Fig. 2: Regulation of inhibitory properties of EXACT-inhibitors.
From: Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors
a–c Simulation of composition of bivalent-binding for all binding complexes (a) enzyme inhibition curves (b) and the maximum inhibition and IC50 (c) with varying KEA,I by EXACT inhibitors with different KEA,I based on the proposed two-step binding model with additional assumptions (see Supplementary Note 2) d–f Synergistic-binding of EXACT inhibitors can be tuned by varying linker length. The inhibition curves of HD22-DAB conjugates with different dA linker lengths in fluorescent peptidyl substrate cleavage assay (d) indicate that the linker length regulates the KEA,I (e), IC50 and inhibition max (f) of HD22-DAB conjugates. g–i, Exosite-binding aptamers regulate the selectivity of EXACT inhibitors. Inhibition of factor Xa (g) and thrombin (h) activity by EXACT inhibitors constructed with thrombin (HD22) or factor Xa (11F7t)-binding aptamer, respectively, demonstrate that the selectivity of the small molecule inhibitor DAB can be dictated by the exosite-binding aptamer to which it is conjugated. The inhibition potency of EXACT inhibitors is not sensitive to the affinity of small molecule active site inhibitors (i). j, k The linker domain modulates the antidote-mediated reversibility of EXACT inhibitors. j Sequence of HD22-7A-DAB and different antidotes. k Thrombin activity (%) of each sample is measured by fluorogenic activity assay and normalized with the thrombin activity in the absence of inhibitor as 100%. Samples AO1-5 show thrombin activity in the presence of 250 nM HD22-7A-DAB and 2 µM of different AOs. Sample No AO and DAB ctrl show thrombin activity in the presence of 250 nM HD22-7A-DAB and DAB, respectively. In panels (d, g–i and k) data are presented as mean values +/‒ SD calculated from three independent experiments (n = 3). In panels e and f, data are presented as mean values +/‒ SE fitted from the data in panel d. In penal (k) One-way ANOVA test followed by Bonferroni’s multiple comparisons test (two-sided) was used to compare between two sets of data. *P < 0.05, ns, not significant. Exact P-values are provided as follows. AO2 vs AO1, AO2 vs AO8, AO2 vs AO9, AO2 vs No AO, AO2 vs DAB ctrl: p < 0.0001. AO2 vs AO3: p = 0.4144; AO2 vs AO4: p = 0.0807; AO2 vs AO5: p = 0.7064; AO2 vs AO7: p = 0.0984; AO2 vs AO6 and AO9 vs No AO: p > 0.9999.
