Peptide antagonists of zonulin and methods for use of the same

Details Peptide antagonists of zonulin and methods for use of the same Peptide antagonists of zonulin and methods for use of the same

1998-08-03

2002-10-01

Davenport, Avis M. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

8 to 10 amino acid residues in defined sequence

C530S300000, C530S324000, C530S329000, C530S350000, C514S002600, C514S012200, C514S016700, C514S015800, C514S021800, C424S185100

Reexamination Certificate

active

06458925

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to peptide antagonists of zonulin, as well as methods for the use of the same. Said peptide antagonists bind to the zonula occludens receptor, yet do not physiologically modulate the opening of mammalian tight junctions.
BACKGROUND OF THE INVENTION
I. Function and Regulation of Intestinal Tight Junctions
The tight junctions (“tj”) or zonula occludens (hereinafter “ZO”) are one of the hallmarks of absorptive and secretory epithelia (Madara,
J. Clin. Invest.,
83:1089-1094 (1989); and Madara,
Textbook of Secretory Diarrhea
Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). As a barrier between apical and basolateral compartments, they selectively regulate the passive diffusion of ions and water-soluble solutes through the paracellular pathway (Gumbiner,
Am. J. Physiol.,
253 (
Cell Physiol.
22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond,
Physiologist,
20:10-18 (1977)).
Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of enterocyte plasma membranes are relatively high (Madara, supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al,
J. Cell Biol.,
101:2124-2133 (1985)).
There is abundant evidence that ZO, once regarded as static structures, are in fact dynamic and readily adapt to a variety of developmental (Magnuson et al,
Dev. Biol.,
67:214-224 (1978); Revel et al,
Cold Spring Harbor Symp. Quant. Biol.,
40:443-455 (1976); and Schneeberger et al,
J. Cell Sci.,
32:307-324 (1978)), physiological (Gilula et al,
Dev. Biol.,
50:142-168 (1976); Madara et al,
J. Membr. Biol.,
100:149-164 (1987); Mazariegos et al,
J. Cell Biol.,
98:1865-1877 (1984); and Sardet et al,
J. Cell Biol.,
80:96-117 (1979)), and pathological (Milks et al,
J. Cell Biol.,
103:2729-2738 (1986); Nash et al,
Lab. Invest.,
59:531-537 (1988); and Shasby et al,
Am. J. Physiol.,
255(
Cell Physiol.,
24):C781-C788 (1988)) circumstances. The regulatory mechanisms that underlie this adaptation are still not completely understood. However, it is clear that, in the presence of Ca
2+
, assembly of the ZO is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of ZO elements, the composition of which has been only partially characterized (Diamond, Physiologist, 20:10-18 (1977)). A candidate for the transmembrane protein strands, occludin, has recently been identified (Furuse et al,
J. Membr. Biol.,
87:141-150 (1985)).
Six proteins have been identified in a cytoplasmic submembranous plaque underlying membrane contacts, but their function remains to be established (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al,
Proc. Natl. Acad. Sci., USA,
88:3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). Most immunoelectron microscopic studies have localized ZO-1 to precisely beneath membrane contacts (Stevenson et al,
Molec. Cell Biochem.,
83:129-145 (1988)). Two other proteins, cingulin (Citi et al,
Nature
(London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et al,
J. Cell Biol.,
120:477-483 (1993)) are localized further from the membrane and have not yet been cloned. Rab 13, a small GTP binding protein has also recently been localized to the junction region (Zahraoui et al,
J. Cell Biol.,
124:101-115 (1994)). Other small GTP-binding proteins are known to regulate the cortical cytoskeleton, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al,
Cell,
70:389-399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al,
Cell,
70:401-410 (1992)). Based on the analogy with the known functions of plaque proteins in the better characterized cell junctions, focal contacts (Guan et al,
Nature,
358:690-692 (1992)), and adherens junctions (Tsukita et al,
J. Cell Biol.,
123:1049-1053 (1993)), it has been hypothesize that tj-associated plaque proteins are involved in transducing signals in both directions across the cell membrane, and in regulating links to the cortical actin cytoskeleton.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the ZO must be capable of rapid and coordinated responses that require the presence of a complex regulatory system. The precise characterization of the mechanisms involved in the assembly and regulation of the ZO is an area of current active investigation.
There is now a body of evidence that tj structural and functional linkages exist between the actin cytoskeleton and the tj complex of absorptive cells (Gumbiner et al, supra; Madara et al, supra; and Drenchahn et al,
J. Cell Biol.,
107:1037-1048 (1988)). The actin cytoskeleton is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actin-binding proteins. An example of how the state of phosphorylation of an actin-binding protein might regulate cytoskeletal linking to the cell plasma membrane is the myristoylated alanine-rich C kinase substrate (hereinafter “MARCKS”). MARCKS is a specific protein kinase C (hereinafter “PKC”) substrate that is associated with the cytoplasmic face of the plasma membrane (Aderem,
Elsevier Sci. Pub.
(UK), pages 438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks to the membrane actin. Thus, it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid (Hartwig et al,
Nature,
356:618-622 (1992)). Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al,
J. Exp. Med.,
172:1211-1215 (1990); and Thelen et al,
Nature,
351:320-322 (1991)). The actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra). These data suggest that the F-actin network may be rearranged by a PKC-dependent phosphorylation process that involves actin-binding proteins (MARCKS being one of them).
A variety of intracellular mediators have been shown to alter tj function and/or structure. Tight junctions of amphibian gallbladder (Duffey et al,
Nature,
204:451-452 (1981)), and both goldfish (Bakker et al,
Am. J. Physiol.,
246:G213-G217 (1984)) and flounder (Krasney et al,
Fed. Proc.,
42:1100 (1983)) intestine, display enhanced resistance to passive ion flow as intracellular cAMP is elevated. Also, exposure of amphibian gallbladder to Ca
2+
ionophore appears to enhance tj resistance, and induce alterations in tj structure (Palant et al,
Am. J. Physiol.,
245:C203-C212 (1983)). Further, activation of PKC by phorbol esters increases paracellular permeability both in kidney (Ellis et al,
C. Am. J. Physiol.,
263 (
Renal Fluid Electrolyte Physiol.
32):F293-F300 (1992)), and intestinal (Stenson et al,
C. Am. J. Physiol.,
265(
Gastrointest. Liver Physiol.,
28):G955-G962 (1993)) epithelial cell lines.
II. The Blood-Brain Barrier
The blood-brain barrier (BBB) is an extremely thin membranous barrier that is highly resistant to solute free diffusion, and separates blood and the brain. In molecular dimensions, the movement of drugs or solute through this membrane is essentially nil, unless the compound has access to one of several specialized enzyme-like transport mechanisms that are embebbed within the BBB membrane. The BBB is composed of multiple cells rather than a single layer of epithelial cells of the four different types of cells that compose the BBB (endothelial cells, perycites, astrocytes, and neurons) the endothelial cel

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