No abstract

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 Å (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI Coagulation factor XI (FXI) 4 is a unique homodimeric coagulation protein, present in human plasma at a concentration of ϳ30 nM, that is essential for normal hemostasis, as evidenced by the fact that FXI deficiency is associated with a hemorrhagic disorder (1). The zymogen FXI can bind to receptors (2-4), consisting of the glycoprotein Ib-IX-V complex (5) on the plasma membranes of activated human platelets, where it can be incorporated into platelet membrane microdomains (i.e. lipid rafts) (6) for efficient activation by thrombin or by FXIIa (7-10). The resulting enzyme, FXIa, can then activate the vitamin K-dependent protein, FIX, to initiate the consolidation phase of blood coagulation (1).A variety of important control mechanisms exist for regulating the activity of coagulation proteases in plasma. Several members of the serpin family have been proposed as physiological regulators of FXIa activity in plasma, including C1 inhibitor (11, 12), ␣-1-protease inhibitor (13, 14), antithrombin III (15), ␣-2-antiplasmin (16), and protease nexin 1 (17). However, a platelet secretory protein and member of the class of Kunitz-type inhibitors, protease nexin 2 (PN2), has recently been shown to be a much more potent and physiologically relevant FXIa inhibitor based on detailed kinetics studies (18 -23). PN2 is a ϳ120-kDa isoform of the Alzheimer ␤-amyloid protein precursor (APP) that contains a Kunitz-type serine protease inhibitor (PN2KPI) domain (22). Platelets are an important source of several isoforms of APP, including the APP 751 isoform of PN2 (23, 24). Full-length APP is membraneassociated (25) and is processed by proteases in platelets (26, 27). Upon platelet activation by physiological stimulators, PN2 is secreted from ␣-granules into plasma and inhibits FXIa (22-24), suggesting a role for this protein in blood coagulation. PN2 is a slow, tight binding inhibitor of FXIa with K i of ϳ300 -500 pM (18,20,22). The KPI domain of PN2 is 57 amino acids in length (Glu 289 -Ile 345 in the 751-amino acid isoform of PN2) and is known to contain the entire FXIa inhibitory function of PN2 (19 -21, 28). Similarly, all of the information r...

Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370 -607 (rhFXI 370 -607 ). Here we report the first crystal structure of rhFXI 370 -607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 Å resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2 in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI 370 -607 -ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.

The structure-based design, chemical synthesis, and biological evaluation of various peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from 60 to 280 000 M-1 s-1 and antiviral EC50's which approach 0.15 microM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] = 800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 = 0.056 microM). A 1.9 A crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.