Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1994-01-11
2002-09-10
Marschel, Ardin H. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C435S006120, C436S501000, C514S002600, C514S04400A, C536S022100, C530S323000, C530S332000, C530S333000
Reexamination Certificate
active
06448373
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to alkylene glycol monomeric units and to oligomers constructed from these units. The oligomers can be synthesized to have either random or predefined sequences of monomeric units and can be joined via phosphate linkages, including phosphorothioate and phosphodiester phosphate linkages. Each of the monomeric units can include a chemical moiety thereon for binding of the oligomeric structures to proteins, nucleic acid, and other biological targets. In preferred embodiments, the compounds of the invention act as inhibitors of enzymes such as phospholipase A
2
and are used for the treatment of inflammatory diseases including atopic dermatitis and inflammatory bowel disease.
BACKGROUND OF THE INVENTION
Phospholipases A
2
(PLA
2
) are a family of enzymes that hydrolyze the sn-2 ester linkage of membrane phospholipids resulting in release of a free fatty acid and a lysophospholipid (see, Dennis, E. A., The Enzymes, Vol. 16, pp. 307-353, Boyer, P. D., ed., Academic Press, New York, 1983). Elevated levels of type II PLA
2
are correlated with a number of human inflammatory diseases. The PLA
2
-catalyzed reaction is the rate-limiting step in the release of a number of pro-inflammatory mediators. Arachidonic acid, a fatty acid commonly linked at the sn-2 position, serves as a precursor to leukotrienes, prostaglandins, lipoxins and thromboxanes. The lysophospholipid can be a precursor to platelet-activating factor. PLA
2
is regulated by pro-inflammatory cytokines and, thus, occupies a central position in the inflammatory cascade (see, e.g., Dennis, ibid.; Glaser, et al.,
TiPs Reviews
1992, 14, 92; and Pruzanski, et al.,
Inflammation
1992, 16, 451).
All mammalian tissues evaluated thus far have exhibited PLA
2
activity. At least three different types of PLA
2
are found in humans: pancreatic (type I), synovial fluid (type II) and cytosolic. Studies suggest that additional isoenzymes exist. Type I and type II, the secreted forms of PLA
2
, share strong similarity with phospholipases isolated from the venom of snakes. The PLA
2
enzymes are important for normal functions including digestion, cellular membrane remodeling and repair, and in mediation of the inflammatory response. Both cytosolic and type II enzymes are of interest as therapeutic targets. Increased levels of the type II PLA
2
are correlated with a variety of inflammatory disorders including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease and septic shock, suggesting that inhibitors of this enzyme would have therapeutic utility. Additional support for a role of PLA
2
in promoting the pathophysiology observed in certain chronic inflammatory disorders was the observation that injection of type II PLA
2
into the footpad of rats (Vishwanath, et al.,
Inflammation
1988, 12, 549) or into the articular space of rabbits (Bomalaski, et al.,
J. Immunol
. 1991, 146, 3904) produced an inflammatory response. When the protein was denatured before injection, no inflammatory response was produced.
The type II PLA
2
enzyme from synovial fluid is a relatively small molecule (about 14 kD) and can be distinguished from type I enzymes (e.g., pancreatic) by the sequence and pattern of its disulfide bonds. Both types of enzymes require calcium for activity. The crystal structures of secreted PLA
2
enzymes from venom and pancreatic PLA
2
, with and without inhibitors, have been reported (Scott, et al.,
Science
1990, 250, 1541). Recently, the crystal structure of PLA
2
from human synovial fluid has been solved (Wery, et al.,
Nature
1991, 352, 79). The structures clarify the role of calcium and amino acid residues in catalysis. The calcium acts as a Lewis acid to activate the scissile ester carbonyl and bind the lipid, and a His-Asp side chain dyad acts as general base catalyst to activate a water molecule nucleophile. This is consistent with the absence of any acyl enzyme intermediates, and is also comparable to the catalytic mechanism of serine proteases. The catalytic residues and the calcium ion are at the end of a deep cleft (ca. 14 Å) in the enzyme. The walls of this cleft contact the hydrocarbon portion of the phospholipid and are composed of hydrophobic and aromatic residues. The positively-charged amino-terminal helix is situated above the opening of the hydrophobic cleft. Several lines of evidence suggest that the N-terminal portion is the interfacial binding site. (see, e.g., Achari, et al.,
Cold Spring Harbor Symp. Quant. Biol
. 1987, 52, 441; Cho, et al.,
J. Biol. Chem
. 1988, 263, 11237; Yang, et al.,
Biochem. J
. 1989, 262, 855; and Noel, et al.,
J. Am. Chem. Soc
. 1990, 112, 3704).
Much work has been reported in recent years on the study of the mechanism and properties of PLA
2
-catalyzed hydrolysis of phospholipids. In in vitro assays, PLA
2
displays a lag phase during which the enzyme adsorbs to the substrate bilayer and a process called interfacial activation occurs. This activation may involve desolvation of the enzyme/lipid interface or a change in the physical state of the lipid around the cleft opening. The evidence favoring this hypothesis comes from studies revealing that rapid changes in PLA
2
activity occur concurrently with changes in the fluorescence of a membrane probe (Burack, et al.,
Biochemistry
1993, 32, 583). This suggests that lipid rearrangement is occurring during the interfacial activation process. PLA
2
activity is maximal around the melting temperature of the lipid, where regions of gel and liquid-crystalline lipid coexist. This is also consistent with the sensitivity of PLA
2
activity to temperature and to the composition of the substrate, both of which can lead to structurally distinct lipid arrangements separated by a boundary region. Fluorescence microscopy was used to simultaneously identify the physical state of the lipid and the position of the enzyme during catalysis (Grainger, et al.,
FEBS Lett
. 1989, 252, 73). These studies clearly show that PLA
2
binds exclusively at the boundary region between liquid and solid phase lipid.
While the hydrolysis of the secondary ester bond of 1,2-diacylglycerophospholipids catalyzed by the enzyme is relatively simple, the mechanistic and kinetic picture is clouded by the complexity of the enzyme-substrate interaction. A remarkable characteristic of PLA
2
is that maximal catalytic activity is observed on substrate that is aggregated (i.e., phospholipid above its critical micelle concentration), while low levels of activity are observed on monomeric substrate. As a result, competitive inhibitors of PLA
2
either have a high affinity for the active site of the enzyme before it binds to the substrate bilayer or partition into the membrane and compete for the active site with the phospholipid substrate. Although a number of inhibitors appear to show promising inhibition of PLA
2
in biochemical assays (see, e.g., Yuan, et al.,
J. Am. Chem. Soc
. 1987, 109, 8071; Lombardo, et al.,
J. Biol. Chem
. 1985, 260, 7234; Washburn, et al.,
J. Biol. Chem
. 1991, 266, 5042; Campbell, et al.,
J. Chem. Soc., Chem. Commun
. 1988, 1560; and Davidson, et al.,
Biochem. Biophys. Res. Commun
. 1986, 137, 587), reports describing in vivo activity are limited (see, e.g., Miyake, et al.,
J. Pharmacol. Exp. Ther
. 1992, 263, 1302).
Traditional structure activity relationship type drug discovery gives unambiguous products but yet requires the preparation of numerous individual test candidates. The preparation of each structure requires significant amounts of time and resources. Another drug discovery approach, de novo design of active compounds based on high resolution enzyme structures, generally has not been successful. Yet another approach involves screening complex fermentation broths and plant extracts for a desired biological activity. The advantage of screening mixtures from biological sources is that a large number of compounds can be screened simultaneously, in some cases leading to the discovery of novel and complex natural products with activity that could not have been predict
Acevedo Oscar L.
Cook Phillip D.
Davis Peter W.
Ecker David J.
Herbert Normand
ISIS Pharmaceuticals Inc.
Marschel Ardin H.
Woodcock & Washburn LLP
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