Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against humoral hypercalcemia of malignancy.

A humanized monoclonal antibody against parathyroid hormone-related protein (PTHrP) was generated from the mouse monoclonal antibody raised against the peptide corresponding to the N-terminal 34 amino acids of the human PTHrP [(PTHrP(1-34)]. The humanized antibody interacted with the PTHrP(1-34) with a kD value of 1.90 x 10(-10) M, and the epitope resides between the amino acids 20 and 30 of the PTHrP. PTHrP(1-34) significantly increased the intracellular cAMP levels in the rat osteosarcoma cells that expressed PTHR1, and the 5 microg/mL or higher concentrations of the humanized antibody almost completely blocked the PTHrP-induced cAMP production even in the presence of 2 microg/mL PTHrP(1-34), demonstrating its ability to fully neutralize PTHrP function. There was no significant difference in the potency of the mouse, chimera, or the humanized antibodies to suppress the PTHrP-induced increase in the intracellular cAMP in ROS cells. Furthermore, at the same doses, the administration of the chimera or the humanized antibody was equally effective in reducing the blood ionized calcium levels of hypercalcemic mice bearing the PAN-7-JCK human pancreatic cancer xenograft or the LC-6-JCK human lung cancer xenograft that secreted PTHrP. Thus, humanized anti-PTHrP may be useful for the treatment of the humoral hypercalcemia of malignancy in humans.

A novel therapeutic approach for thrombocytopenia by minibody agonist of the thrombopoietin receptor.

Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis. These agonist antibodies, however, should be further improved to exert more potent biologic activities and appropriate serum half-life depending upon the disease indications. Here, we report that IgG antibodies against the thrombopoietin receptor (Mpl), which have an absence or very weak agonist activity, can be engineered to be agonist minibodies, which include diabody or sc(Fv)2 as potent as natural ligand. Through this technological development, minibodies have been successfully constructed to bind and activate 2 types of dysfunctional mutant Mpls that cause congenital amegakaryocytic thrombocytopenia (CAMT). This drastic conversion of biologic activities by designing minibodies can be widely applicable to generate agonist minibodies for clinical application, which will constitute a new paradigm in antibody-based therapeutics.

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.

Structure-based design of P3 moieties in the peptide mimetic factor VIIa inhibitor.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein.

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor.

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.