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468.23. (4-((4-((3-(4-Fluorobenzyl)-2-oxo-2,3-dihydrothiazol-5-yl)methyl)phenoxy) methyl)phenyl)boronic Acidity (23) To a solution of compound 1 (45.7 mg, 0.0989 mmol) in dimethyl sulfoxide (0.75 mL), sodium borohydride (29.1 mg, 0.769 mmol) was slowly added. responsible for the hydrolysis of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, as depicted in Plan 1.1,2 The bioactive lipid LPA stimulates migration, proliferation and survival of cells by activating specific G protein-coupled receptors.(3) The ATX-LPA signaling axis is usually involved in malignancy, swelling and fibrotic disease.4?6 Potent and Nr2f1 selective ATX inhibitors are needed to elucidate the contribution of ATX action to signaling cascades that may result in disease in case of malfunction. Open in a separate window Plan 1 Autotaxin (ATX) is Responsible for Hydrolyzing the Lipid Lysophosphatidylcholine (LPC) into Lysophosphatidic Acid (LPA) and Choline ATX, also known as eNPP2, is a unique member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (eNPP) family of proteins. It is the only family member capable of generating LPA by hydrolysis of LPC.(7) Recently reported crystal structures of mouse(8) and rat(9) ATX confirmed that a threonine residue and two zinc ions are necessary for activity of ATX.(10) From these structures, it could be concluded that ATX hydrolyzes its substrates through a typical alkaline phosphatase/phosphodiesterase mechanism.11,12 Furthermore, these constructions showed that ATX specifically binds its lipid substrates inside a hydrophobic pocket extending from your active site of ATX. This pocket accommodates the alkyl chain of the lipids in different poses as was also demonstrated in various crystal constructions.(8) Recently, we described the discovery of a boronic acid-based ATX Z-YVAD-FMK inhibitors that helped to reveal the short half-life (5 min) of LPA in vivo.13,14 We introduced a boronic acid moiety in the inhibitor structure to rationally target the threonine oxygen nucleophile of ATX with a hard matching Lewis acid. The crystal structure of ATX in complex with HA155 (1)(9) confirmed our hypothesis that this inhibitor focuses on the threonine oxygen nucleophile in the ATX active site via the boronic acid moiety, while the hydrophobic 4-fluorobenzyl moiety of inhibitor 1 focuses on the hydrophobic pocket responsible for lipid binding (Number ?(Figure11). Open in a separate window Number 1 ATX structure liganded with inhibitor 1 (PDB ID 2XRG). (A) Surface representation of ATX with inhibitor 1 (magenta). (B) Binding of inhibitor 1 to the threonine oxygen nucleophile and two zinc ions. (C) Visualizing the ether linker of inhibitor 1 bound to ATX. (D) Visualizing the degree of freedom for the thiazolidine-2,4-dione core of inhibitor 1 in the ATX binding site. Here, we statement a number of synthetic routes, systematically substituting linkers and the thiazolidine-2,4-dione core in 1, while keeping the boronic acid moiety untouched. The observed structureCactivity relations could well be explained from your ATX structure in complex with inhibitor 1. A remarkable binding pose Z-YVAD-FMK of a novel inhibitor, as expected from molecular docking experiments, suggests additional avenues for further inhibitor design. Results and Discussion Design of Inhibitors The structure of inhibitor 1 bound to the ATX active site (Number ?(Number1)1) showed that its 4-fluorobenzyl moiety binds into the hydrophobic lipid binding pocket of ATX (Number ?(Number11C,D).(9) This pocket also accommodates the lipid tail of LPA, the hydrolysis product of LPC.(8) The thiazolidine-2,4-dione core of 1 1 and the conjugated aromatic ring are located between the hydrophobic pocket and the catalytic site (Figure ?(Figure1D).1D). The ether linker, bridging the two aromatic rings in 1, and especially a methylene and arylboronic acid moiety are well accessible to solvent (Number ?(Number1C).1C). Binding of inhibitor 1 to the ATX active site is definitely predominately driven by hydrophobic relationships (the interaction interface is approximately 500 ?2) and by the boronic acid binding to the threonine oxygen nucleophile of ATX.(9) The boronCoxygen range observed is 1.6 ?, which is consistent with a covalent relationship. As expected, this binding is definitely reversible evidenced by the fact that ATX activity Z-YVAD-FMK can be fully restored upon washing out the inhibitor.(13) In addition, one of the boronic acid hydroxyl moieties is usually tethered by the two zinc ions in the ATX active site. Therefore, the boronic acid moiety focuses on not Z-YVAD-FMK only the threonine oxygen nucleophile, but also the two zinc ions that are essential for catalytic activity of ATX (Number ?(Figure1B).1B). Amazingly, there are no hydrogen bonds or salt bridges that participate in binding of inhibitor 1 to ATX. Inhibitor 1 is definitely locked inside a pose with reduced molecular flexibility, forming an ideal starting point for.