Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile

Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 μM, efficacy = 100%). Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%–100%), an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design

The putative anion-binding allosteric site of human FXIIIa.

<p>(A) The electrostatic potential of the surface exposed anion-binding site of FXIII (PDB ID: 1GGU). (B) The basic residues in the site are shown as spheres. The residues matching the heparin-binding site of transglutaminase are K61, K73, R303, and K678. (C) The electrostatic potential of human thrombin is shown (PDB ID: 1XMN). (D) The basic residues of thrombin’s exosite 2 are shown in spheres. Positive and negative potentials are colored in blue and red, respectively.</p

Michaelis−Menten kinetics of dansylcadaverine and <i>N</i>,<i>N</i>-dimethylcasein conjugation by human FXIIIa in the presence of NSGM 13.

<p>The initial rate of conjugation at (A) various dansylcadaverine concentrations (0–750 μM) and fixed dimethylcasein concentration (5 mg/mL) or (B) various dimethylcasein concentrations (0–5 mg/mL) and fixed dansylcadaverine concentration (250 μM) was measured spectrofluorometrically in pH 8.0 buffer at 37°C. Solid lines represent nonlinear regressional fits to the data by the standard Michaelis− Menten <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#pone.0160189.e004" target="_blank">Eq 4</a> to yield <i>K</i>_<i>M</i> and <i>V</i>_<i>MAX</i>. See details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#sec011" target="_blank">Materials and Methods</a>.</p

Inhibition Percent of Human FXIIIa by Sulfated Small Molecules.^a

<p>Inhibition Percent of Human FXIIIa by Sulfated Small Molecules.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#t001fn001" target="_blank">^a</a></p

Interaction of human FXIIIa and α-thrombin (α-Th) with NSGM 13 and UFH.

<p>(A) The inhibition of FXIIIa (●) and α-Th (○) by NSGM <b>13</b> was measured spectrofluorometrically through a bisubstrate, fluorescence-based transglutamination assay (FXIIIa) or chromogenic substrate assay (α-Th) at pH 7.4/8.0 and 37°C. Solid lines represent sigmoidal fits to the data to obtain <i>IC</i>_<i>50</i>, <i>HS</i>, <i>Y</i>_<i>M</i>, <i>and Y</i>_<i>O</i> using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#pone.0160189.e001" target="_blank">Eq 1</a>. (B) Spectrofluorometric measurement of the affinity of human FXIIIa for inhibitor <b>13</b> at pH 8.0 and 37°C using the intrinsic tryptophan fluorescence (λ_EM = 348 nm, λ_EX = 280 nm). Solid lines represent nonlinear regressional fits using quadratic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#pone.0160189.e002" target="_blank">Eq 2</a>. (C) Spectrofluorimetric measurement of the affinity of human FXIIIa for UFH at pH 8.0 and 37°C using the intrinsic tryptophan fluorescence (λ_EM = 348 nm, λ_EX = 280 nm). Solid lines represent nonlinear regressional fits using the standard Hill <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#pone.0160189.e003" target="_blank">Eq 3</a>. See details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#sec011" target="_blank">Materials and Methods</a>.</p

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

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 <i>V</i>_max of substrate hydrolysis without influencing the <i>K</i>_M. Mutagenesis of residues of the heparin-binding site (HBS) 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 suggests 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

Inhibition Profiles of Human Factor XIIIa (FXIIIa), Human α-Thrombin (α-Th), Human Factor Xa (FXa), and Papain by Iodoacetamide (IAA) and the NSGMs 13 and 14.^a

<p>Inhibition Profiles of Human Factor XIIIa (FXIIIa), Human α-Thrombin (α-Th), Human Factor Xa (FXa), and Papain by Iodoacetamide (IAA) and the NSGMs 13 and 14.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160189#t002fn001" target="_blank">^a</a></p