The objective of this proposal is to obtain a deep and fundamental understanding of the molecular behavior of the human cytosolic sulfotransferases. This 13-member enzyme family regulates the receptor interactions of hundreds of small molecules by transferring the sulfuryl-group (-SO3) from a nucleotide donor (PAPS, 3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyl- or amine-moieties of small-molecule acceptors. Understanding the molecular interactions between SULTs and their substrates and allosteric modulators will substantially deepen our understanding of the roles of these enzymes in biology and provide a means of controlling SULT activity in-vivo.
Aim I. We have discovered that SULT1A1 uses positive synergy to enhance the catalytic efficiencies of select substrates 103-104-fold. This is the first example of positive synergy in the SULT field. The molecular basis of these stunning catalytic enhancements will be determined, and the substrate features that elicit positive synergy will be identified with the goal of understanding how SULT-substrate reactivity is controlled.
Aim II focuses on an important and virtually unexplored area in sulfur metabolism - the allosteric regulation of SULT function. The literature describes a small number of important drugs and nutrients (aspirin, Celebrex (r), Ponstel (r) and epigallocatechin gallate - which comprises ~ 12% of the mass of dry tea leaves) that regulate SULTs by binding at sites separate from those of substrates. Binding is tight, isozyme specific and physiologically relevant. Certain compounds inhibit while others change the specificity and activate turnover of the enzyme. We will determine the first allostere-bound SULT structures - the crystals needed to do this are in-hand. Seeing these ligand-bound allosteric pockets at atomic resolution will change our perceptions of SULT metabolism and provide novel opportunities to control SULT activity.
Aim III. Hundreds of FDA-approved drugs are inactivated by sulfation. Preventing this modification is expected to increase the concentration and half-lives of the active forms of these compounds in-vivo. Classical inhibition strategies are detrimental because they prevent essential SULT functions. Consequently, no means of achieving this end is described in the literature. Our recent insights into the molecular basis of SULT-substrate selectivity lay the foundations for a novel strategy to prevent sulfation without inhibiting SULTs or altering a compound's receptor-binding affinity. We will develop this strategy and demonstrate its therapeutic potential. Sidechains that prevent sulfation will be identified and inserted into two FDA-approved drugs whose bioactivities are potently suppressed by sulfation: apomorphine, used to treat late-stage Parkinson Disease, and ethinyl estradiol, the active estrogen in most oral contraceptives. The receptor affinities of these new compounds will be tested in mammalian cells and their metabolism will be evaluated using primary human hepatocytes.
The ligand/receptor interactions of hundreds of small molecules (metabolites, drugs and other xenobiotics) are regulated by the human cytosolic sulfotransferases. This project will explore and define the molecular interactions that determine SULT behavior with the goal of using the findings to control SULT activity in-vivo. These efforts will significantly deepen our understanding of SULT biology and lead to improved therapeutics.
|Wang, Ting; Cook, Ian; Leyh, Thomas S (2016) Design and Interpretation of Human Sulfotransferase 1A1 Assays. Drug Metab Dispos 44:481-4|
|Cook, Ian; Wang, Ting; Wang, Wei et al. (2016) Controlling Sulfuryl-Transfer Biology. Cell Chem Biol 23:579-86|
|Cook, Ian; Wang, Ting; Leyh, Thomas S (2015) Sulfotransferase 1A1 Substrate Selectivity: A Molecular Clamp Mechanism. Biochemistry 54:6114-22|
|Cook, Ian; Wang, Ting; Falany, Charles N et al. (2015) The allosteric binding sites of sulfotransferase 1A1. Drug Metab Dispos 43:418-23|
|Englander, Michael T; Avins, Joshua L; Fleisher, Rachel C et al. (2015) The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. Proc Natl Acad Sci U S A 112:6038-43|
|Kang, Soosung; Watanabe, Mizuki; Jacobs, J C et al. (2015) Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability. Eur J Med Chem 90:448-61|
|Rodriguez, Sofia B; Leyh, Thomas S (2014) An enzymatic platform for the synthesis of isoprenoid precursors. PLoS One 9:e105594|
|Wang, Ting; Cook, Ian; Leyh, Thomas S (2014) 3'-Phosphoadenosine 5'-phosphosulfate allosterically regulates sulfotransferase turnover. Biochemistry 53:6893-900|
|Wang, Ting; Cook, Ian; Falany, Charles N et al. (2014) Paradigms of sulfotransferase catalysis: the mechanism of SULT2A1. J Biol Chem 289:26474-80|