Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1999-11-29
2004-05-11
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S184000
Reexamination Certificate
active
06733988
ABSTRACT:
BACKGROUND OF THE INVENTION
Fibrosis and Scarring
Normal wound healing involves the formation of scars and fibrous tissues. Their structures consist largely of collagen fibrils. Although collagen is required in wound repair, collagen often accumulates in excessive amounts and impairs the normal function of the affected tissue. Such excessive amounts of collagen become an important event in scarring of the skin following burns or other traumatic injury, as well as in fibrosis of the liver, lungs and other organs following injury. Because of the central role of collagen in the pathogenesis of fibrosis, there has been considerable interest in agents capable of inhibiting collagen accumulation in fibrotic diseases. Potential target sites for inhibiting collagen accumulation include transcription of the genes, translation of the mRNAs, and some of the post-translational enzymes involved in the biosynthesis of collagen [G. C. Fuller, 1981, J. Med. Chem., 24: 651-658; M. Trojanowska et al., 1998, J. Mol. Med., 76: 266-274]. Here we will focus our attention to circumvent collagen accumulation in fibrosis by inhibiting cross-link levels. To understand the rationale behind attempts to circumvent collagen accumulation by inhibiting cross-link levels it is necessary to get an insight into the way cross-linking of collagen molecules occurs.
Hydroxylation and Cross-linking of Collagen: Some Basic Concepts
Biosynthesis of collagen is a multistep process, resulting in extensive modification of the molecule (FIG.
1
). The different modifications of the molecule are catalyzed by various enzymes with an intra- or extracellular localization [K. Kadler, 1994, Protein Profile, 1: 515-638]. One of the steps in the biosynthesis of collagen is hydroxylation of certain proline residues in the triple helix by prolyl 4-hydroxylase (EC 1.14.11.2) and prolyl 3-hydroxylase (EC 1.14.11.7) as well as hydroxylation of certain lysine residues in the triple helix and telopeptides by lysyl hydroxylase (EC 1.14.11.4). In a next step, hydroxylysine residues in the triple helix can be subjected to glycosylation by glycosyl transferases. Hydroxylation of proline and lysine are essential for a proper functioning of collagen. There is hardly any variation in the level of prolyl hydroxylation within a specific collagen type. In fact, the hydroxylation level of prolyl residues of each fibrillar collagen type is close to a maximum; underhydroxylation of proline residues results in a non-functional molecule with a weakened triple helix that can easily be degraded. In contrast, there are large variations in lysyl hydroxylation within the same collagen type (e.g. type I collagen) between the different tissues. The functional significance of this variation is unknown, but under- and overhydroxylation of lysine residues is associated with several connective tissue disorders, such as Ehlers-Danlos type VI syndrome [A. Ihme et al., 1984, J. Invest. Dermatol., 83: 161-165] and osteogenesis imperfecta [W. G. Cole, 1994, Bone Miner. Res., 8: 167-2041].
Once the collagen molecule is secreted, the propeptides are cleaved off by procollagen N-peptidase (EC 3.4.24.14) and procollagen C-peptidase (EC 3.4.24.19), resulting in a mature molecule consisting of a triple helix with a short telopeptide at both termini. The mature molecules aggregate spontaneously into microfibrils. Further stabilization of the molecules occurs by means of cross-links. Cross-linking is initiated by conversion of specific lysine or hydroxylysine residues of the telopeptides into the aldehydes allysine and hydroxyallysine, respectively, by the enzyme lysyl oxidase (EC 1.4.3.13) [H. M. Kagan, 1994, Path. Res. Pract., 190: 910-919]. The aldehydes subsequently react with lysine or hydroxylysine residues of the triple helix to give characteristic difunctional cross-links. These cross-links eventually mature into tri- or tetra-functional cross-links [D. R. Eyre, 1987, Meth. Enzymol., 144: 115-139; A. J. Bailey et al., 1998, Mech. Ageing Developm., 106: 1-56]. Two related routes for the formation of cross-links have been described, one based on allysine from the telopeptides, the other based on the hydroxyallysine of the telopeptides. Each route results in chemically distinct cross-links. For the nomenclature and origin of some of the most common cross-links we refer to FIG.
2
.
Hydroxylation of lysine in the triple helix of collagen occurs exclusively on lysine present in the helical amino acid sequence Gly-X-Lys-Gly; a lysine in the X position is not hydroxylated [K. Kadler, 1994, Protein Profile, 1: 515-638]. The hydroxylated lysine in the telopeptides is embedded in an entirely different amino acid sequence; in view thereof, the existence of another enzyme, whose substrate would be the non-helical telopeptide region, has occasionally been proposed [J. E. Gerriets et al., 1996, Biochim. Biophys. Acta, 1316: 121-131; J. E. Gerriets et al., 1993, J. Biol. Chem., 286: 25553-25560; L. Knott et al, 1997, Biochem. J., 322: 535-542; M. J. Barnes et al., 1974, Biochem. J., 139: 461-468; P. M. Royce & M. J. Barnes, 1985, Biochem. J., 230: 475-480]. However, the circumstantial evidence that an enzyme exists that specifically hydroxylates the telopeptide lysine has mostly been ignored and/or questioned. We will show later on that hydroxylation of telopeptide lysine residues is indeed under separate control from that of helical lysine residues, i.e. hydroxylation of telopeptide lysine and helical lysine is independently and specifically controlled.
Suppression of Cross-link Formation as an Antifibrotic Treatment
Essentially all stages of collagen biosynthesis have been used as targets for the pharmacological control of collagen accumulation in fibrosis, the most important ones being the inhibition of prolyl hydroxylation (e.g. by incorporation of prolyl analogues into the triple helix instead of prolyl) [H. M. Hanauske-Abel, 1991, J. Hepatol., 13 Suppl. 3: S8-S16] and cross-linking. Here we will focus our attention to the inhibition of cross-link formation as a tool to decrease collagen accumulation in fibrotic tissues. Collagen molecules that are not cross-linked are more easily degraded by proteinases [C. A. Vater et al., 1979, Biochem. J., 181: 639-645], thus facilitating the removal of collagen. Two routes have been described to achieve lower cross-link levels: compounds that are able to inhibit lysyl oxidase (so-called lathyrogens, an example being &bgr;-aminopropionitrile) and compounds that essentially block aldehydes (such as penicillamine, cysteine, and other analogues) [M. E. Nimni, 1983, Sem. Arthr. Rheum., 13: 1-86]. Both routes result in a decrease of both allysine and hydroxyallysine derived cross-links. The consequence of inhibition of lysyl oxidase is that aldehydes cannot be formed in the telopeptides; as a result, cross-link formation does not take place (FIG.
1
-
2
). Blocking the aldehydes also effectively inhibits cross-link formation, as blocked aldehydes cannot participate in the formation of intramolecular and intermolecular cross-links. Although lathyrogens and compounds that react with aldehydes are potent antifibrotic agents, concentrations needed to display antifibrotic effects have toxic effects, thus limiting its clinical use as antifibrotic drugs. An additional drawback is, that unphysiological collagen is formed, showing inferior biomechanical properties due to the lower cross-link levels. This unphysiological collagen is not only formed by proliferating fibroblasts but also by the cells surrounding the injury, thus impairing the biomechanical quality of healthy tissue as well. In this invention we will describe a method that does not show this drawback: agents can be developed that selectively inhibit the formation of unwanted hydroxyallysine cross-links in collagen of the fibrotic lesions, leaving synthesis of allysine cross-links in the intact tissue and healing wound unaffected.
In yet another approach minoxidil, an agent capable
Bank Rudolf Antonius
TeKoppele Johannes Maria
Nederlandse Organisatie voor toegepast-natuurwetenschappelijk On
Norris & McLaughlin & Marcus
Patterson Jr. Charles L.
LandOfFree
Inhibition of telopeptide lysyl hydroxylase activity or... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Inhibition of telopeptide lysyl hydroxylase activity or..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inhibition of telopeptide lysyl hydroxylase activity or... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3219776