two synthetic peptides containing lysine or cysteine residues, which is labor-intensive and time-consuming. To improve assay throughput, sensitivity, and accuracy, we have developed a spectrophotometric assay for skin sensitization potential based on MIE measurement-the ProtReact assay. ProtReact is also a cheaper, faster, simpler, and more accessible alternative for the DPRA, giving comparable results. A set of 106 chemicals was tested with ProtReact and the peptide depletion values compared with those reported for the DPRA. The predictive capacity of both assays was evaluated with human reference data. ProtReact and DPRA assays show similar predictive capacities for hazard identification (75% and 74%, respectively), although ProtReact showed a higher specificity (86% versus 74%, respectively) and lower sensitivity (69% versus 73%). Overall, the results show that ProtReact assay described here represents an efficient, economic, and accurate assay for the prediction of skin sensitization potential of chemical haptens.

SUBMITTER: Ferreira I

PROVIDER: S-EPMC10404171 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

Ferreira Isabel I Brites Gonçalo G Silva Ana A Caramelo Francisco F Oliveiros Bárbara B Neves Bruno Miguel BM Cruz Maria Teresa MT

Archives of toxicology 20230719 9

It is well established that chemical-peptide conjugation represents the molecular initiating event (MIE) in skin sensitization. This MIE has been successfully exploited in the development of in chemico peptide reactivity assays, with the Direct Peptide Reactivity Assay (DPRA) being validated as a screening tool for skin sensitization hazard as well as an OECD test guideline. This test relies on the use of a high-performance liquid chromatography/ultraviolet detection method to quantify chemical- ...[more]

PMID: 37466788

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Project description:BackgroundImportant clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus.ResultsWe have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct). Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct). Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP) fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products.ConclusionOur reporter-based system can rapidly screen gene products for their ability to be translocated to the nucleus. Large-scale applications of the system presented herein should provide invaluable information for a more complete biological atlas.

Dataset Information

Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

Publications

Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

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