Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.

Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker.

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.

A hexapeptide VTKFYF from C-terminal part of interleukin-1 receptor antagonist, an inhibitor of IL-1-IL-1 receptor interaction.

In order to find the low-molecular-weight interleukin-1 (IL-1) inhibitors we synthesized a series of peptides, derived from three regions of interleukin-1 receptor antagonist (IL-1ra): N-terminal (residues 5-9), central (90-98) and C-terminal (143-148). The decision was based on the thorough analysis of structural and functional properties of IL-1 proteins and the resemblance of some fragments of IL-1ra to well-known immunomodulators, like thymopentin and tuftsin. The competition between our peptides and IL-1 was measured as the inhibition of IL-1 induced IL-2 production in LBRM/CTLL cell line system. The activity of tuftsin (TKPR), a peptide immunomodulator derived from IgG molecule, was also examined. All peptides presented some activity, although the most interesting results (when the range of activity and dose-dependence were taken into account) were obtained for tuftsin and peptide VTKFYF from the C-terminal part of IL-1ra, which is in agreement with the latest reports on the structure of IL-1ra-receptor complex.

Immunomodulatory activity of oligopeptides related to interleukin 1 receptor antagonist sequence.

We examined the immunomodulatory properties of peptides from interleukin 1 receptor antagonist (IL-1Ra) with regard to the humoral (plaqueforming cells-PFC) and cellular (delayed type hypersensitivity-DTH) immune response and GvH reaction. It was found that peptide RKSSK (II) from the N-terminal part of IL-1Ra, although inactive with regard to the inhibition of IL-1-IL-1 receptor interaction, reduces immune response in a manner similar to cyclosporin A (DTH, PFC in vivo). Peptide GRKSSK (III) was even more potent, whereas peptides from respective fragment of mouse IL-1Ra were weaker immunosuppressants than II. Peptide VTKFYF (VII) from the C-terminal part of IL-1Ra, very active as IL-1 inhibitor, and its analog VIII with Asp residue, characteristic for IL-1, instead of Lys from IL-1Ra, showed only limited activity despite the previously observed competition with IL-1 for the cellular receptor. Thus, no correlation between the inhibitory and immunomodulatory properties of peptides derived from IL-1Ra was observed.

Nonpolar interactions of thrombin and its inhibitors at the fibrinogen recognition exosite: thermodynamic analysis.

Nonpolar interactions play a major role in the association of the fibrinogen recognition exosite of thrombin with the C-terminal fragment (55-65), Asp-Phe-Glu-IIe-Pro-Glu-Glu-Tyr-Leu-Gln, of hirudin, which is a naturally occurring thrombin inhibitor. The thermodynamic details (free energy, enthalpy, entropy, and heat capacity) of the molecular recognition are studied by using five analogs of a synthetic bivalent thrombin inhibitor (P552), tert-butylbenzensulfonyl-Arg-(D-pipecolic acid)-(12-amino-dodecanoic acid)-(gamma-aminobutyric acid)-hirudin55-65. The residue of PheH56, IleH59, ProH60, TyrH63, or LeuH64 in hirudin 55-65 segment is substituted by Gly in each analog in order to elucidate the contributions of these nonpolar side chains. The results show that the interactions of these nonpolar side chains with thrombin are enthalpy-driven, except for the contribution of the PheH56 side chain which is entropy-driven. Interestingly, molecular modeling predicts a large conformational change due to the Gly substitution of PheH56. In analyzing the correlation among the thermodynamic and structural properties of the nonpolar interaction, a good correlation is observed between the binding free energy and the hydrophobicity of the molecular surface; i.e., tighter binding is observed as more nonpolar atoms are buried and more polar atoms are exposed upon molecular association.

The search for inhibitors of interleukin-1 based on the sequence of interleukin-1 receptor antagonist.

In order to find the low-molecular-weight interleukin-1 (IL-1) inhibitors, we synthesised a series of peptides, derived from three regions of interleukin-1 receptor antagonist (IL-1ra): N-terminal (residues 5-9), central (90-98) and C-terminal (143-148). The decision was based on the thorough analysis of structural and functional properties of IL-1 proteins and the resemblance of some fragments of IL-1ra to well-known immunomodulators, like thymopentin and tuftsin. The competition between our peptides and IL-1 were measured as the inhibition of IL-1 induced IL-2 production in LBRM/CTLL cell line system. All peptides presented some activity, although the most interesting results (when the range of activity and dose-dependence were taken into account) were obtained for tuftsin and peptide VTKFYF from the C-terminal part of IL-1ra.