Bivalent inhibitors

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Bivalirudin, another bivalent inhibitor of thrombin, is administered intravenously, with a rapid onset and offset of action. 

Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin. Incorporation of a ketomethylene pseudo peptide bond (3-4) resulted in a noncleavable bivalent inhibitor that retained high thrombin affinity [24]. Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character continues to be observed. 

Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (d-Phe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (3-17, [27]) or 4-tert-butylbenzenesulfonyl-Arg-(D-pipecolic acid) (3-18, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Arg-containing inhibitors make similar interactions with the aspartic acid within the SI specificity pocket, the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors. 

Bivalirudin, another bivalent inhibitor of thrombin, is administered intravenously, with a rapid onset and offset of action. The drug has a short half-life with clearance that is 20% renal and the remainder metabolic. Bivalirudin inhibits platelet activation and been FDA-approved for use in percutaneous coronary angioplasty. 

Independent molecular modeling studies of HIV-1 gpl20 inhibitors have appeared. The synthetic bivalent inhibitor 17, constructed from two molecules of 8 tethered at the C-4 positions, was reported [75]. However, the potency of this compound was not significantly enhanced when compared to 8 although it was more potent than a monomer core with the linker attached. 

The distance of 28 A between the pi-pi and pV-pT active sites corresponds almost exactly to a nonapeptide in extended conformation. The X-ray structure of 20S proteasome containing the ThrlAla mutant of the p subunit revealed the binding mode of the non-processed propeptide to the substrate binding cleft up to the adjacent p2 active site (Fig. 2.4) [63]. Correspondingly, this propeptide sequence was used in a first approach to design bivalent inhibitors bearing a glutamic acid aldehyde on the C-terminal position to address in a more selective maimer the p active site... 

To allow for an access of two anchor groups to two identical or different active sites from the non-primed S subsites, the crystal structure of Ac-Leu-Leu-Nle-H bound to pS and pS of the yeast 20S proteasome was used as a template [34]. The entry of substrates into the proteolytic chamber is restricted by the bottle-neck of the a ring, which recruits from outside only fully unfolded linear polypeptides for digestion. This fact significantly restricts the choice of spacers for bivalent inhibitor constructs. Such a spacer should mimic as much as possible the unstructured polypeptide chain of an unfolded protein, and reach a length of about 50 A. Peptides of appropriate size are known to be rapidly degraded by the yeast proteasome, and thus linear polyoxyethylene (PEG) chains were selected as mimic of random-coiled polypeptide chains [37, 64], since this polymer is known to be highly solvated and... 

Homo- and Heterobivalent Inhibitors of the Yeast 20S Proteasome 401 Table 2.2 Inhibition of 20S proteasome by mono- and bivalent inhibitors (/C50, tM)... 

Well-defined electron density maps were obtained for the tripeptide -Leu-Leu-Nle-H head groups by X-ray analysis of the 20S proteasome complexed with the bivalent inhibitor 5, whereas the PEG spacer could not be identified, thus confirming degrees of (Eig. 2.5). This flexibility allows the head groups to reach the Thrl residues from the S subsites and thus concomitant formation of the hemiacetal bonds at two active sites is achieved. 

Since the tripeptide moieties of the inhibitor 5 were identified in all six active sites, as in the case of the acetylated tripeptide aldehyde, in the absence of substrate and at the high concentration of inhibitor used for the soaking experiments, the pi, and fS active sites are indeed insufficiently selective to discriminate the C-terminal norleucinal as the PI residue. Conversely, the bivalent inhibitor 8 containing the tripeptide aldehyde -Arg-Val-Arg-H was detected only in the two tryp-sin-like and fl active sites, despite the high concentration used. This observation confirms a significant degree of selectivity of this bivalent ligand for the trypsin-like active sites. 

Table 2.5 Inhibition of human y -tryptase with y -cyclodextrin-based monovalent and bivalent inhibitors...

Fig. 2.11 X-ray structure of the two subunits A (green) and D (yellow) of the tetra-meric / -tryptase complexed with the bivalent inhibitor 16.

A method for the design of inhibitors for such proteases was published in Proceedings of the National Academy of Sciences of the USA by another colleague from the Max-Planck-Institute. Professor Luis Moro-der, who is also Professor of Biochemistry and Biotechnology and the Technical University Munich, presents the principle of polyvalency by structure-based design of mono- and bivalent inhibitors for tryptase, proteasome and serine proteases, e.g., FXa. 

As a proof of principle, the authors synthesized by click chemistry the triazole-linked bivalent inhibitor syn-4 with site-specific ligands 2 and 3 as building blocks (Scheme 15.3) [27]. 

GammUl RB, Bell FP, Bell LT, Bisaha SN, Wilson GJ (1990) Antiatherosclerotic agents. A structurally novel bivalent inhibitor of AcylCoA cholesterol O-acyltransferase with systemic activity. J Med Chem 33 2685-2687... 

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