Controlling Sulfuryl-Transfer Biology

Abstract

In humans, the cytosolic sulfotransferases (SULTs) catalyze regiospecific transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate to thousands of metabolites, including numerous signaling small molecules, and thus regulates their activities and half-lives. Imbalances in the in vivo set points of these reactions leads to disease. Here, with the goal of controlling sulfonation in vivo, molecular ligand-recognition principles in the SULT and nuclear receptor families are integrated in creating a strategy that can prevent sulfonation of a compound without significantly altering its receptor affinity, or inhibiting SULTS. The strategy is validated by using it to control the sulfonation and estrogen receptor (ER) activating activity of raloxifene (a US Food and Drug Administration-approved selective estrogen receptor modulator) and its derivatives. Preventing sulfonation is shown to enhance ER-activation efficacy 10(4)-fold in studies using Ishikawa cells. The strategy offers the opportunity to control sulfuryl transfer on a compound-by-compound basis, to enhance the efficacy of sulfonated drugs, and to explore the biology of sulfuryl transfer with unprecedented precision.

Keywords: derivatives; estrogen receptor; inhibition; mechanism; raloxifene; selectivity; structure; sulfation; sulfonation; sulfotransferase; synthesis.

Figure 1. Cap Closure Sterically Restricts Active Site Access

(A.) DHEA binds in a reactive conformation in the open and closed conformations of SUL2A1. (B.) The Raloxifene (RAL) R-group (see, Fig 2A) prevents access to the active site when the cap is closed. The active site cap of SULT2A1 is shown in the open (orange) and closed (blue) positions. With and without nucleotide bound, the enzyme spends 95 and ≤ 5% of its time in the closed conformation, respectively. Nucleotide is believed to be saturating in vivo.

Figure 2. Raloxifene Bound to the Estrogen Receptor

(A.) The Structure of Raloxifene. Raloxifene is composed of a steroid-like base and a large R-group inserted at C3 of the benzothiophene ring. The R-group prevents binding to the closed form of SULT2A1 (see, Fig 1B). (B.) Raloxifene Bound the Human Estrogen Receptor α.

Figure 3. The Binding of Benzoyl-Ral to the E and E·PAP forms of SULT2A1

Binding was monitored via intrinsic fluorescence of the enzyme (λ_ex = 290 nm, λ_em = 340 nm). Data are plotted as a ratio of fluorescence intensity at a given ligand concentration to that in the absence of ligand (I/I₀). Solution composition: SULT2A1 (0.50 µM), PAP [0 µM (Red), 125 µM (Black)], MgCl₂ (5.0 mM), KPO₄ (50 mM), pH 7.4, 25 ± 2 °C. Titrations were performed in triplicate and averaged. Lines through the data represent the behavior predicted by a best-fit, single-site binding model.

Figure 4. ER activation in Ishikawa Cells

(A.) ER Activation in (−) SULT Control Cells. ER activation was monitored via alkaline phosphatase levels. EC₅₀ values are similar (~ 0.2 nM) for each of the five derivatives indicated (see, Table 2). The Ishikawa cells were stably transfected with vector that did not contain a SULT coding region. SULT activity toward DHEA is not detected in extracts of these cells (Materials and Methods). (B.) ER Activation in (+) SULT2A1 Cells. The Ishikawa cells were stably transfected with vector containing the SULT2A1 coding region. SULT activity in extracts of these cells was comparable to that seen in human liver extracts (Materials and Methods). The EC₅₀ values of cells too large to pass through the acceptor pore of SULT2A1 are not affected by 2A1 expression, while those of the compounds that can pass through the pore increase ~ 10⁴-fold.

Fig 5. Sulfonation of Raloxifene and an Acetyl-derivative of the Raloxifene Base by SULT2A1 (+) and (−) Ishikawa Cells

Cells were grown to ~70% confluency in MEM + 10% FBS media, washed, and media containing 25 µM radiolabeled acceptor ([³H]-RAL or [¹⁴C]-acetyl-derivative, SA 0.65 and 0.13 Ci/mmol, respectively) was added. Formation of sulfonated acceptor in the medium was monitored via scintillation counting after chloroform extraction. Measurements were performed in duplicate and averaged. Acetyl-derivative data were fit using a single exponential. The acetyl derivative is rapidly sulfonated in the SULT2A1 (+) cell line and slowly in (−) line. Raloxifene is not detectibly sulfonated by either cell line.