Fig. 1: Rational design of thrombin-binding EXACT inhibitor.
From: Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors
a, b Crystal structures of hirudin-thrombin complex (a) in comparison to HD22-7A-DAB-thrombin complex (b). In both inhibitors, the active site-binding domain (magenta) and the exosite binding domain (green) are connected by a linker domain (gray), which allows synergistic binding to thrombin (tan). In b, the active site was zoomed in to show thrombin’s interacting residues (red) with dabigatran. The composite omit 2Fo-Fc map contoured at 1.0 σ shows the electron density for DAB as gray mesh. c Thrombin inhibition by HD22-7A-DAB and other inhibitors in fluorescent peptidyl substrate cleavage assay. HD22-7A-DAB showed a significantly higher inhibitory effect of thrombin than DAB, HD22, or their equimolar mixture, and can be efficiently reversed by an antisense oligonucleotide (AO2). Data are presented as mean values +/- SD calculated from three independent experiments (n = 3). d two-step binding model of EXACT inhibitor (AI) to protease (E) determined by dissociation constants KE,A, KE,I, and KEA,I. e Characterization of binding kinetics of biotinylated HD22-7A-DAB and its derivatives to thrombin (1.6 nM) by bio-layer interferometry. The illustration in panel (d) was created using Biorender.com.
