In vitro characterization of MOD-5014, a novel long-acting carboxy-terminal peptide (CTP)-modified activated FVII.

INTRODUCTION: Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. AIM: The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. METHODS: The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. RESULTS: Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. CONCLUSION: The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.

Folding and activity of recombinant human procollagen C-proteinase enhancer.

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

Interaction of myosin subfragment 1 with two non-nucleotide ATP analogues.

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40 degreesC and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeFx), aluminum fluoride (AlF4-), or vanadate (Vi) phosphate analogues, while the S1.PhAEDP complex is stable in the presence BeFx but much less stable with AlF4- and Vi. These results indicate that the S1.PhAEDP.Pi state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.