Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein–Protein Interaction

Abstract

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein−protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC 50 of 0.44 μM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

“…These approaches include substrate-based design of linear and cyclic peptidomimetics [10][11][12][13][14], mutagenesis of key residues to engineer Kunitz-and Kazal-type protein/peptide inhibitors [15][16][17], covalent inhibition through a reactive nitrile or aldehyde warhead [18,19], and structure-based computational inhibitor design [20,21]. Each of these approaches typically targets the enzyme's active site.…”

“…The monomeric scaffolds included chalcones (compounds 1-10), flavonoids (11)(12)(13)(14)(15)(16) [30][31][32], sucrose octasulfate (17) [40], quinazolinones (18 and 19) [37], and tetrahydro-isoquinolines (20)(21)(22)(23)(24)(25)(26)(27) [36], whereas the dimeric scaffolds comprised flavonoid-quinazolinone heterodimers (28)(29)(30)(31)(32)(33)(34) [37], bis-quinazolinones homodimers (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) [37], and bis-flavonoid homodimers (48-55). In addition to the inherent diversity of the scaffolds in this library, NSGMs also differed in the number (1 to 8) and orientation of the sulfate groups.…”

Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines

“…15,16 Very recently, new TXA analogues based on an isoxazolone scaffold have been reported which possess an enhanced potency and improved overall profile. 17 In addition to these developments targeting the plasmin formation, a direct and selective plasmin inhibition may provide a more rapid and stronger effect to reduce bleeding. The work on synthetic plasmin inhibitors was recently reviewed.…”

Plasminogen binding inhibitors demonstrate unwanted activities on GABA A and glycine receptors in human iPSC derived neurons

“…This has been associated to an unwanted selectivity towards inhibitory neuronal receptors GABAA and glycine (307)(308)(309) which is also observed for more potent inhibitors as 4-PIOL (310). To identify PBIs which do not show GABAA receptor activity potent inhibitors are counter screened for this effect (311).…”

“…Studying receptor functionality in hiPSC derived neurons is performed by using various techniques such as patch clamping (312), calcium imaging (313), MEA, used to study neuronal circuitconnectivity (314). One of the previous techniques in the pharmaceutical industry to measure binding activity to neuronal derived receptors is based on isolation of neuronal rat membrane (310,311). This requires animal and is both time consuming and does not show compound mode of action, if it behaves as an agonist or antagonist.…”