Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-06-02
2002-12-03
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S440000, C435S471000, C435S218000, C435S325000, C435S252300, C536S023100, C536S023500
Reexamination Certificate
active
06489143
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention in the field of biochemistry and medicine relates to compositions comprising mutant proteins of plasminogen activator inhibitor-type 1 (PAI-1) which have the capacity to inhibit the enzyme elastase and to inhibit vitronectin (Vn)-dependent migration of cells. This invention also relates to uses of these proteins for the treatment of diseases and disorders associated with elastase activity or in which migration and migration-driven proliferation of cells have pathophysiologic consequences.
2. Description of the Background Art
1. PLASMINOGEN ACTIVATORS
Plasminogen activators (PAs) are specific serine proteinases that activate the proenzyme plasminogen, by cleavage of a single Arg-Val peptide bond, to the enzyme plasmin (Saksela O,
Biochim Biophys Acta
(1985) 823:35-65). Two plasminogen activators are found in mammals, tissue-type PA (tPA) and urokinase-type PA (uPA) (Saksela O et al,
Annu Rev Cell Biol
(1988) 4:93-126). These enzymes are thought to influence critically many biological processes, including vascular fibrinolysis (Bachmann E,
Thromb Haemost
(1987) 10:227-265), ovulation (Hsuch A J W et al, In: Haseltine FP et al, eds,
Meiotic Inhibition: Molecular Control of Meiosis
New York: Liss 1988:227-258), inflammation (Pollanen J et al.,
Adv Cancer Res
(1991) 57:273-328), tumor metastasis (Dano K et al.,
Adv Cancer Res
(1985) 44:139-266), angiogenesis (Moscatelli D et al.,
Biochim Biophys Acta
(1988) 948:67-85), and tissue remodeling (Saksela, supra).
The regulation of PAs is a complex process controlled on many levels. The synthesis and release of PAs are governed by various hormones, growth factors, and cytokines (Saksela, supra; Dano et al., supra). Following secretion, PA activity can be regulated both positively and negatively by a number of specific protein-protein interactions. Activity can be enhanced or concentrated by interactions with fibrin (Hoylaerts M et al.,
J Biol Chem
(1982) 257:2912-2919), the uPA receptor (uPAR) (Ellis V et al.,
Semin Thromb Hemost
(1991) 17:194-200), the tPA receptor (tPAR) (Hajjar K A et al,
J Biol Chem
(1990) 265:2908-2916), or the plasminogen receptor (Plow E F et al.,
Thromb Haemost
(1991) 66:32-36).
PA activity can be downregulated by specific PA inhibitors (PAIs) (Lawrence, D. A et al., In:
Molecular Biology of Thrombosis and Hemostasis
, Roberts, H. R. et al., (Eds.), Marcel Dekker Inc., New York, chapter 25, pp. 517-543 (1995)). In addition, PA activity is dependent on its location or microenvironment and may be different in solution (e.g., circulating blood) as compared to a solid-phase (e.g., on a cell surface or in the extracellular matrix (ECM)). The overall activity of the PA system is determined by the interactions among these various elements and the balance between the opposing activities of enzymes and inhibitors.
The PAIs have become recognized as critical regulators of the PA system. The identification of an efficient inhibitor of tPA in endothelial cells (ECs) was first reported in 1983 (Loskutoff D J et al.,
Proc Natl Acad Sci USA
(1983) 80:2956-2960). Four kinetically relevant PAIs are currently recognized: PAI type 1 (PAI-1), initially described as the endothelial cell PAI; PAI type 2 (PAI-2), also referred to as placental PAI, PAI type 3 (PAI-3), also. known as activated protein C (APC) inhibitor and proteinase nexin 1 (PN-1), also called glia-derived neurite-promoting factor. The present invention is directed in particular to PAI-1.
2. OTHER SERINE PROTEINASES
Elastase is a serine proteinase released by activated neutrophils and macrophages and monocytes. During inflammatory responses, neutrophils are activated and release elastase leading to tissue destruction through proteolysis. In the lung, elastase degrades elastic tissues and leads to emphysema. Elastase is also a compounding factor in cystic fibrosis (CF) and in both adult and infant acute respiratory distress syndrome (ARDS). Elastase has also been implicated in TNF-mediated inflammation (Massague, J. et al.,
Annu. Rev. Biochem
. 62:515-541 (1993) and HIV infection (Bristow, C. L. et al.,
International Immunol
. 7:239-249 (1995)).
Elastase has a broader spectrum of reactivity than plasminogen activators each of which acts preferentially on a precursor substrate to activate it.
The natural defense to elastase is a protein called &agr;
1
anti-trypsin (&agr;
1
AT) or &agr;
1
proteinase inhibitor ((&agr;
1
PI). Patients who are deficient in &agr;
1
AT are prone to emphysema, especially smokers. Furthermore, smoking provokes inflammation. In such &agr;
1
AT deficiencies, the enzyme is present (CRM
+
) but is functionally impaired. In addition, even in individuals with normal enzyme, smoking directly inactivates &agr;
1
AT. Therefore, an improved inhibitor of elastase would be highly desirable for the prevention of emphysema in susceptible subjects or for reversal of the pathophysiological process leading to this an other related diseases.
3. SERPINS
The major PAIs belong to the serine proteinase inhibitor (serpin) gene superfamily which includes many proteinase inhibitors in blood as well as other proteins with unrelated or unknown function (Huber R et al.,
Biochemistry
(1989) 28:8951-8966). The serpins share a common tertiary structure and have evolved from a common ancestor. Serpins regulate many processes including coagulation, fibrinolysis, complement activation, ovulation, angiogenesis, inflammation, neoplasia, viral pathogenesis and allergic reactivity.
Current models of serpin structure are based on x-ray crystallographic studies of one member of the family, &agr;
1
AT (reviewed in Huber et al., supra). An interesting feature of the structure of a modified form of &agr;
1
AT, cleaved in its reactive center (Loebermann H et al.,
J Mol Biol
(1984) 177:531-557), is that the two amino acid residues that normally constitute the reactive center (Met-Ser bond), are found on opposite ends of the molecule, separated by almost 70 Å. This is shown for PAI-1 in FIG.
2
and can be compared to the active structure modeled in FIG.
1
. Relaxation of a strained configuration takes place upon cleavage of the reactive-center peptide bond, rather than a major rearrangement of the inhibitor structure. In this model, the reactive center is part of an exposed loop, also called the strained loop. Upon cleavage, this loop moves or “snaps back,” becoming one of several central strands in a major &bgr; sheet structure. This transformation is accompanied by a large increase in thermal stability, presumably as a result of the reconstitution of the six-stranded &bgr; sheet A.
Synthetic peptides homologous to the reactive-center loops of serpins, when added in trans, incorporate into their respective molecules, presumably as a central strand of the major &bgr; sheet structure and increase the thermal stability of the molecule like that observed after cleavage at the reactive center. This structural change converts the serpin from an inhibitor to a substrate for its target proteinase (Carrell R W et al.,
Nature
(1991) 353:576-578, Bjork I et al.,
J Biol Chem
(1992) 267:1976-1982).
Serpins act as suicide inhibitors, reacting only once with their target proteinase to form a sodium dodecyl sulfate (SDS)-stable complex. These complexes can dissociate to yield free active enzyme together with a cleaved inhibitor similar to that seen in the &agr;
1
AT crystal structure (Carrell R W et al., In: Barrett A J, et al. eds.,
Proteinase Inhibitors
. Amsterdam: Elsevier Science Publishers 1986:403-420) and modeled in
FIGS. 1 and 2
for PAI-1.
Serpins interact with their target proteinase by providing a “bait” amino acid residue in the reactive center which is thought to mimic the normal substrate of the enzyme and to associate via its side-chain atoms with the specificity crevice, or S1 site, of the enzyme (Huber et al., supra.; Carrell et al., supra; Shubeita H E et al.,
J Biol Chem
(1990) 265:18379-18385; York J D et al.,
J. Biol Chem
(1991) 266:8495-8500; Sherman P M et al.
Lawrence Daniel A.
Stefansson Steingrimur P.
American National Red Cross
Foley & Lardner
Low Christopher S. F.
Schnizer Holly
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