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In silico Identification of Thyroid Disrupting Chemicals: among industrial chemicals and household dust contaminants
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Thyroid disruptions by xenobiotics have been associated with a broad spectrum of severe adverse human health effects, such as impaired brain development and metabolic syndrome. Ingestion of indoor dust and contact with industrial chemicals are two significant human exposure routes of thyroid hormone disrupting chemicals (THDCs), raising serious concerns for human health. However, it is a laborious and costly process to identify THDCs using conventional experimental methods, due to the number of chemicals in commerce and the varieties of potential disruption mechanisms.

In this thesis, we are aimed at in silico identification of novel THDCs targeting transthyretin (TTR) and thyroid hormone receptor (THR) among dust contaminants and commonly used industrial chemicals. In vitro assays were used to validate the in silico prediction results. Co-crystallization and molecular dynamics (MD) simulations were applied to reveal binding modes of THDCs at the studied biological targets and to explain their intermolecular recognition.

The main findings presented in this thesis are:

1. Over 144 environmental pollutants have been confirmed as TTR-binders in vitro and these cover a wide range of environmental pollutants and show distinct chemical profiles including a large group of halogenated aromatic compounds and a second group of per- and polyfluoroalkyl substances. (Paper I)

2. In total 485 organic contaminants have been reported to be detected in household dust. The developed QSAR classification model predicted 7.6% of these dust contaminants and 53.1% of their metabolites as potential TTR-binders, which emphasizes the importance of metabolic bioactivation. After in vitro validation, four novel TTR binders with IC50 ≤ 10 µM were identified, i.e. perfluoroheptanesulfonic acid, 2,4,2',4'-tetrahydroxybenzophenone (BP2), 2,4,5-trichlorophenoxyacetic acid, and 3,5,6-trichloro-2-pyridinol. (Paper II)

3. The development of a robust structure-based virtual screening (VS) protocol resulted in the prediction of 31 dust contaminants as potential binders to THRβ1 including musk compounds, PFASs, and bisphenol A derivatives. The in vitro experiments confirmed four compounds as weak binders to THRβ1, i.e. 2,4,5-trichlorophenoxyacetic acid, bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether, 2,4,2',4'-tetrahydroxybenzophenone, and 2,4-dichlorophenoxyacetic acid. (Paper III)

4. We revealed the binding conformations of perfluorooctanesulfonic acid, perfluorooctanoic acid, and BP2 in the thyroxine binding sites (TBSs) of TTR by co-crystallizing TTR with the three compounds. A VS protocol was developed based on the TTR complex structures that predicted 192 industrial chemicals as potential binders to TTR. Seven novel TTR binders were confirmed by in vitro experiments including clonixin, 2,6-dinitro-p-cresol (DNPC), triclopyr, fluroxypyr, bisphenol S, picloram, and mesotrione. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr, and their complex structures showed that the compounds bind in the TBSs as proposed by the VS protocol.

In summary, 13 indoor dust contaminants and industrial chemicals were identified as THDCs using a combination of in silico and in vitro approaches. To the best of our knowledge, none of these compounds has previously been reported to bind to TTR or THR. The identifications of these THDCs improve our understanding on the structure-activity relationships of THDCs. The crystal structures of TTR-THDC complexes and the information on THDC-Target intermolecular interactions provide a better understanding on the mechanism-of-actions behind thyroid disruption. The dataset compiled and in silico methods developed serve as a basis for identification of more diverse THDCs in the future and a tool for guiding de novo design of safer replacements.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 54 p.
Keyword [en]
Thyroid disruption chemicals, virtual screening, tranthyretin, thyroid hormone receptor, QSAR modeling, molecular docking, molecular dynamics
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-125631ISBN: 978-91-7601-551-3OAI: oai:DiVA.org:umu-125631DiVA: diva2:968922
Public defence
2016-10-07, KB3B1, KBC-huset, Umeå, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council Formas, 210-2012-131Swedish Research Council, 521-2011-6427
Available from: 2016-09-16 Created: 2016-09-13 Last updated: 2016-09-15Bibliographically approved
List of papers
1. Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment
Open this publication in new window or tab >>Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment
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2015 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 19, 5625-5634 p.Article in journal (Refereed) Published
Abstract [en]

A variety of anthropogenic compounds has been found to be capable of disrupting the endocrine systems of organisms, in laboratory studies as well as in wildlife. The most widely described endpoint is estrogenicity, but other hormonal disturbances, e.g., thyroid hormone disruption, are gaining more and more attention. Here, we present a review and chemical characterization, using principal component analysis, of organic compounds that have been tested for their capacity to bind competitively to the thyroid hormone transport protein transthyretin (TTR). The database contains 250 individual compounds and technical mixtures, of which 144 compounds are defined as TTR binders. Almost one third of these compounds (n = 52) were even more potent than the natural hormone thyroxine (T-4). The database was used as a tool to assist in the identification of thyroid hormone-disrupting compounds (THDCs) in an effect-directed analysis (EDA) study of a sediment sample. Two compounds could be confirmed to contribute to the detected TTR-binding potency in the sediment sample, i.e., triclosan and nonylphenol technical mixture. They constituted less than 1 % of the TTR-binding potency of the unfractionated extract. The low rate of explained activity may be attributed to the challenges related to identification of unknown contaminants in combination with the limited knowledge about THDCs in general. This study demonstrates the need for databases containing compound-specific toxicological properties. In the framework of EDA, such a database could be used to assist in the identification and confirmation of causative compounds focusing on thyroid hormone disruption.

Keyword
Thyroid hormone-disrupting compound (THDC), Transthyretin (TTR), Database, Structure-activity lationship (SAR), Effect-directed analysis (EDA), Sediment
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-107176 (URN)10.1007/s00216-015-8736-9 (DOI)000358136900008 ()25986900 (PubMedID)
Available from: 2015-09-02 Created: 2015-08-19 Last updated: 2016-09-13Bibliographically approved
2. In Silico Approach To Identify Potential Thyroid Hormone Disruptors among Currently Known Dust Contaminants and Their Metabolites
Open this publication in new window or tab >>In Silico Approach To Identify Potential Thyroid Hormone Disruptors among Currently Known Dust Contaminants and Their Metabolites
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2015 (English)In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 49, no 16, 10099-10107 p.Article in journal (Refereed) Published
Abstract [en]

Thyroid hormone disrupting chemicals (THDCs) interfere with the thyroid hormone system and may induce multiple severe physiological disorders. Indoor dust ingestion is a major route of THDCs exposure in humans, and one of the molecular targets of these chemicals is the hormone transporter transthyretin (TTR). To virtually screen indoor dust contaminants and their metabolites for THDCs targeting TTR, we developed a quantitative structure activity relationship (QSAR) classification model. The QSAR model was applied to an in-house database including 485 organic dust contaminants reported from literature data and their 433 in silico derived metabolites. The model predicted 37 (7.6%) dust contaminants and 230 (53.1%) metabolites as potential TTR binders. Four new THDCs were identified after testing 23 selected parent dust contaminants in a radio-ligand TTR binding assay; 2,2',4,4'-tetrahydroxybenzophenone, perfluoroheptanesulfonic acid, 3,5,6-trichloro-2-pyridinol, and 2,4,5-trichlorophenoxyacetic acid. These chemicals competitively bind to TTR with 50% inhibition (IC50) values at or below 10 mu M. Molecular docking studies suggested that these THDCs interacted similarly with TTR via the residue Ser117A, but their binding poses were dissimilar to the endogenous ligand T4. This study identified new THDCs using an in silico approach in combination with bioassay testing and highlighted the importance of metabolic activation for TTR binding.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-108461 (URN)10.1021/acs.est.5b01742 (DOI)000359891700082 ()26207645 (PubMedID)
Funder
Swedish Research Council, 521-2011-6427
Available from: 2015-09-22 Created: 2015-09-11 Last updated: 2016-09-13Bibliographically approved
3. Identification and Molecular Interaction Studies of Thyroid Hormone Receptor Disruptors among Household Dust Contaminants
Open this publication in new window or tab >>Identification and Molecular Interaction Studies of Thyroid Hormone Receptor Disruptors among Household Dust Contaminants
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2016 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 29, no 8, 1345-1354 p.Article in journal (Refereed) Published
Abstract [en]

Thyroid hormone disrupting chemicals (THDCs), often found abundantly in the environment, interfere with normal thyroid hormone signaling and induce physiological malfunctions, possibly by affecting thyroid hormone receptors (THRs). Indoor dust ingestion is a significant human exposure route of THDCs, raising serious concerns for human health. Here, we developed a virtual screening protocol based on an ensemble of X-ray crystallographic structures of human THRβ1 and the generalized Born solvation model to identify potential THDCs targeting the human THRβ1 isoform. The protocol was applied to virtually screen an in-house indoor dust contaminant inventory, yielding 31 dust contaminants as potential THRβ1 binders. Five predicted binders and one negative control were tested using isothermal titration calorimetry, of which four, i.e., 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether (BADGE-HCl-H2O), 2,2',4,4'-tetrahydroxybenzophenone (BP2), and 2,4-dichlorophenoxyacetic acid (2,4-D), were identified as THRβ1 binders with binding affinities ranging between 60 μM and 460 μM. Molecular dynamics (MD) simulations were employed to examine potential binding modes of these binders and provided a rationale for explaining their specific recognition by THRβ1. The combination of in vitro binding affinity measurements and MD simulations allowed identification of four new potential THR-targeting THDCs that have been found in household dust. We suggest using the developed structure-based virtual screening protocol to identify and prioritize testing of potential THDCs.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-125629 (URN)10.1021/acs.chemrestox.6b00171 (DOI)27410513 (PubMedID)2-s2.0-84982300220 (ScopusID)
External cooperation:
Available from: 2016-09-13 Created: 2016-09-13 Last updated: 2016-09-14Bibliographically approved
4. Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin
Open this publication in new window or tab >>Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-125630 (URN)
External cooperation:
Available from: 2016-09-13 Created: 2016-09-13 Last updated: 2016-09-14

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