Allosteres to regulate neurotransmitter sulfonation

Abstract

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 μm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.

Keywords: SULT1A3; allosteric regulation; allostery; catecholamine; dopamine; enzyme inhibitor; enzyme kinetics; enzyme mechanism; enzyme structure; epinephrine; inhibition; mechanism; neurotransmitter; norepinephrine; nuclear magnetic resonance (NMR); serotonin; spin label; sulfotransferase.

Figure 1.

Inhibition of SULT isoforms by CMP8. The initial rates of SULT catalyzed 1-HP sulfonation are plotted as a function of inhibitor concentration and are normalized relative to [inhibitor] = 0. Enzyme activity was monitored by the sulfonation dependent change in 1-HP fluorescence (λ_ex = 325 nm, λ_em = 370 nm). Less than 5% of the concentration-limiting substrate converted at the reaction end point was consumed during initial rate measurements. Each point is the average of three independent determinations. Conditions were as follows: SULT (20 nm, active sites), PAPS (0.50 mm, 17 × K_m), 1-HP (5.0 μm, 61 × K_m), KPO₄ (50 mm), pH 7.5, 25 ± 2 °C.

Figure 2.

The NMR measurements. A, structure and 600-MHz ¹H NMR spectrum of CMP8. The protons of CMP8 are labeled in the spectrum and structure. Red labels identify the proton positions used in NMR distance measurements. Conditions were as follows: CMP8 (1.9 mm), DMSO (0.50 mm), TMS (0.50 mm), D₂O (>95%), 25 ± 1 °C. B, spin label effects on the H5 proton peak of CMP8. The solution ¹H NMR spectrum (600 MHz) of the H5 peak of CMP8 is shown as a function of percentage of CMP8 bound to spin-labeled Cys-234-SULT1A3. Peak amplitudes were normalized to reflect 1.0 mm CMP8. Conditions were as follows: CMP8 (1.0 mm (purple), 400 μm (green), 200 μm (blue), 100 μm (red)), spin-labeled Cys-234-SULT1A3 (20 μm, active site), PAP (500 μm, 17 × K_d), KPO₄ (50 mm), pD 7.4, 25 ± 1 °C. Conditions associated with the black and purple peaks were identical except that the black spectrum conditions lacked enzyme. CMP8 is saturating at all concentrations (i.e. ≥2300 K_d). C, line width versus fraction of inhibitor bound. The effects of paramagnetic spin labels (4-maleimido-PROXYL attached at Cys-234 (red), Cys-198 (black), or Cys-116 (blue)) and diamagnetic control (N-cyclohexylmaleimide attached at Cys-234 (green)) on the line width of the H5 proton peak are plotted as a function of fraction of enzyme-bound CMP8. Fractions of CMP8 bound were 0.02, 0.05, 0.10, and 0.20. Conditions were as described in B. Diamagnetic controls for Cys-198 and Cys-116 were indistinguishable from the Cys-234.

Figure 3.

Structure of the CMP8·SULT1A3·PAPS·dopamine complex. A, structure at a distance. CMP8 is labeled and shown in white. Carbon atoms of residues in direct contact with CMP8 are blue. The cap (orange) is shown in the closed conformation and sitting above the substrates, PAPS and dopamine (Dop). B, CMP8-binding pocket. CMP8 is shown interacting with six direct-contact residues, shown in blue. The refined structure (shown) was generated by molecular dynamics energy minimization of the NMR distance–constrained structure (see “Results and discussion”).

Figure 4.

Mechanism of inhibition. The effect of CMP8 on PAP binding to SULT1A3 was monitored using a stopped-flow spectrofluorimeter (λ_ex = 290 nm, λ_em ≥ 330 nm (cutoff filter)). Reactions were initiated by rapidly mixing (1:1, v/v) a solution containing SULT1A3 (25 nm, dimer), CMP8 (0 (blue) or 1.7 μm, 50 × K_d (red)), KPO₄ (50 mm), pH 7.5, 25 ± 2 °C, with a solution that was identical except that it lacked SULT1A3 and contained PAP at twice the indicated concentrations. k_obs values were obtained by fitting the average of 6–9 progress curves to a single exponential. Each k_obs value was determined in triplicate, and the averaged values are shown. k_on and k_off are given, respectively, by the slopes and intercepts obtained from linear least-squares fitting of the k_obs versus [PAP] plots.

Figure 5.

Free energy correlation plot. ΔΔG values were either calculated ab initio, using molecular dynamics simulations, or obtained from experimentally determined k_cat values (see “Results and discussion”). The data show strong linear correlation, slope = 1.2 ± 0.1. Numbers associated with data points correspond to the compound numbering in Table 3. Error bars, ± 1 standard unit. Error in the calculated (y axis) values is vanishingly small.