Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes

Risto O. Juvonen; Pekka A. Postila; Pankaj Kumar Singh; Juhani Huuskonen; Juri Timonen; Muluneh Fashe; Rasikh Hussain; Zaeema Aqip; Olli Kärkkäinen; Hannu Raunio; Olli T. Pentikäinen

doi:10.1016/j.ejps.2025.107249

Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes

Risto O. Juvonen^*
, Pekka A. Postila
, Pankaj Kumar Singh
, Juhani Huuskonen
, Juri Timonen
, Muluneh Fashe
, Rasikh Hussain
, Zaeema Aqip
, Olli Kärkkäinen
, Hannu Raunio
, Olli T. Pentikäinen

^*Corresponding author for this work

Research output: Contribution to journal › Article › Scientific › peer-review

5 Downloads (Pure)

Abstract

Sulfonation is one of drug metabolism reactions affecting homeostasis of estrogens. C-3 aryl substituted 7-hydroxycoumarins are fluorescent estrogen mimetics; i.e., the hydroxyl groups of both estrogens and 7-hydroxycoumarins are conjugated by human sulfotransferases (SULTs). Sulfonation of the 7‑hydroxyl group by SULTs decreases the fluorescence of 7-hydroxycoumarins. Sulfonation of a series of 7-hydroxycoumarins by human SULTs was determined based on this property. SULT subtype-specific binding interactions of 7-hydrocoumarins were assessed against the modelled optimal arrangement needed for sulfonation. 3-(4-Methoxyphenyl)-7-hydroxycoumarin (11) and 3-(4-hydroxyphenyl)-7-hydroxycoumarin (9) were selective substrates for SULT1E1, whereas 3-(1H-1,2,4-triazol-1-yl)-7-hydroxycoumarin (14) was a selective SULT1A1 substrate. Other tested 7-hydroxycoumarin were sulfonated by more than two SULTs. Sulfonation of most 7-hydroxycoumarins by SULT1A1 or SULT1C4 followed Michaelis-Menten kinetics, while substrate inhibition kinetics occurred in sulfonation of several derivatives by SULT1E1. Selective sulfonation of derivatives 9 and 11 by SULT1E1 was due to the enzyme's long and cylindrical active site that assures optimal 7‑hydroxyl group placement in the precursory reaction state. SULT1A1 and SULT1C4 preferred smaller derivatives as substrates than the SULT1E. Estrogens potently inhibited the sulfonation of 3,4-dimethyl-7-hydroxycoumarin (4) by SULT1E1 (IC₅₀ below 1 µM). SULT1A1 and SULT1C4 were less potently inhibited by the estrogens. Several 7-hydroxycoumarin derivatives share common binding interaction patterns with the estrogens at SULT1E1 and SULT1A1 active sites. Fluorescent 7-hydroxycoumarins could serve as convenient probe substrates for SULTs to evaluate their inhibition by new chemical entities during drug development. 7-Hydroxycoumarins 9 or 11 could be used as selective probe substrates for SULT1E1 and 14 for SULT1A1.

Original language	English
Article number	107249
Journal	European Journal of Pharmaceutical Sciences
Volume	213
DOIs	https://doi.org/10.1016/j.ejps.2025.107249
Publication status	Published - 1 Oct 2025
MoE publication type	A1 Journal article-refereed

Funding

The authors wish to acknowledge Ms. Hannele Jaatinen for technical help, Biocenter Finland/DDCB for financial support. The Finnish IT Center for Science (CSC) is acknowledged for generous computational resources (O.T.P.: Project Nos. jyy2516 and jyy2585; P.A.P.: Project No. tty3975), and the Turku Screening Unit of the Drug Discovery and Chemical Biology platform of Biocentre Finland is acknowledged for support. Novo Nordisk Foundation (O.T.P.; Pioneer Innovator Grant, grant number 0068,926; Distinguished Innovator Grant; grant number 0075,825) funded the research. This research was also supported by the Research Council of Finland's Flagship InFLAMES (P.A.P.). The funding decision numbers are 337,530 and 357,910. This work was supported by the National Institutes of Health (grant number: Z01ES1005–01) (MF). Novo Nordisk Foundation (O.T.P.; Pioneer Innovator Grant, grant number 0068,926; Distinguished Innovator Grant; grant number 0075,825) funded the research. This research was also supported by the Research Council of Finland’s Flagship InFLAMES (P.A.P.). The funding decision numbers are 337,530 and 357,910. This work was supported by the National Institutes of Health (grant number: Z01ES1005–01 ) (MF).

Keywords

7-hydroxycoumarin
Active site
Estrogen
Human
Structure-activity relationship (SAR)
Sulfotransferase (SULT)

Access to Document

10.1016/j.ejps.2025.107249Licence: CC BY

1-s2.0-S0928098725002477-mainFinal published version, 7.55 MBLicence: CC BY

https://urn.fi/URN:NBN:fi-fe2026021313227

Cite this

Juvonen, R. O., Postila, P. A., Singh, P. K., Huuskonen, J., Timonen, J., Fashe, M., Hussain, R., Aqip, Z., Kärkkäinen, O., Raunio, H., & Pentikäinen, O. T. (2025). Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes. European Journal of Pharmaceutical Sciences, 213, Article 107249. https://doi.org/10.1016/j.ejps.2025.107249

@article{92eff70e76fa4258ab30590fd149fee8,

title = "Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes",

abstract = "Sulfonation is one of drug metabolism reactions affecting homeostasis of estrogens. C-3 aryl substituted 7-hydroxycoumarins are fluorescent estrogen mimetics; i.e., the hydroxyl groups of both estrogens and 7-hydroxycoumarins are conjugated by human sulfotransferases (SULTs). Sulfonation of the 7‑hydroxyl group by SULTs decreases the fluorescence of 7-hydroxycoumarins. Sulfonation of a series of 7-hydroxycoumarins by human SULTs was determined based on this property. SULT subtype-specific binding interactions of 7-hydrocoumarins were assessed against the modelled optimal arrangement needed for sulfonation. 3-(4-Methoxyphenyl)-7-hydroxycoumarin (11) and 3-(4-hydroxyphenyl)-7-hydroxycoumarin (9) were selective substrates for SULT1E1, whereas 3-(1H-1,2,4-triazol-1-yl)-7-hydroxycoumarin (14) was a selective SULT1A1 substrate. Other tested 7-hydroxycoumarin were sulfonated by more than two SULTs. Sulfonation of most 7-hydroxycoumarins by SULT1A1 or SULT1C4 followed Michaelis-Menten kinetics, while substrate inhibition kinetics occurred in sulfonation of several derivatives by SULT1E1. Selective sulfonation of derivatives 9 and 11 by SULT1E1 was due to the enzyme's long and cylindrical active site that assures optimal 7‑hydroxyl group placement in the precursory reaction state. SULT1A1 and SULT1C4 preferred smaller derivatives as substrates than the SULT1E. Estrogens potently inhibited the sulfonation of 3,4-dimethyl-7-hydroxycoumarin (4) by SULT1E1 (IC50 below 1 µM). SULT1A1 and SULT1C4 were less potently inhibited by the estrogens. Several 7-hydroxycoumarin derivatives share common binding interaction patterns with the estrogens at SULT1E1 and SULT1A1 active sites. Fluorescent 7-hydroxycoumarins could serve as convenient probe substrates for SULTs to evaluate their inhibition by new chemical entities during drug development. 7-Hydroxycoumarins 9 or 11 could be used as selective probe substrates for SULT1E1 and 14 for SULT1A1.",

keywords = "7-hydroxycoumarin, Active site, Estrogen, Human, Structure-activity relationship (SAR), Sulfotransferase (SULT)",

author = "Juvonen, \{Risto O.\} and Postila, \{Pekka A.\} and Singh, \{Pankaj Kumar\} and Juhani Huuskonen and Juri Timonen and Muluneh Fashe and Rasikh Hussain and Zaeema Aqip and Olli K{\"a}rkk{\"a}inen and Hannu Raunio and Pentik{\"a}inen, \{Olli T.\}",

note = "Publisher Copyright: {\textcopyright} 2025",

year = "2025",

month = oct,

day = "1",

doi = "10.1016/j.ejps.2025.107249",

language = "English",

volume = "213",

journal = "European Journal of Pharmaceutical Sciences",

issn = "0928-0987",

publisher = "Elsevier",

}

TY - JOUR

T1 - Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes

AU - Juvonen, Risto O.

AU - Postila, Pekka A.

AU - Singh, Pankaj Kumar

AU - Huuskonen, Juhani

AU - Timonen, Juri

AU - Fashe, Muluneh

AU - Hussain, Rasikh

AU - Aqip, Zaeema

AU - Kärkkäinen, Olli

AU - Raunio, Hannu

AU - Pentikäinen, Olli T.

PY - 2025/10/1

Y1 - 2025/10/1

N2 - Sulfonation is one of drug metabolism reactions affecting homeostasis of estrogens. C-3 aryl substituted 7-hydroxycoumarins are fluorescent estrogen mimetics; i.e., the hydroxyl groups of both estrogens and 7-hydroxycoumarins are conjugated by human sulfotransferases (SULTs). Sulfonation of the 7‑hydroxyl group by SULTs decreases the fluorescence of 7-hydroxycoumarins. Sulfonation of a series of 7-hydroxycoumarins by human SULTs was determined based on this property. SULT subtype-specific binding interactions of 7-hydrocoumarins were assessed against the modelled optimal arrangement needed for sulfonation. 3-(4-Methoxyphenyl)-7-hydroxycoumarin (11) and 3-(4-hydroxyphenyl)-7-hydroxycoumarin (9) were selective substrates for SULT1E1, whereas 3-(1H-1,2,4-triazol-1-yl)-7-hydroxycoumarin (14) was a selective SULT1A1 substrate. Other tested 7-hydroxycoumarin were sulfonated by more than two SULTs. Sulfonation of most 7-hydroxycoumarins by SULT1A1 or SULT1C4 followed Michaelis-Menten kinetics, while substrate inhibition kinetics occurred in sulfonation of several derivatives by SULT1E1. Selective sulfonation of derivatives 9 and 11 by SULT1E1 was due to the enzyme's long and cylindrical active site that assures optimal 7‑hydroxyl group placement in the precursory reaction state. SULT1A1 and SULT1C4 preferred smaller derivatives as substrates than the SULT1E. Estrogens potently inhibited the sulfonation of 3,4-dimethyl-7-hydroxycoumarin (4) by SULT1E1 (IC50 below 1 µM). SULT1A1 and SULT1C4 were less potently inhibited by the estrogens. Several 7-hydroxycoumarin derivatives share common binding interaction patterns with the estrogens at SULT1E1 and SULT1A1 active sites. Fluorescent 7-hydroxycoumarins could serve as convenient probe substrates for SULTs to evaluate their inhibition by new chemical entities during drug development. 7-Hydroxycoumarins 9 or 11 could be used as selective probe substrates for SULT1E1 and 14 for SULT1A1.

AB - Sulfonation is one of drug metabolism reactions affecting homeostasis of estrogens. C-3 aryl substituted 7-hydroxycoumarins are fluorescent estrogen mimetics; i.e., the hydroxyl groups of both estrogens and 7-hydroxycoumarins are conjugated by human sulfotransferases (SULTs). Sulfonation of the 7‑hydroxyl group by SULTs decreases the fluorescence of 7-hydroxycoumarins. Sulfonation of a series of 7-hydroxycoumarins by human SULTs was determined based on this property. SULT subtype-specific binding interactions of 7-hydrocoumarins were assessed against the modelled optimal arrangement needed for sulfonation. 3-(4-Methoxyphenyl)-7-hydroxycoumarin (11) and 3-(4-hydroxyphenyl)-7-hydroxycoumarin (9) were selective substrates for SULT1E1, whereas 3-(1H-1,2,4-triazol-1-yl)-7-hydroxycoumarin (14) was a selective SULT1A1 substrate. Other tested 7-hydroxycoumarin were sulfonated by more than two SULTs. Sulfonation of most 7-hydroxycoumarins by SULT1A1 or SULT1C4 followed Michaelis-Menten kinetics, while substrate inhibition kinetics occurred in sulfonation of several derivatives by SULT1E1. Selective sulfonation of derivatives 9 and 11 by SULT1E1 was due to the enzyme's long and cylindrical active site that assures optimal 7‑hydroxyl group placement in the precursory reaction state. SULT1A1 and SULT1C4 preferred smaller derivatives as substrates than the SULT1E. Estrogens potently inhibited the sulfonation of 3,4-dimethyl-7-hydroxycoumarin (4) by SULT1E1 (IC50 below 1 µM). SULT1A1 and SULT1C4 were less potently inhibited by the estrogens. Several 7-hydroxycoumarin derivatives share common binding interaction patterns with the estrogens at SULT1E1 and SULT1A1 active sites. Fluorescent 7-hydroxycoumarins could serve as convenient probe substrates for SULTs to evaluate their inhibition by new chemical entities during drug development. 7-Hydroxycoumarins 9 or 11 could be used as selective probe substrates for SULT1E1 and 14 for SULT1A1.

KW - 7-hydroxycoumarin

KW - Active site

KW - Estrogen

KW - Human

KW - Structure-activity relationship (SAR)

KW - Sulfotransferase (SULT)

UR - https://www.scopus.com/pages/publications/105014497146

U2 - 10.1016/j.ejps.2025.107249

DO - 10.1016/j.ejps.2025.107249

M3 - Article

C2 - 40885279

AN - SCOPUS:105014497146

SN - 0928-0987

VL - 213

JO - European Journal of Pharmaceutical Sciences

JF - European Journal of Pharmaceutical Sciences

M1 - 107249

ER -

Designing fluorescent estrogen mimetic 7-hydroxycoumarin probe substrates for human sulfotransferase enzymes

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this