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Allosteric inhibition of human factor XIa: discovery of monosulfated benzofurans as a class of promising inhibitors.

Argade MD, Mehta AY, Sarkar A, Desai UR - J. Med. Chem. (2014)

Bottom Line: Factor XIa (fXIa) is being recognized as a prime target for developing safer anticoagulants.Of these, monosulfated benzofurans were the only group of molecules found to inhibit fXIa (∼100% efficacy) and led to the identification of monosulfated trimer 24 (IC50 0.82 μM) as the most potent inhibitor.Although monosulfated, the inhibitors did not compete with unfractionated heparin alluding to a novel site of interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.

ABSTRACT
Factor XIa (fXIa) is being recognized as a prime target for developing safer anticoagulants. To discover synthetic, small, allosteric inhibitors of fXIa, we screened an in-house, unique library of 65 molecules displaying many distinct scaffolds and varying levels of sulfation. Of these, monosulfated benzofurans were the only group of molecules found to inhibit fXIa (∼100% efficacy) and led to the identification of monosulfated trimer 24 (IC50 0.82 μM) as the most potent inhibitor. Michaelis-Menten kinetics studies revealed a classic noncompetitive mechanism of action for 24. Although monosulfated, the inhibitors did not compete with unfractionated heparin alluding to a novel site of interaction. Fluorescence quenching studies indicated that trimer 24 induces major conformational changes in the active site of fXIa. Docking studies identified a site near Lys255 on the A3 domain of fXIa as the most probable site of binding for 24. Factor XIa devoid of the A3 domain displayed a major defect in the inhibition potency of 24 supporting the docking prediction. Our work presents the sulfated benzofuran scaffold as a promising framework to develop allosteric fXIa inhibitors that likely function through the A3 domain.

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Genetic algorithm-based docking studies to identifya putativebinding site of 24 on factor XIa. (A) Plausible sitesof binding were identified by searching for hydrophobic subdomainsin the vicinity of a basic (Arg/Lys) residue that could engage thesulfate group and aromatic scaffold of 24. Eight plausiblesites in the vicinity of Lys8, Arg136, Gln153, Lys252, Lys325, Lys357,Asn566, and Arg584 (shown as blue van der Waals atom spheres) wereidentified by generating a chimeric model from the crystal structureof the catalytic domain (PDB ID: 1ZOM) and the heavy chain of factor XI (thezymogen, PDB ID: 2F83). (B) One specific site of docking near Lys255 was identified byGOLD as the most probable binding site of 24. GOLD predicteddocking solutions within 1.6 Å RMSD suggesting highly selectiverecognition. Rings A, B, and C refer to the three benzofuran ringsof 24. (C) Close-up of the 24–factorXIa docked complex showing engagement of Lys255, Asn189, Tyr278, andGln226, each of which shows ionic or hydrogen bond interactions, andplausible π–cation interaction with Arg210 and π–πinteraction with Phe206. The site is adjacent to Arg184 known to playan important role in factor IX activation by factor XIa. See textfor details.
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fig7: Genetic algorithm-based docking studies to identifya putativebinding site of 24 on factor XIa. (A) Plausible sitesof binding were identified by searching for hydrophobic subdomainsin the vicinity of a basic (Arg/Lys) residue that could engage thesulfate group and aromatic scaffold of 24. Eight plausiblesites in the vicinity of Lys8, Arg136, Gln153, Lys252, Lys325, Lys357,Asn566, and Arg584 (shown as blue van der Waals atom spheres) wereidentified by generating a chimeric model from the crystal structureof the catalytic domain (PDB ID: 1ZOM) and the heavy chain of factor XI (thezymogen, PDB ID: 2F83). (B) One specific site of docking near Lys255 was identified byGOLD as the most probable binding site of 24. GOLD predicteddocking solutions within 1.6 Å RMSD suggesting highly selectiverecognition. Rings A, B, and C refer to the three benzofuran ringsof 24. (C) Close-up of the 24–factorXIa docked complex showing engagement of Lys255, Asn189, Tyr278, andGln226, each of which shows ionic or hydrogen bond interactions, andplausible π–cation interaction with Arg210 and π–πinteraction with Phe206. The site is adjacent to Arg184 known to playan important role in factor IX activation by factor XIa. See textfor details.

Mentions: To assess the hypothesisof the dual element recognition more quantitatively,we studied the full-length fXIa model and identified eight sites onthe protein surface displaying a relatively higher positive chargedensity and adjacent to a hydrophobic patch. These sites were centeredaround residues Lys8, Arg136, Gln153, Lys252, Lys325, Lys357, Asn566,and Arg584 (Figure 7A), which were stochasticallyselected as possible binding sites of inhibitor 24. Allresidues within 24 Å around the identified Arg/Lys were definedas the binding site for molecular docking purposes. This operationcovered practically the entire protein surface, thus ensuring exhaustiveexploration for identification of possible binding site(s). Inhibitor 24 was docked at each of these eight sites using a geneticalgorithm-based docking and scoring technique, as developed in theliterature.43,44 We focused primarily on consistencyof docking, as evident by the RMSD between docked poses, followingmultiple docking runs to derive meaningful results, as suggested inearlier studies with sulfated molecules.32,40,44


Allosteric inhibition of human factor XIa: discovery of monosulfated benzofurans as a class of promising inhibitors.

Argade MD, Mehta AY, Sarkar A, Desai UR - J. Med. Chem. (2014)

Genetic algorithm-based docking studies to identifya putativebinding site of 24 on factor XIa. (A) Plausible sitesof binding were identified by searching for hydrophobic subdomainsin the vicinity of a basic (Arg/Lys) residue that could engage thesulfate group and aromatic scaffold of 24. Eight plausiblesites in the vicinity of Lys8, Arg136, Gln153, Lys252, Lys325, Lys357,Asn566, and Arg584 (shown as blue van der Waals atom spheres) wereidentified by generating a chimeric model from the crystal structureof the catalytic domain (PDB ID: 1ZOM) and the heavy chain of factor XI (thezymogen, PDB ID: 2F83). (B) One specific site of docking near Lys255 was identified byGOLD as the most probable binding site of 24. GOLD predicteddocking solutions within 1.6 Å RMSD suggesting highly selectiverecognition. Rings A, B, and C refer to the three benzofuran ringsof 24. (C) Close-up of the 24–factorXIa docked complex showing engagement of Lys255, Asn189, Tyr278, andGln226, each of which shows ionic or hydrogen bond interactions, andplausible π–cation interaction with Arg210 and π–πinteraction with Phe206. The site is adjacent to Arg184 known to playan important role in factor IX activation by factor XIa. See textfor details.
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Related In: Results  -  Collection

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fig7: Genetic algorithm-based docking studies to identifya putativebinding site of 24 on factor XIa. (A) Plausible sitesof binding were identified by searching for hydrophobic subdomainsin the vicinity of a basic (Arg/Lys) residue that could engage thesulfate group and aromatic scaffold of 24. Eight plausiblesites in the vicinity of Lys8, Arg136, Gln153, Lys252, Lys325, Lys357,Asn566, and Arg584 (shown as blue van der Waals atom spheres) wereidentified by generating a chimeric model from the crystal structureof the catalytic domain (PDB ID: 1ZOM) and the heavy chain of factor XI (thezymogen, PDB ID: 2F83). (B) One specific site of docking near Lys255 was identified byGOLD as the most probable binding site of 24. GOLD predicteddocking solutions within 1.6 Å RMSD suggesting highly selectiverecognition. Rings A, B, and C refer to the three benzofuran ringsof 24. (C) Close-up of the 24–factorXIa docked complex showing engagement of Lys255, Asn189, Tyr278, andGln226, each of which shows ionic or hydrogen bond interactions, andplausible π–cation interaction with Arg210 and π–πinteraction with Phe206. The site is adjacent to Arg184 known to playan important role in factor IX activation by factor XIa. See textfor details.
Mentions: To assess the hypothesisof the dual element recognition more quantitatively,we studied the full-length fXIa model and identified eight sites onthe protein surface displaying a relatively higher positive chargedensity and adjacent to a hydrophobic patch. These sites were centeredaround residues Lys8, Arg136, Gln153, Lys252, Lys325, Lys357, Asn566,and Arg584 (Figure 7A), which were stochasticallyselected as possible binding sites of inhibitor 24. Allresidues within 24 Å around the identified Arg/Lys were definedas the binding site for molecular docking purposes. This operationcovered practically the entire protein surface, thus ensuring exhaustiveexploration for identification of possible binding site(s). Inhibitor 24 was docked at each of these eight sites using a geneticalgorithm-based docking and scoring technique, as developed in theliterature.43,44 We focused primarily on consistencyof docking, as evident by the RMSD between docked poses, followingmultiple docking runs to derive meaningful results, as suggested inearlier studies with sulfated molecules.32,40,44

Bottom Line: Factor XIa (fXIa) is being recognized as a prime target for developing safer anticoagulants.Of these, monosulfated benzofurans were the only group of molecules found to inhibit fXIa (∼100% efficacy) and led to the identification of monosulfated trimer 24 (IC50 0.82 μM) as the most potent inhibitor.Although monosulfated, the inhibitors did not compete with unfractionated heparin alluding to a novel site of interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.

ABSTRACT
Factor XIa (fXIa) is being recognized as a prime target for developing safer anticoagulants. To discover synthetic, small, allosteric inhibitors of fXIa, we screened an in-house, unique library of 65 molecules displaying many distinct scaffolds and varying levels of sulfation. Of these, monosulfated benzofurans were the only group of molecules found to inhibit fXIa (∼100% efficacy) and led to the identification of monosulfated trimer 24 (IC50 0.82 μM) as the most potent inhibitor. Michaelis-Menten kinetics studies revealed a classic noncompetitive mechanism of action for 24. Although monosulfated, the inhibitors did not compete with unfractionated heparin alluding to a novel site of interaction. Fluorescence quenching studies indicated that trimer 24 induces major conformational changes in the active site of fXIa. Docking studies identified a site near Lys255 on the A3 domain of fXIa as the most probable site of binding for 24. Factor XIa devoid of the A3 domain displayed a major defect in the inhibition potency of 24 supporting the docking prediction. Our work presents the sulfated benzofuran scaffold as a promising framework to develop allosteric fXIa inhibitors that likely function through the A3 domain.

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