The structure of a designed peptidomimetic inhibitor complex of alpha-thrombin.

Thrombin displays remarkable specificity, effecting the removal of fibrinopeptides A and B of fibrinogen through the selective cleavage of two Arg-Gly bonds between the 181 Arg/Lys-Xaa bonds in fibrinogen. Significant advances have been made in recent years towards understanding the origin of the specificity of cleavage of the Arg16-Gly17 bond of the A alpha-chain of human fibrinogen. We have previously proposed a model for the bound structure of fibrinopeptide A7-16 (FPA), based upon NMR data, computer-assisted molecular modeling and the synthesis and study of peptidomimetic substrates and inhibitors of thrombin. We now report the structure of the ternary complex of an FPA mimetic (FPAM), hirugen and thrombin at 2.5 A resolution (R-factor = 0.138) and specificity data for the inhibition of thrombin and related trypsin-like proteinases by FPAM. The crystallographic structures of FPA and its chloromethyl ketone derivative bound to thrombin were determined. Although there are differences between these structures in the above modeled FPA structure and that of the crystal structure of FPAM bound to thrombin, the phi, psi angles in the critical region of P1-P2-P3 in all of the structures are similar to those of bovine pancreatic trypsin inhibitor (BPTI) in the BPTI-trypsin complex and D-Phe-Pro-Arg (PPACK) in the PPACK-thrombin structure. A comparison between these and an NMR-derived structure is carried out and discussed.

Design, synthesis and conformational analysis of gamma-turn peptide mimetics of bradykinin.

Gamma-turns are regular secondary structure elements, found with some frequency in small peptides, that have been implicated in the biologically active conformations of several systems. This report describes the design, synthesis and conformational analysis of a non-peptide gamma-turn mimetic. Low energy conformations of the mimetic system exhibit good conformational agreement with an experimentally observed peptide gamma-turn. The mimetics were incorporated into the nonapeptide bradykinin, for which a gamma-turn, formed by residues Ser 6 to Phe 8, has been hypothesized to be a bioactive conformation. The results indicate that a bioactive conformation of bradykinin may include a reverse turn at this position.

Loops and secondary structure mimetics: development and applications in basic science and rational drug design.

One goal of protein design and structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high resolution analysis and rapid modification. We have developed a variety of small molecules which biochemically and biologically mimic the combining sites of proteins of the immunoglobulin superfamily. The chemical and biological properties of peptide mimetics suggest that these analogs can be used as indicators for new pharmaceutical agents. Mimetics are powerful tools for the study of molecular recognition since they are small in size, maintain solubility in physiologic fluids and are amenable to detailed structural studies. As such, they represent a step toward the rational design of low molecular weight non-peptide pharmaceutical agents devoid of some of the shortcomings of conventional peptides. Here we discuss the rationale and approaches for the development of these molecules, and their current and future applications.

Design and synthesis of a CD4 beta-turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes.

Poor bioavailability, rapid degradation, antigenicity, and high cost often limit the use of proteinaceous pharmaceuticals. One goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of complex proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. We have developed a reverse-turn mimetic system to explore peptide and protein structure-function relationships. We now report the design and synthesis of a small molecule (M(r) 810, as its trifluoroacetate salt), water soluble, proteolytically stable mimetic of residues Gln40-Thr45 of the complementarity-determining 2-like region of CD4. This mimetic has a low micromolar Kd for human T-lymphotropic virus type IIIB gp120 and reduces syncytium formation.

Peptide mimetics of the thrombin-bound structure of fibrinopeptide A.

Recent work has suggested that the thrombin-bound conformation of fibrinopeptide A exhibits a strand-turn-strand motif, with a beta-turn centered at residues Glu-11 and Gly-12. Our molecular modeling analysis indicates that the published fibrinopeptide conformation cannot bind reasonably to thrombin but that reorientation of two residues by alignment with bovine pancreatic trypsin inhibitor provides a good fit within the deep thrombin cleft and satisfies all of the experimental nuclear Overhauser effect data. Based on this analysis, we have successfully designed and synthesized hybrid peptide mimetic substrates and inhibitors that mimic the proposed beta-turn structure. The results indicate that the turn conformation is an important aspect of thrombin specificity and that our turn mimetic design successfully mimics the thrombin-bound conformation of fibrinopeptide.