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Designing allosteric inhibitors of factor XIa. Lessons from the interactions of sulfated pentagalloylglucopyranosides.

Al-Horani RA, Desai UR - J. Med. Chem. (2014)

Bottom Line: Variation in the anomeric configuration did not affect potency.Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa.Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , 800 E. Leigh Street, Suite 212, Richmond, Virginia 23219, United States.

ABSTRACT
We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem. 2013, 56, 867-878). To better understand the SPGG-FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG's sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies.

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Quenching of dansyl fluorescenceof DEGR-factor XIa by acrylamidein the absence (□) and presence of 20 μM β-SPGG-8(●) and 20 μM UFH (Δ). Fluorescence intensity at547 (λEX = 345 nm) was recorded following sequentialaddition of acrylamide. Solid lines represents fits to the data usingeither eq 2 (●, Δ) or 3 (□).
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fig4: Quenching of dansyl fluorescenceof DEGR-factor XIa by acrylamidein the absence (□) and presence of 20 μM β-SPGG-8(●) and 20 μM UFH (Δ). Fluorescence intensity at547 (λEX = 345 nm) was recorded following sequentialaddition of acrylamide. Solid lines represents fits to the data usingeither eq 2 (●, Δ) or 3 (□).

Mentions: The kineticmechanism of inhibition supports the hypothesis that SPGG variantsappear to remotely affect the conformation of the catalytic triadof FXIa. We predicted that this effect may extend to regions beyondthe catalytic triad. To assess this, we studied the quenching of fluorescenceof DEGR-FXIa, a dansyl-labeled variant, by acrylamide in the presenceand absence of β-SPGG-8 (4f). DEGR-FXIa containsthe fluorophore at the end of the EGR tripeptide (P1–P3 residues),which is covalently attached to the catalytic Ser. This implies thatthe dansyl group senses the electrostatics and dynamics around theP4 position. Dextran sulfate and hypersulfated heparin have been earliershown to reduce the quenching of DEGR-FXIa by acrylamide.26 Figure 4 shows the quenchingof DEGR-FXIa fluorescence by acrylamide with and without 20 μMβ-SPGG-8 or 20 μM UFH. Acrylamide quenches FXIa’sfluorescence both in the absence and presence of ligands in a dose-dependentmanner. Yet, the efficiency of quenching is dramatically different.Whereas considerable saturation is observed for FXIa alone with increasingquencher concentrations, no such effect is noted in the presence ofthe two allosteric ligands. Considering that FXIa is a physiologicaldimer,18,19 the significant nonlinearity of quenchingsuggests the possibility of two slightly different fluorophores, whichare being differentiated by the quencher. Indeed, it is possible toisolate FXIa with only half-functional unit.18,19 This implies that acrylamide is able to sense protein dynamics fordimeric FXIa. In contrast, both β-SPGG-8 and UFH stem quenchingto only about 50% of that observed in their absence at 350 mM acrylamide.At the same time, essentially no saturation of quenching is observedin their presence. In fact, the profiles follow the traditional one-fluorophoreStern–Volmer linear relationship well. This suggests that eitherone or both dansyl fluorophore(s) is(are) sterically less accessibleto the quencher all or part of the time. A simple explanation forthis behavior is that both β-SPGG-8 and UFH induce conformationalchanges in and around the active site that reduce steric and dynamicaccessibility to probes as small as the acrylamide.


Designing allosteric inhibitors of factor XIa. Lessons from the interactions of sulfated pentagalloylglucopyranosides.

Al-Horani RA, Desai UR - J. Med. Chem. (2014)

Quenching of dansyl fluorescenceof DEGR-factor XIa by acrylamidein the absence (□) and presence of 20 μM β-SPGG-8(●) and 20 μM UFH (Δ). Fluorescence intensity at547 (λEX = 345 nm) was recorded following sequentialaddition of acrylamide. Solid lines represents fits to the data usingeither eq 2 (●, Δ) or 3 (□).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216218&req=5

fig4: Quenching of dansyl fluorescenceof DEGR-factor XIa by acrylamidein the absence (□) and presence of 20 μM β-SPGG-8(●) and 20 μM UFH (Δ). Fluorescence intensity at547 (λEX = 345 nm) was recorded following sequentialaddition of acrylamide. Solid lines represents fits to the data usingeither eq 2 (●, Δ) or 3 (□).
Mentions: The kineticmechanism of inhibition supports the hypothesis that SPGG variantsappear to remotely affect the conformation of the catalytic triadof FXIa. We predicted that this effect may extend to regions beyondthe catalytic triad. To assess this, we studied the quenching of fluorescenceof DEGR-FXIa, a dansyl-labeled variant, by acrylamide in the presenceand absence of β-SPGG-8 (4f). DEGR-FXIa containsthe fluorophore at the end of the EGR tripeptide (P1–P3 residues),which is covalently attached to the catalytic Ser. This implies thatthe dansyl group senses the electrostatics and dynamics around theP4 position. Dextran sulfate and hypersulfated heparin have been earliershown to reduce the quenching of DEGR-FXIa by acrylamide.26 Figure 4 shows the quenchingof DEGR-FXIa fluorescence by acrylamide with and without 20 μMβ-SPGG-8 or 20 μM UFH. Acrylamide quenches FXIa’sfluorescence both in the absence and presence of ligands in a dose-dependentmanner. Yet, the efficiency of quenching is dramatically different.Whereas considerable saturation is observed for FXIa alone with increasingquencher concentrations, no such effect is noted in the presence ofthe two allosteric ligands. Considering that FXIa is a physiologicaldimer,18,19 the significant nonlinearity of quenchingsuggests the possibility of two slightly different fluorophores, whichare being differentiated by the quencher. Indeed, it is possible toisolate FXIa with only half-functional unit.18,19 This implies that acrylamide is able to sense protein dynamics fordimeric FXIa. In contrast, both β-SPGG-8 and UFH stem quenchingto only about 50% of that observed in their absence at 350 mM acrylamide.At the same time, essentially no saturation of quenching is observedin their presence. In fact, the profiles follow the traditional one-fluorophoreStern–Volmer linear relationship well. This suggests that eitherone or both dansyl fluorophore(s) is(are) sterically less accessibleto the quencher all or part of the time. A simple explanation forthis behavior is that both β-SPGG-8 and UFH induce conformationalchanges in and around the active site that reduce steric and dynamicaccessibility to probes as small as the acrylamide.

Bottom Line: Variation in the anomeric configuration did not affect potency.Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa.Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , 800 E. Leigh Street, Suite 212, Richmond, Virginia 23219, United States.

ABSTRACT
We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem. 2013, 56, 867-878). To better understand the SPGG-FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG's sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies.

Show MeSH
Related in: MedlinePlus