Nonpolar Interactions of Thrombin S‘ Subsites with Its Bivalent Inhibitor:  Methyl Scan of the Inhibitor Linker

Abstract

We have designed bivalent thrombin inhibitors, consisting of a nonsubstrate type active site blocking segment, a hirudin-based fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The inhibition provided by the bivalent inhibitors with various linker lengths revealed that a minimum of 15 atoms was required for simultaneous binding of the two blocking segments of the inhibitor to thrombin without significant distortion. The crystal structure of the inhibitors with a 16-atom linker showed some conformational flexibility in the linker portion which still lies deep in the groove joining the active site and the fibrinogen recognition exosite. Since the thrombin S' subsites are not well characterized, we designed a new strategy to search for possible nonpolar interactions between the linker and the thrombin S' subsites. This strategy, the "methyl scan", is based on the incorporation of a methyl side chain at each atom position of the linker by using sarcosine, D,L-alanine, D,L-3-aminoisobutyric acid, or N-methyl-beta-alanine. The methyl groups on the second and the eighth atom positions of the linker, which correspond to the side chains of the P1' and the P3' residues, respectively, improved the affinity of the inhibitors significantly. Further study of the stereospecificity showed that L-Ala at the P1' residue and D-Ala at the P3' residue preferably improved the affinity of the inhibitors 20- and 25-fold, respectively. Molecular modeling calculations using a methyl probe were also carried out to identify favorable nonpolar interacting sites on the thrombin surface. Two sites were identified in the vicinity of the P1' and the P3' residues, supporting the validity of the methyl scan method. Thus, this study has improved our understanding of the interactions taking place in this groove. In particular, we have been able to show that some specific structural features, such as hydrophobic complementarity between the linker and the thrombin S' subsites, could be exploited and make these inhibitors trivalent.

Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors

“…The S′ subsites' interactions between the inhibitors and protease are important for binding [41]. A systematic probing of thrombin S′ subsites with ‘methyl scan’ also has demonstrated the potential for targeting prime subsites in the design of inhibitors [42]. However, unlike variegin, other naturally occurring thrombin inhibitors, including hirudin [31], rhodniin [43], ornithodorin [44] and boophilin [45], are not inserted into the canyon-like cleft (the prime subsites) connecting the active site and exosite-I.…”

“…In these cases, the S′ subsite interactions are sub-optimal due to the lack of specific side chain interactions. As a result, extensive and lengthy optimizations, through synthetic chemistry using multiple and unnatural amino acids, were necessary to produce inhibitors with enhanced binding to the prime subsites [42], [53], [54]. In contrast, tight binding of s-variegin to the prime subsites is achieved through specific interactions involving the side chains of natural amino acids.…”

Direct thrombin inhibitors – a survey of recent developments

“…Furthermore, a series of bivalent thrombin inhibitors termed hirunorms was generated that contain a peptidic module that blocks the active-site in a nonsubstrate mode [82,83]. Other variations of the initially developed hirulogs include modifications in the linker [84,85] and the fibrinogen exosite-binding region [86,87] as well as size reduction of the hirudin-derived part, as in BCH-2763 [88]. More recently, a chimera of hirudin and dipetalogastin II, a potent peptidic thrombin inhibitor from the bug Dipetalogaster maximus was described which exhibited an inhibition equilibrium constant (K i ) of 45 fM [89] which is close to that of natural hirudin.…”

The pharmacophore of a peptoid VEGF receptor 2 antagonist includes both side chain and main chain residues

“…With peptide-based agents, this exercise is simplified by the regular structure of the molecule. A common practice is to evaluate a series of derivatives in which each residue in turn is replaced with a glycine or alanine (alanine scanning) 1, 2. Recently, we reported the effective application of glycine scanning to a peptoid (N-substituted oligoglycine) inhibitor of the 19S regulatory particle of the proteasome.…”