The crystal structures of human α‐thrombin complexed with active site‐directed diamino benzo[b] thiophene derivatives: A binding mode for a structurally novel class of inhibitors

Abstract

The crystal structures of four active site-directed thrombin inhibitors, 1-4, in a complex with human alpha-thrombin have been determined and refined at up to 2.0 A resolution using X-ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3-disubstituted benzo[b]thiophene structure. Compared to traditional active-site directed inhibitors, the X-ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C-3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C-2 was found to point away from the active site, occupying a location between the S1 and S1' sites. Together, the aromatic rings of the C-2 and C-3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence "Tyr-Pro-Pro-Trp." [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323-373).] In contrast to the binding mode of more classical thrombin inhibitors (D-Phe-Pro-Arg-H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three-dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure-based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900-fold more potent than the screening hit 1.

Discrimination of α-Thrombin and γ-Thrombin Using Aptamer-Functionalized Nanopore Sensing

“…In this work, we aim to discriminate two closely related proteins, α-thrombin and γ-thrombin, using aptamer-functionalized nanopores. Human α-thrombin (PDB reference 1D3T ) is a spherical protein with a molecular weight of 36,230 Da and a volume of 49.6 nm 3 , which specifically binds to the selected aptamer. Human γ-thrombin (PDB reference 2HNT ) is a modified α-thrombin lacking the aptamer binding epitope .…”

In Silico Study Reveals How E64 Approaches, Binds to, and Inhibits Falcipain-2 of Plasmodium falciparum that Causes Malaria in Humans

“…3a–c ) used. Indeed, the ΔG binding = −12.2 ± 1.1 kJ/mol reflects a strong binding and is comparable to the typical ΔG binding that accompanies strong protein-ligand binding 28 – 31 . Please, refer to the methods section and to the caption of Fig.…”

Privileged Structural Motif Detection and Analysis Using Generative Topographic Maps

“…Furthermore, RECAP cores of P_PRP 2 and 3 included lysine and arginine residues and heterocyclic-substituted ketones, providing an activated warhead for irreversible protease inhibition. These basic aliphatic motifs are highly common among inhibitors of thrombin-like serine proteases, for example, in argatroban, an approved competitive thrombin inhibitor . Thrombin-like enzymes display a primary substrate specificity for basic amino acids in the P1 position, i.e., the amino acid N-terminal of the scissile peptide bond.…”

“…These basic aliphatic motifs are highly common among inhibitors of thrombin-like serine proteases, for example, in argatroban, an approved competitive thrombin inhibitor. 33 Thrombin-like enzymes display a primary substrate specificity for basic amino acids in the P1 position, i.e., the amino acid Nterminal of the scissile peptide bond. The corresponding residues such as arginine are recognition elements for the interaction with the S1 binding pocket.…”