Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates

Abstract

This work focuses on the development of specific substrates for estrogen sulfotransferase (SULT1E1) to produce molecular imaging probes for this enzyme. SULT1E1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after beta-naphthol (betaN), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULT1E1 over SULT1A1*1, SULT1A1*2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity--V(max)/K(m) ratios-and specificity for SULT1E1. K(m) values ranged from 0.12-2.36 microM. A strong correlation was observed between polarity of the 4'-sustituent on the 2-aryl moiety (Hammett sigma(p)) and the log(V(max)/K(m)) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its (1)H NMR chemical shift (delta(OH)) with the log(V(max)/K(m)) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4'-substituent outlined by the correlation of the C-2 (13)C NMR chemical shift (delta(C2)) with the log(V(max)/K(m)) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 ((11)C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled (11)C-(2b)-6-O-sulfate and the SULT1E1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans.

Fig. 1.

(A) Interplay of estrogen sulfation and estrogen receptor (ER). Tissue levels of E₂ are regulated by SULT1E1 and STS, thus, modulating E₂ interaction with the estrogen receptor (ER). (B): Active site of human SULT1E1 with E₂ (Green) bound in the active site. Amino acid residues that form the binding pocket are show in Beige (oxygen, Red; nitrogen, Blue; sulfur, Red; and free water molecures, Cyan) [Modified with permission from (25).© National Institute of Environmental Health Sciences, 2003].

Fig. 2.

(A) Linear correlation between log(V_max/K_m) and Hammett σ_p (r = 0.964) for 2-arylsubstituted-6-hydroxybenzothiazole derivatives. Compounds 2g (4^′ = NHCOCH₃) and 2k (SO₂-CH₃) are apparent outliers due to possible steric hindrance of the large 4^′-substituents affecting binding with SULT1E1. (B) Linear correlation between δ_C2 and the log(V_max/K_m) (r = 0.987). Compounds 2g and 2k are apparent outliers due to steric hindrance. (C) 6-Hydroxy proton chemical shifts (δ_OH) and the log(V_max/K_m) (r = 0.963). Compounds 2g and 2k are outliers due to steric hindrance. All numbers represent the average of triplicate values.

Scheme 1.

Fig. 3.

(Top) Representative microPET scans in rat brain (Top, Left) and whole-body mice (Top, Right) with ¹¹C-(2b) co-registered with microCT images. The animals were scanned in a dynamic mode for 25 min. (Bottom): Western blot data obtained from the same tissues. Lane 1: 80 μg frontal cortical area (Cortex 1); Lane 2: 80 μg superior cortical areas (Cortex 2); Lane 3: 80 μg subcortical areas; Lane 4: 80 μg cerebellum; Lane 5: 80 μg brain stem; Lane 6: 50 μg testis; Lane 7 100 μg testis; and Lane 8: 150 μg testis

Scheme 2.