Qudsia Rashid, Md. Asim Azhar, Asma Naseem, Md. Sazzad Khan, Poonam Singh, Mohamad Aman Jairajpuri
Protein Conformation and Enzymology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India.


An elevated prothrombotic state is a major risk factor for venous thromboembolism, atrial fibrillation and cardiac strokes. The regulation of various coagulation cascade proteases plays an important role in determining a prothrombotic state. Clinically used anticoagulants are inhibitors of enzymes involved in the coagulation pathway, primarily thrombin and factor Xa. The conformational activation of antithrombin by heparin is a critical step in the inhibition of factor Xa by antithrombin. Despite heparin being the most potent physiological activator which enhances the otherwise very lethargic antithrombin inhibition of factor Xa by approximately 1000 folds, the conventional heparin therapy poses serious complications because of heparin`s polyanionic nature and its cross-reactivity. Out of the numerous attempts to design or discover new molecules as anticoagulants, designing molecules that may activate antithrombin is quite promising. A theoretical screening approach for identifying alternative non-heparin conformational activators of antithrombin for factor Xa inhibition is an ideal strategy to identify lead compounds and modify them for an appreciable activation of antithrombin. Binding specificity of various polyphenolic scaffolds to antithrombin at the heparin binding site is the best indicator of its conformational activation potential. A screening strategy has been applied using a blind docking protocol to find the binding energy and interactions of the modified versus unmodified organic scaffolds. We choose some representative organic compounds that have been shown to be involved in coagulation modulation by affecting antithrombin. Flavonoids, Xanthones, Dihydroxybenzofurans (DHP) and Tetrahydoisoquinoline were sulfated at specific location to target ATIII to compare their binding energies and specific interactions with their corresponding non-sulfated molecules. The interactions computed using Autodock has shown that blind docking can easily distinguish between the changes in specificity due to the sulfation. Non-sulfated hesperetin binds antithrombin in strand 2A in the extended heparin binding site region which is involved in the propagation of conformational change on account of heparin binding. Sulfation of hesperetin switches the affinity away from the heparin binding site where it now binds the C-sheet with a binding energy of -6.8 kcal/mol. Effect of sulfated hesperetin on coagulation, antithrombin and factor Xa inhibition will be checked after synthesis. Querecitin, rutin, mangiferin and benzofuran bind away from the heparin binding site in the native state but a sulfation induced binding specificity switch takes place where they now bind specifically in the heparin binding site. Significant increase in the binding energy is observed in Rutin for the heparin binding site on account of sulfation. These results clearly indicated that a sulfation based specificity switch either inside or away from the heparin binding site can be used as an initial screening to test a range of organic scaffolds. Specific increase in the affinity on account of sulfation or other modifications can also be detected. The results also emphasize that blind docking is a much better screening strategy to isolate lead compound. Read More …

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