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.
Highlights
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
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 sulfotransferase 1A3 (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
We present the structure of that allostere bound to SULT1A3 and demonstrate that the allostere slows turnover by stabilizing the enzyme’s active-site cap, a conserved stretch of ϳ30 residues that is intimately involved in SULT substrate selection and turnover
Summary
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. 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. We present the structure of that allostere bound to SULT1A3 and demonstrate that the allostere slows turnover by stabilizing the enzyme’s active-site cap, a conserved stretch of ϳ30 residues that is intimately involved in SULT substrate selection and turnover. These are the first man-made, isoform-specific allosteric SULT inhibitors; they bound tightly, and their inhibition characteristics correlated remarkably well with the predicted values. The behavior of this small, catalytically important active-site cap is predictable and can be reliably incorporated into SULT1A3 inhibitor designs
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