Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to Its Heparin-Binding Site

Abstract

To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the VMAX of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively-charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggest the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs.

Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reaction: Scope, Mechanism, and Applications

“…A diverse set of 1,5-triazole products can be found in these reports which cover a wide range of targets and target classes including various enzyme inhibitors, [203][204][205][206][207][208][209][210][211][212][213] kinases, [214][215][216][217] proteases, 110,112 antivirals 218 (see also chapter 5.3) G-protein coupled receptors (GPCRs), 219 ion channels, [220][221][222] heat shock proteins, 95 and tRNA ligands. 223 1,5-Triazole derivatives have shown activity against numerous cancer cell lines, 205,215,[224][225][226][227] and against the parasites Trypanosoma cruzi 70,228 and Plasmodium falciparum.…”

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

“…We reasoned that small, synthetic, homogenous, non-saccharide GAG mimetics (NSGMs) may offer an avenue for discovering novel plasmin inhibitors. In fact, Desai and co-workers have developed a sizeable number of NSGMs based on various scaffolds including sulfated flavonoids [30][31][32][33], sulfated benzofurans [34,35], sulfated tetrahydroisoquinolines [36], sulfated quinazolinones [37] and sulfated galloyl glucopyranosides [38,39] as modulators of a range of coagulation proteins. The NSGMs resemble sulfated GAGs in the form of presenting one or more sulfate groups to interact with GAG-binding domains on targeted proteins.…”

“…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.…”

“…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. NSGMs 11-30, and 35-41 were synthesized as reported earlier [30][31][32][33][34][35][36][37].…”

“…Briefly, the synthesis of these NSGMs was achieved in 4 to 8 steps using traditional protection-deprotection chemistry following construction of the base scaffold with appropriate substitution pattern. The final step for the generation of each NSGM was chemical sulfation using trimethylamine-sulfur trioxide at elevated temperature, as described in our studies earlier [30][31][32][33][34][35][36][37][38][39][40][41][42]. For example, the synthesis of two promising plasmin inhibitors (Scheme 1) involved exploitation of the intramolecularly hydrogen bonded 5-OH group of quercetin 60 to selectively introduce a reactive handle (propargyl group in 63 or in situ alkyl bromide) that can be coupled with either an alkyl azide containing quinazolinone unit 32a or MOM-protected quercetin 61 to eventually give inhibitors 32 and 52, respectively.…”