US20110130344A1

US20110130344A1 - Tight junction protein modulators and uses thereof

Info

Publication number: US20110130344A1
Application number: US13/024,187
Authority: US
Inventors: Margaret C. Nerville; Robert S. Hodges; Neal Edward Beeman
Original assignee: University of Colorado Denver
Current assignee: University of Colorado Denver
Priority date: 2007-11-19
Filing date: 2011-02-09
Publication date: 2011-06-02
Also published as: US20140045768A1; WO2009102367A2; US8563515B2; US20110263511A1; US9067968B2; WO2009102367A3

Abstract

The invention provides tight junction protein modulators, compositions comprising the same, and uses thereof. In particular, the invention provides tight junction protein modulators that modulate the second extracellular loop of tight junction proteins, such as occludin or claudin.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Continuation-In-Part Application of U.S. patent application Ser. No. 12/743,816, filed May 19, 2010, which is a U.S. national phase application of PCT Patent Application No. PCT/US08/84100, filed Nov. 19, 2008, which claims the priority benefit of U.S. Provisional Application No. 60/988,865, filed Nov. 19, 2007, all of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number DOD DAMD17-00-1-0210 awarded by the Department of Defense and grant number PO1-HD38129 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to tight junction protein modulators, compositions comprising the same, and uses thereof. In particular, the invention relates to peptide modulators of tight junction proteins that mimic or modulate the second extracellular loop of claudins or occludins.

BACKGROUND OF THE INVENTION

Organs and fluid spaces throughout the bodies of animals are lined by polarized epithelia that serve, in part, to modify the apical and basolateral fluid compartments. In order for these organs and fluid spaces to function, it is necessary that the apical and basolateral fluid compartments be kept physiologically separated, so as to maintain the modifications imparted by the epithelia. This physiological separation is maintained by the tight junction, classically known as the zonula occludens.
The tight junction is an unbroken intercellular junction formed by an anastomosing network of protein and lipid strands that apically circumscribes every luminal epithelial cell. This intercellular adhesion complex is a continuous molecular seal between all of the luminal epithelial cells and forms a selective barrier to paracellular, and thereby transepithelial, solute flux and ionic current (i.e., the gate function). The tight junction is also believed to maintain the distinct lipid and protein composition of the apical and basolateral cellular membranes (i.e., the fence function). The gate and fence functions of the tight junction thus polarize the unit epithelial cell and the epithelium itself at the same physical plane. Occludin and the claudins help to form, and are localized within, tight junction strands where they participate in homophilic and/or heterophilic interactions between adjacent cells. Occludin and the claudins are tetraspanin proteins, having intracellular N and C termini, two extracellular loops, and four transepithelial domains, and have been shown to be involved in establishing and maintaining the physiological properties of the tight junction.
In addition to physiological barrier roles, the tight junction regulates many aspects of intracellular behavior. For example, the tight junction has been shown to be involved in the cell cycle arrest attendant on contact inhibition. Tight junction disruption induces epithelial to mesenchymal transition, increases cellular motility, produces overgrowth of cultured cells, and increases tumorigenicity of cells transplanted into animals. Several tight junction proteins regulate transcription. Tight junction formation is also involved in the inter-related phenomena of development of cellular polarity and epithelial differentiation.
The physiological barrier functions as well as the regulatory activities of the tight junction must be maintained in epithelia whose cellular populations are undergoing constant turnover throughout the life of the organism. In this process individual epithelial cells are extruded apically and undergo apoptosis in a manner that does not alter the electrical resistance or tracer flux across the epithelium.
Higher levels of apoptosis induced in epithelia during experiments or during pathological states have been shown to alter barrier properties. Disruption of occludin has been shown in a number of studies to disrupt the physiological and structural properties of the tight junction. A 19 amino acid second extracellular loop sequence peptide mimic of occludin impeded recovery of tight junction structure following a short period of incubation with a calcium-free solution (the calcium switch) in T84 intestinal epithelial cells.
Isolated patches of cells throughout treated monolayers showed punctate, intracellular distributions of the tight junction proteins ZO-1, occludin, claudin-1, and JAM-A. A similar peptide was used to treat the EPH4 mammary epithelial cell line leading to punctate, intracellular, non-tight junctional distribution of occludin in isolated patches of cells throughout treated monolayers. Another similar peptide was shown to disrupt barrier function in a sertoli cell line. This same peptide shut down spermatogenesis and decreased testicular weight when injected into the testicular lumen of rats. The relationship between occludin disruption and cellular survival has not been widely studied. However, disruption of several types of cellular adhesion proteins; integrins, cadherins, and connexins has been reported to stimulate apoptosis. Interestingly, epithelial cell lines derived from the occludin knockout mouse showed decreased survival signaling and increased apoptotic rates.
Although most tumors are devoid of tight junctions, many tight junction proteins, particularly claudins 3, 4 and 7 are found at high levels in tumors of epithelial origin where their function and localization are presently unknown.
Although tight junction protein modulators are known, these are not without any problems. Therefore, there is a continuing need for other tight junction protein modulators.

SUMMARY OF THE INVENTION

Some aspects of the invention provide tight junction protein modulators, compositions comprising the same, and uses thereof. In other aspects, the invention provides peptide modulators of tight junction proteins that mimic or modulate the second extracellular loop of claudins or occludin, and methods for using the same.
In some embodiments of the invention provide a method for modulating apoptosis or abnormal growth or development of cells, such as epithelial cells, of a subject comprising administering to the subject a therapeutically effective amount of a tight junction protein modulator.
In other embodiments, the tight junction protein modulator causes internalization of the tight junction protein occludin or claudin. Still in other embodiments, the tight junction protein modulator stimulates or causes apoptosis of epithelial cells.
There are various clinical conditions associated with abnormal apoptosis, growth or development of epithelial cells including cancer, such as skin cancer, breast cancer, ovarian cancer, and metastases thereof. Thus, in some embodiments, methods of the invention are useful in treating a subject who suffers from cancer.
Yet in other embodiments, the tight junction protein modulator modulates an adhesion protein. Exemplary adhesion proteins that can be modulated by methods of the invention include occludin, claudin, junction adhesion molecule, integrins, or a combination thereof. In some embodiments, the tight junction protein modulator modulates occludin, claudin, or a combination thereof.
In other embodiments, the tight junction protein modulator comprises at least 3 consecutive amino acid sequences of the second extracellular loop sequence of occludin or claudin, or a derivative thereof. Within these embodiments, in some instances, the tight junction protein modulator comprises D-amino acid sequences or retro-inverso D-amino acid sequences of LYHY, DFYNP, or a derivative thereof. Still in other instances, the tight junction protein modulator comprises about 20 amino acid sequences or less. Yet in other instances, the tight junction protein modulator is a cyclic peptide or a linear peptide, or a derivative thereof.
In one particular embodiments, the tight junction protein modulator is of the formula:
wherein

- each of X¹and X⁴is independently cysteine, glutamic acid, aspartic acid, lysine, or ornithine;
- each of L¹and L⁴is independently the corresponding functional group of the amino acid side chain of X¹and X⁴, respectively, or —(CH₂)_m—;
- each of X²and X³is glycine;
- m is an integer from 1 to 4;
- each n is independently 0, 1 or 2;
- Z is glycine, isoleucine, alanine, leucine, lysine, methionine, cysteine, phenylalanine, threonine, tryptophan, valine, proline, serine, tyrosine, arginine, or histidine;
- F is phenylalanine, tyrosine, tryptophan, or leucine;
- Y is tyrosine, phenylalanine, tryptophan, or leucine;
- N is asparagine or glutamine; and
- P is proline.

It should be appreciated that amino acids are often identified by a single letter codes such as P for proline and C for cysteine. As shown above, however, in some instances within this disclosure, such a single code can also represent amino acids that are known to be “interchangeable” or have a similar side-chain. Thus, while F typically represents phenylalanine, it can also represent tyrosine, tryptophan, or glutamine. Thus, unless explicitly stated or the context requires otherwise it is to be understood that the single letter amino acid codes includes amino acids having a similar side-chain or are known to be interchangeable by one skilled in the art.
Within Compounds of Formula I, in some instances Z is glycine, F is phenylalanine, Y is tyrosine, and N is asparagine. In other instances, X¹and X⁴are cysteine. In such instances, often L¹and L²together is a moiety of the formula —S—S—. It should be appreciated that in such instances where L¹and L²together is a moiety of the formula —S—S—, the sulfur atoms are side chain functional groups of the corresponding cysteine of X¹and X⁴.
Other aspects of the invention provide methods for removing at least a portion of epithelia cells from a subject. Generally such methods include causing apoptosis of the epithelia cells; however, it should be appreciated that methods can also simply involve disrupting the tight junction protein to cause removal of adhesion between epithelial cells. Such methods typically comprise administering to the subject in need of such treatment a tight junction protein inhibitor. In some embodiments, the epithelia cells are in hyperplastic stages. In one particular instance, the epithelia cells are in the subject's breast. In such instance, the tight junction protein can be administered to the subject by intraductal injection.
Still other aspects of the invention provide a compound comprising 20 (typically 10, often 6) amino acid sequences or less and at least 3 consecutive amino acid sequences of the second extracellular loop sequence of occludin or claudin, or a derivative thereof. In some embodiments, the compound comprises amino acid sequences of LYHY, ZFYNP, or a derivative or a combination thereof.
Still in other embodiments, the compound comprises a cyclic peptide, D-amino acid sequences, retro-inverso D-amino acid sequences, or a combination thereof. Within these embodiments, in some instances the cyclic peptide is of the formula:
wherein

Yet other aspects of the invention provide methods for treating a clinical condition associated with abnormal apoptosis, growth or development of epithelial cells in a subject, said method comprising administering to the subject in need of such a treatment a composition a therapeutically effective amount of a tight junction protein modulator. In some embodiments, the tight junction protein modulator stimulates apoptosis of epithelial cells.
Any clinical condition associated with abnormal apoptosis, growth or development of epithelial cells can be treated by methods of the invention. In some embodiments, methods of the invention are used to treat a clinical condition that comprises skin cancer, breast cancer, ovarian cancer, or metastases thereof.
In other embodiments, the tight junction protein modulator modulates an adhesion protein. Suitable adhesion proteins include, but are not limited to, occludin, claudin, junction adhesion molecule, integrins, or a combination thereof. In some instances, the adhesion protein comprises occludin, claudin, or a combination thereof. In other instances, the tight junction protein modulator comprises at least 3 consecutive amino acid sequences of the second extracellular loop sequence of occludin or claudin, or a derivative thereof. In some cases, the amino acid comprises at least one D-amino acid, at least one retro-inverso D-amino acid, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show diagram of claudin structure and conserved sequence in the second extracellular loop selected for peptide, respectively. Note that claudin 5 differs in only two amino acid residues.

FIGS. 2A and 2B show the effect of DFYNP on indicators of apoptosis and transepithelial resistance. EPH4 cells were treated with 400 μM claudin peptide or a control peptide, for 8, 16, and 24 hours. Cells were TUNEL stained (A) or loaded with a fluorescent probe-linked indicator for active caspase-8 (B, carboxyfluorescein-LETD-fluoromethyl ketone, dashed lines) or -3 (B, sulphorhodamine-DEVD-fluoromethyl ketone, solid lines). Mean±S.E.M, n=3-6, *p<0.05

FIG. 3A is a schematic illustration of structural variations of cysteine residues on peptide 1. For peptide 2 an acyl group was substituted for the NH₂on the N-terminal cysteine; for peptide 3 an amide group was additionally placed on the COOH terminus.

FIG. 3B shows the effect of altering the cysteine side groups as shown in FIG. 3A. EPH4 cells were treated with

claudin peptides

1, 2 and 3 for 16 hours prior to fixation. To visualize apoptosis cells were incubated with rabbit anti-cleaved caspase-3 and rat anti-ZO-1 primary antibodies, followed by Cy3- and FITC-conjugated secondary antibodies, respectively. Mean±S.E.M, n=3-6, *p<0.05.

FIG. 4 shows dose response curves of L and D peptides. After treatment of EPH4 cells with the indicated dose of the D- or L-forms of the DFYNP peptide for 16 hours, cells were fixed and the caspase-3 positive cells were detected with an antibody to caspase-3. At 2 mM the D-peptide produced 100% cell death in 16 hours. Mean±S.E.M, n=3-6 wells per concentration.

FIG. 5 shows comparison of apoptotic effects of linear D-peptide with cyclic L and D peptides. EPH4 cells were treated with claudin peptide in either the cyclic D-amino acid form (200 μM), the linear D-amino acid form (200 μM), or the cyclic L-amino acid form (2 mM) for 16 hours prior to fixation. Apoptosis was scored with an antibody to active caspase-3 as in methods and the percentage of positive cells plotted. Mean±S.E.M., n=3-6, *p<0.05.

FIG. 6 shows the effect of labeling the D-peptide with FITC. EPH4 cells were treated for 16 hours with 800 μM of the D-form of the DFYNP peptide linked to FITC. Apoptosis was detected by caspase-3 activation as in FIG. 5. Claudin-4 localization was imaged in the absence and presence of the FITC-DFYNP (data not shown). Mean±S.E.M., n=3-6.

FIG. 7 shows the effect of replacing each amino acid of the D-peptide with glycine. EPH4 cells were treated with the D-forms of the DFYNP peptide with a glycine substituted for each of the 5 residues for 16 hours prior to fixation. Apoptosis was detected as in FIG. 5. Mean±S.E.M., n=3-6, *p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “retro” in reference to a peptide refers to a peptide that is made up of L-amino acids in which the amino acid residues are assembled in opposite direction to the native peptide.
The term “inverso modified” refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the same direction (i.e., from the amino terminal to the carboxy terminal of the peptide) as the native peptide.
The term “retro-inverso modified” refers to a peptide which is made up of D-amino acids in which the amino acid residues are assembled in the opposite direction to the native peptide.
The term “native peptide” refers to any sequence of L amino acids used as a starting sequence or a reference for the preparation of partial or complete retro, inverso or retro-inverso analogues.
The term “peptide” as used throughout the specification and claims is to be understood to include amino acid chain of any length.
Thus, if the native peptide (L-amino acids, N—*C direction) is: ZFYNP, the retro-inverso Link-N peptide (D-amino acids, C—*N direction) is: ZFYNP; retro peptide (L-amino acids, C—*N direction) is: ZFYNP; and inverso peptide (D-amino acids, N—*C direction) is: ZFYNP.
The terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

Overview

The tight junction regulates inter alia permeability across epithelia and maintains distinct apical and basal regions of unit epithelial cells. While known to interact with various signaling molecules and to regulate intracellular behavior, the tight junction has not been reported to regulate apoptosis. The present inventors have found that disrupting the tight junction protein occludin, e.g., by dominant-negative occludin or claudin expression and externally applied function-blocking peptides, mimicked loss of occludin function in otherwise normal epithelial cells that are unable to form or maintain occludin binding or have undergone cellular dysregulation disruptive of occludin function. This disruption increased apoptosis. Caspases-8 and 3 were activated within minutes of occludin disruption. Caspase-8 inhibition, but not caspase-9 inhibition, prevented caspase-3 activation, indicating that apoptosis was induced via elements of the death receptor pathway. Occludin disruption lead to movement of endogenous occludin out of the junctional complex where it localized with elements of the death inducing signaling complex; Fas, FADD, and activated caspase-8. Cells showing tight junctional disruption were extruded from the monolayer without any significant loss of transepithelial electrical resistance during a several fold increase in apoptotic rate.
The present inventors have also found that occludin disruption led to an increase in the rate of cellular removal and apoptosis. Moreover, the present inventors discovered that caspase-8 was activated within minutes of occludin disruption and that caspase-8 activation preceded caspase-3 activation during treatment with an occludin function blocking peptide. As disclosed herein, activation of caspase-8 was achieved by elements of the death inducing signaling complex (DISC). Moreover, the physiological property of transepithelial electrical resistance remained stable during the several-fold increase of cellular extrusion and apoptosis.
Disruption of endogenous occludin function was used to mimic loss of occludin function in otherwise normal epithelial cells that are spatially unable to form or maintain occludin binding or have undergone any cellular dysregulation disruptive of occludin function. The present inventors have discovered that occludin disruption in cultured epithelial monolayers led to an increase in the rate of cellular removal and apoptosis. In addition, the present inventors discovered that caspase-8 was activated within minutes of occludin disruption. Moreover, in many instances caspase-8 activation preceded caspase-3 activation during treatment with an occludin function blocking peptide. Furthermore, the present inventors observed activation of caspase-8 by elements of the death inducing signaling complex (DISC). It was also observed that the physiological property of transepithelial electrical resistance remained stable during the several-fold increase of cellular extrusion and apoptotic rate seen during the experiments.
Two occludin-disrupting and two claudin-disrupting tools each were used to disrupt occludin function in three epithelial cell lines; it was found that this disruption increased apoptosis in treated cells. Apoptotic cells were lost from the monolayer with no significant change in TER. Movement of occludin out of the junctional complex in response to an occludin peptide mimic led to activation of caspase-8 in these same regions that were enriched in displaced occludin and DISC proteins.
It is believed that otherwise normal epithelial cells that are unable to form, or that lose, any of the more widely studied forms of cellular attachments undergo apoptosis. Accordingly, it is expected that disruption of integrin-mediated cell-to-substratum attachment, disruption of cadherin-mediated adherens junctions, and disruption of connexin-mediated gap junctions all stimulate apoptosis. These apoptotic responses to the disruption of cellular attachments provide an adaptive advantage to the host allowing epithelia to remove malfunctioning cells. The present inventors observed that disruption of occludin or claudin function by several means in confluent epithelial monolayers likewise leads to apoptosis.
Occludin was the first tight junction adhesion protein discovered. However, until the discovery by the present inventors, no one has recognized disruption of tight junction adhesion protein leads to apoptosis. When intact monolayers of the EPH4 cell line were treated with an occludin second extracellular loop peptide, isolated patches of cells throughout the monolayer showed a punctate, intracellular, non-junctional distribution of occludin. While these peptide treated cells maintained the majority of TER seen in untreated controls, TER was reduced. An occludin second extracellular loop peptide mimic impeded recovery of TER and caused internalization of several tight junction transepithelial proteins following the calcium switch in human intestinal epithelial cells. Similar results were obtained in a rat sertoli cell line. Moreover, intratesticular injection of an occludin mimic peptide shut down spermatogenesis and decreased testis weight more than three fold.
Cell death was recently reported in epithelial cells treated with an occludin first extracellular loop peptide mimic Peptide treatment reduced TER and increased solute permeability of treated cells. Cell death was ascertained via increased release of lactose dehydrogenase in treated cells. No attempt was made to ascertain either the rate of cell death or whether cell death was necrotic or apoptotic. More significantly, no report of apoptosis by disrupting the second extracellular loop of occludin has been reported to date.
Cells expressing dominant negative occludin appeared to migrate out of the monolayer prior to becoming TUNEL positive. The present inventors have observed that many of the occludin peptide treated cells that showed non-junctional occludin distribution and caspase-8 activation also showed the distinctive morphology of cellular extrusion. The finding that the several fold increase in apoptosis did not decrease the trans-epithelial resistance indicates that cell loss proceeds by an orderly biological process that maintains rather than disrupts epithelial barrier properties. Similarly, cultured monolayers of intestinal epithelial cells were able to maintain about 50% of basal TER when treated with a Fas crosslinking antibody that led to the loss of half of the cells in the culture in only 24 hours.
Occludin and claudin peptides caused occludin to stain in intracellular, punctate, nonjunctional patches in islands of cells throughout the monolayers, while the majority of treated cells showed normal occludin localization. In several of the experiments, dominant negative occludin was expressed in only a minority of cells. These conditions should mimic loss of occludin function in otherwise normal epithelial cells that are spatially unable to form or maintain occludin binding or have undergone any cellular dysregulation disruptive of occludin function, indicating that an endogenous pathway is triggered to remove epithelial cells unable to maintain occludin function. Without being bound by any theory, it is believed that this pathway is an adaptive defense against epithelial disruption.
It is believed that disruption of occludin function initiates the death receptor pathway of apoptosis. Several studies showed that disruption of integrin function and disruption of cadherin function caused caspase-8 activation. The present inventors have discovered that elements of the death receptor or extrinsic apoptotic pathway function generally to trigger apoptosis in epithelial cells that have lost normal cell-cell attachments. The present inventors have also discovered that a specific caspase-containing complex was formed during loss of normal cellular attachment occurring prior to changes that lead to nuclear condensation and cell extrusion.
The localization of activated caspase-8 in regions of non-junctional occludin indicates that displaced occludin acts to promote caspase-8 activation, as appears to be the case for unligated integrin in detachment-induced apoptosis or anoikis. The Akt antagonist, lipid phosphatase PTEN is another candidate molecule for linking loss of occludin function to apoptosis. It has been demonstrate that the PIPS phosphatase PTEN binds to the tight junction associated MAGI, PAR, and DLG proteins. The lipid phosphatase activity of PTEN correlates positively with the stability of the apical junction complex. Moreover, PTEN plays a role in activation of the death receptor pathway of apoptosis under various conditions. The present inventors have also discovered that PTEN is associated with the tight junction (data not shown).
One of the necessary steps in the metastatic process is disruption of intercellular junctions without subsequent apoptosis. Without being bound by any theory, it is believed that the molecular pathway(s) linking occludin or claudin disruption to apoptosis is attenuated or absent in epithelial cancers, as is the case with more widely studied cellular adhesion proteins. The molecular pathways linking occludin or claudin dysregulation to metastases can be augmented by drug therapies to induce apoptosis or halt epithelial to mesenchymal transformation specifically in cells that have lost normal occludin or claudin based adhesion.

Pharmaceutical Compositions

The compounds of the present invention can be administered to a patient to achieve a desired physiological effect. Preferably the patient is an animal, more preferably a mammal, and most preferably a human. The compound can be administered in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally. Parenteral administration in this respect includes administration by the following routes: intravenous; intramuscular; subcutaneous; intraocular; intrasynovial; transepithelially including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation and aerosol; intraperitoneal; and rectal systemic.
The active compound can be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it can be enclosed in hard or soft shell gelatin capsules, or it can be compressed into tablets, or it can be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparation can contain at least 0.1% of active compound. The percentage of the compositions and preparation can, of course, be varied and can conveniently be between about 1 to about 10% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared such that an oral dosage unit form contains from about 1 to about 1000 mg of active compound.
The tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin can be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the active compound, sucrose as a sweetening agent, methyl and propylparabens a preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and formulation.
The active compound can also be administered parenterally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacterial and fungi. The carrier can be a solvent of dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, e.g., sugars or sodium chloride. Prolonged absorption of the injectable compositions of agents delaying absorption, e.g., aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
The therapeutic compounds of the present invention can be administered to a mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
Furthermore, compounds of the invention can be administered in combination with other pharmaceutically active compound, such as other anticancer compound, anti-inflammatory compound, or a combination thereof. Such co-administration of pharmaceutically active compound can lead to synergistic effects.
The physician will determine the dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment and it will vary with the form of administration and the particular compound chosen, and also, it will vary with the particular patient under treatment. The physician will generally wish to initiate treatment with small dosages by small increments until the optimum effect under the circumstances is reached. The therapeutic dosage can generally be from about 0.1 to about 1000 mg/day, and preferably from about 10 to about 100 mg/day, or from about 0.1 to about 50 mg/Kg of body weight per day and preferably from about 0.1 to about 20 mg/Kg of body weight per day and can be administered in several different dosage units. Higher dosages, on the order of about 2× to about 4×, may be required for oral administration.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.

EXAMPLES

Materials and Methods


	ABBREVIATION	NAME

	Fmoc	Fluorenylmethoxycarbonyl
	HOBt	1-Hydroxybenzotriazole monohydrate
	DIC	Diisopropylcarbodiimide
	DMF	N,N-Dimethylformamide
	TFA	Trifluoroacetic acid
	Trt	Trityl
	tBu	t-Butyl
	OtBu	t-Butoxy

Antibodies:

Various antibodies were obtained as follows: Anti occludin, Zymed® clone OCOC-3F10; anti Fas, BD Biosciences clone 13; anti ZO-1, Chemicon® MAB 1520; anti FADD, USBiological clone 12E7; anti MUC1, Abcam Inc. clone EP1024Y; anti-caspase-3, Cell Signaling Technology® (8G10); anti-caspase-8, Axxora® (1G12).

Cells and Cell Culture:

CIT3 mouse mammary epithelial cells were grown as described by Toddywalla V S et al., J. Pharmacol. Exp. Ther., 1997, 280, 669-676. To differentiate cells, this growth medium was modified by removal of EGF and addition of 3 μg/ml each of ovine prolactin and hydrocortisone (differentiation media).
EPH4 mouse mammary epithelial cells were grown as described by Reichmann E et al., J. Cell Biol., 1989, 108, 1127-1138. To differentiate cells, this growth medium was modified by addition of 3 μg/ml each of ovine prolactin and hydrocortisone (differentiation media).
MDCK cells were grown in MEM with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. To differentiate cells, this growth medium was modified by addition of 3 μg/ml each of ovine prolactin and hydrocortisone (differentiation media) to that described by Peixoto E B et al., Cell Biol. Int., 2006, 30, 101-113.
For all images shown cells were trypsinized from polycarbonate cultures plated at 1:2 and grown for 7 days then plated at 2× confluent density on FBS treated Lab Tek II, CC2 glass chamber slides (Nunc) or Transwell® filters (Product #3413). Cells were grown 3 days then switched to differentiation media for 2 days prior to the beginning of experiments.
Cultures were maintained at 5% CO₂and 37° C.

Expression Constructs and Transient Transfection:

Mouse occludin ATCC #MGC-5797 was cut with BsaA1 in the second extracellular loop and Bcl1 in the 5′ UTR and inserted in pFLAG-CMV-1 (Sigma), placing it downstream of secretory signal peptide and N-terminal FLAG epitope tag (FAOcc). F-AOcc or pFLAG-CMV-1-BAP construct (Sigma) were transfected into cells using FuGENE® HD (Roche). Cells were plated in the morning at confluent density, transfected in the evening, and transfection media was replaced with differentiation media the following morning. pFLAG-CMV-1-BAP is the identical vector encoding secretory, N-terminal FLAG tagged, bacterial alkaline phosphatase.

Adenoviral Synthesis and Transduction:

The F-ΔOcc was cut out of pFLAG-CMV-1 using available restriction enzyme sites and used to construct an adenovirus (Ad-F-ΔOcc) following the AdEasy system. See, for example, He T C et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 2509-2514. GFP only adenovirus and Ad-F-ΔOcc were titered together by transducing CIT3monolayers and counting GFP positive cells at 9 hours post transduction. Adjusted viral stocks were used at equal effective titers.

Western Blotting:

Cells were lysed for 1 hour 40 then scraped in 50 mM tris pH 6.8, 2% SDS, 20% glycerol, 2.5 mM DTT, containing phosphatase inhibitor cocktail (cat.#P2850, Sigma) and protease inhibitor cocktail (cat.#P8340, Sigma) both 1:200. Lysate was passed through a tuberculin syringe and centrifuged 30 minutes at 16,000 rcf, 4° C. 40 μg supernatant protein per sample was separated (10-20% gradient gels BioRad). Gels were transferred onto nitrocellulose and antibody stained. Stained blots were visualized using HRP-anti-host IgG (Pierce) with enhanced chemiluminescence (Amersham).

Peptide Synthesis:

The claudin mimic peptides, were synthesized using standard solid phase peptide synthesis methodology and Fmoc chemistry on preloaded Fmoc-amino acid-Wang resin (substitution of amino acid was 0.5 mmol/g). The side-chain protecting groups were Cys(Trt), Asp(OtBu), Asn(Trt), and Tyr(tBu). A 5 molar excess of Fmoc-amino acid, HOBt and DIC (1:1:1) was used for the coupling. Completion of the coupling was monitored with the Kaiser test. Cleavage of the peptide from the resin and removal of protecting groups was carried out with TFA, water, ethanedithiol, and triisopropylsilane as scavengers (90:5:3:2 v/v). Crude peptide was purified by reversed-phase HPLC using a linear AB gradient where eluent A is 0.2% aqueous TFA and eluent B is 0.18% TFA in acetonitrile at a flow rate of 2 ml/min, and a gradient rate of 0.1% acetonitrile/min See Chen et al. in J. Chromatography A, 2007, 1140, 112-120. The column used was Zorbax SB-300 C18, 9.4 mm I.D.×250 mm Fractions were characterized by electrospray mass spectrometry (Perseptive Biosystems Mariner Biospectrometry work station) and analytical reversed-phase HPLC using a Zorbax SB-300 C18, 2.1 mm I.D.×150 mm column on an Agilent 1100 Series liquid chromatograph (Agilent Technologies). Pure fractions were pooled and lyophilized Air oxidation of the cysteine to form a disulfide bond was achieved by stirring the peptide at room temperature in 0.1M ammonium bicarbonate, pH 8.0, at a concentration of 0.5 mg per ml for 18 hours. The reaction was monitored by analytical reversed-phase HPLC. The intra-chain disulfide bridged peptide had a lower retention time than the reduced sulfhydryl form. Upon completion of disulfide bond formation, the solution was lyophilized, then relyophilized from water and lyophilized again. The oxidized peptide was used without further purification since only a single component was obtained.
Peptides synthesized include: the linear form of the L-amino acid peptide, NH₂-G-DFYNP-G-OH; the cyclic peptide flanked by different end-groups, Ac-C-DFYNP-C-OH and Ac-C-DFYNP-C-amide; the original peptide in the D-amino acid form, both cyclic and linear; the linear D-amino acid form linked to FITC, and a glycine scan (substituting each amino acid with glycine) of the linear D-amino acid form. All peptides were dissolved in a 30% DMSO in water stock solution just before use and added to culture medium to bring the final concentration of DMSO to <0.5% final volume of media. Cells were treated with the various forms of the peptide for times up to 16 hours before cell fixation.

Formation of the Disulfide Bond

Air oxidation of the cysteine residues to form the intra-chain disulfide bond was achieved by stirring the peptide at room temperature in 0.1 M ammonium bicarbonate, pH 8.0, at a peptide concentration of 0.5 mg/mL for 18 hours. The reaction was monitored by analytical reversed-phase HPLC. The oxidized peptide had a lower retention time than the reduced form. Upon completion of disulfide bond formation, the solution was lyophilized and reophilized twice from water to remove the ammonium bicarbonate. The oxidized peptide was used without further purification.

Peptide Treatment

Peptides were solubilized in 30% DMSO in water just before the experiment. Cells were treated with 10 μM-5 mM of peptide for 2-16 hours at 37° C.

Tissue Culture

The mouse mammary epithelial cell line, EPH4, was used for examination of apoptosis in normal mammary epithelial cells in vitro. EPH4 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 10 mM Hepes (all from GibcoBRL, Grand Island, USA). Growth media was refreshed every 3-5 days and cells were trypsinized (0.25% Trypsin, EDTA, GibcoBRL) and plated 1:2 every 7 days. Cells were plated with a 1:1 surface area onto Lab-Tek glass 8-chamber slides (NUNC, Rochester, N.Y., USA) for experiments.
The mouse embryonic fibroblast cell line, 3T3L1, was also used to test the specificity of the claudin mimic peptide. 3T3L1 cells were grown in DMEM/F12 medium supplemented with 10% Bovine Calf Serum (B CS, Sigma, St. Louis, Mo., USA) and 1% penicillin/streptomycin. Cells were trypsinized (0.25% Trypsin, EDTA) and plated 1:8 every 2-3 days. Cells were plated with a 1:1 surface area onto Lab-Tek glass 8-chamber slides as EPH4 cells above.

Immunofluorescence

Cell monolayers were fixed with 2% paraformaldehyde for 15 mM at room temperature at various time points after treatment with the claudin mimic peptide, then permeabilized with 0.5% Triton X-100 for 5 minutes before blocking with 2% BSA for an hour. Cells were then treated with mouse anti-claudin-4 (1:200, Zymed, Carlsbad Calif.), rabbit anti-cleaved caspase-3 (1:100, Cell Signaling Technologies, Danvers, Mass.), rat anti-ZO-1 (1:50, Santa Cruz Biotechnology, Santa Cruz, Calif.), rabbit anti-occludin (Invitrogen, Carlsbad, Calif.), and/or rat cleaved caspase-8 (Enzo Life Sciences, San Diego, Calif.) primary antibodies for 1 hour. After washing five times, five minutes each, with Phosphate Buffered Saline (PBS), cells were treated with donkey anti-mouse-FITC (1:150, Jackson ImmunoResearch Laboratories, West Grove, Pa.), donkey anti-rabbit-CY3 (1:150, Jackson ImmunoResearch Laboratories), donkey anti-rat-CY5 (1:150, Jackson ImmunoResearch Laboratories) and/or donkey anti-rat-FITC (1:150, Jackson ImmunoResearch Laboratories) for 45 minutes. Monolayers were then washed five times, five minutes each, with PBS and OPDA was applied before addition of a coverslip. Fluorescence was imaged on a Nikon Diaphot TMD microscope or an Olympus Spinning Disk confocal microscope, using SlideBook software (Intelligent Imaging Innovations, Inc.).

Caspase Activation

At various time points after incubation with peptide, apoptosis was measured in live cells using Image-iT®LIVE Red Caspase-3 and -7 Detection Kit or caspase-8 activation using Image-iT®LIVE Green Caspase-8 Detection Kit (Molecular Probes/Invitrogen, Eugene, Calif., USA). After treatment with the claudin mimic peptide, cells were washed and growth media containing 1× fluorescent inhibitor of caspase (FLICA, a sulforhodamine group reporter, company) reagent was added to cell chambers and incubated for 1 hour at 37° C. Cells were then treated with growth media containing 1 μM Hoechst 33342 for 5 minutes at 37° C. Cells were washed in Hanks Buffer Saline Solution (HBSS) two times and then fixed with 2% paraformaldehyde in HBSS for 15 minutes at room temperature. Caspase-3 activation was also measured in fixed cells using a mouse anti-cleaved caspase-3 antibody.
Apoptosis was also measure by TUNEL assay. TUNEL staining was performed using the Roche In Situ Cell Death Detection Kit, TMR red. Cells were fixed in 2% paraformaldehyde and permeabilized in 1% sodium citrate (trisodium salt) containing 0.1% Triton X-100. Staining was performed per manufacturer's instructions.

Statistics

Data are presented as means±Standard Error of the Mean (SEM). An unpaired Student t test was used for statistical comparison between control and treatment groups. A p value of <0.05 was considered significant.

Results

Treatment of Eph4 Mammary Cells with a 5 Amino acid L-Peptide
To test the prediction that amino acids F147, Y148, and P150 in claudin-5 and its congeners are involved in mediation of cell-cell interactions by claudins, a five amino acid peptide mimic containing the DFYNP sequence was designed. This sequence is highly conserved in the second extracellular loop of claudins-3, -4, -7, and -8, among the claudins most highly expressed in epithelial tissues and tumors (FIGS. 1A and B). To stabilize the peptide, cysteine residues were added to both ends of the peptide which was then cyclized with air oxidation.
To confirm that the DFNYP peptide disrupts normal claudin interactions at the tight junction, a normal mammary epithelial cell line (EPH4) was treated with media containing 400 μM of the cyclic, L-amino acid form of the DFYNP peptide. After 16 hours of treatment, cells were fixed and stained with an antibody against claudin-4. In untreated cell monolayers, claudin-4 localization was restricted to the tight junctions (data not shown). After treatment with the DFYNP peptide, claudin-4 localization changed in two ways. In some cells it was distributed uniformly in the cytoplasm; in most cells, however, it appeared to vesiculate along the tight junction (data not shown). The mis-localization of claudin-4 in response to the peptide indicates that the peptide is indeed disrupting normal claudin binding at the tight junction. That this reaction was specific was shown by the observation that a control peptide, LYQY, had no effect (data not shown).
Disruption of tight junctions is normally thought to occur downstream of apoptotic signaling. However, the present inventors observed increased apoptosis after tight junctions were disrupted with the DFYNP peptide. EPH4 cells treated with 400 μM of this peptide or a control peptide, LYQY, were tested for TUNEL staining after 8, 16, or 24 hours of treatment. TUNEL staining significantly increased and appeared to peak at 16 hours of treatment with the peptide compared to untreated cells (FIG. 2A).
Apoptosis was also assessed by measuring caspase activation in similar experiments (FIG. 2B). Caspase activation was measured using a fluorescent probe-linked indicator of active caspase-8 or caspase-3, added during the last hour of incubation with the DFYNP or control peptide. After 16 hours of DFYNP peptide treatment, a significant number of cells showed activated caspase-8 as well as caspase-3 compared to control (FIG. 2B). These data further confirm that apoptosis is induced in response to the claudin mimic peptide disrupting normal claudin interactions. In many of the subsequent experiments the activity of various peptides was assessed by indicators of apoptosis.
The initial DFYNP peptide had an NH₂group on the N-terminal cysteine and an —OH group on the C-terminal cysteine (labeled “Peptide 1”, FIG. 3A). To confirm that these groups did not influence the function of the peptide, substitutions were made as shown in FIG. 3A, and function assessed by using immunohistochemistry to assess caspase-3 activation. FIG. 3B shows that caspase-3 was activated to the same extent as peptide 1 (7.17±1.24) when the NH₂group was replaced with an acetyl group (9.22±0.65, p=0.2210) or the —OH group was replaced with an amide (7.96±0.75, p=0.7059). This finding shows that the DFYNP sequence is the sole determinant of the apoptotic effect of the peptide.

The D-Form of the Peptide has Increased Activity.

A cyclic, D-amino acid form of the peptide was synthesized to test the specificity of the DFYNP peptide and was, surprisingly and unexpectedly, more potent at inducing apoptosis than the cyclic, L-amino acid form (FIG. 4). The dose-response curves show that 1 μM of the D-peptide was as active as 300 μM of the L-peptide. At 2 mM, a concentration of L-peptide that induced apoptosis in about 7% of the cells by 16 hours, the D-peptide induced apoptosis in 100% of the cell population. These results indicate, as expected, that the D-amino acid form of the peptide is more stable to proteolysis than the L-amino acid form since peptidases in the extracellular milieu of the cells attack L-peptide linkages but not D-linkages. They also suggest that the interaction of the peptide with the second extracellular loop of claudin is not stereospecific. For this reason it was believed that removing the disulfide bridge (making the peptide linear) would still result in a more potent inducer of apoptosis than the cyclic L-form peptide. FIG. 5 shows that after 16 hours of treatment 200 μM, the linear D-peptide induced 12.27±0.72% apoptosis, which was significantly more apoptotic than the cyclic L-peptide (7.17±1.24%, p=0.020584) at a 10 fold higher concentration although somewhat less than the 16.46±0.51% (p=0.0115) apoptosis induced by the cyclic D-peptide at this concentration. Because synthesis of the linear peptide is significantly easier than that of the cyclic peptide the linear peptide was used in all the rest of the experiments reported here.

Use of a FITC-Labeled Peptide to Localize Peptide in Eph4 Cells.

To investigate potential interactions of the peptide with the tight junction, a fluorescent probe (FITC) was linked to the N-terminus of the linear, D-form of the DFYNP peptide, referred to as “FITC-DFYNP”. FIG. 6 shows that the FITC-DFYNP peptide was able to induce apoptosis (10.58±0.83%, p=0.1769) to a extent similar to that of the unlinked linear D-peptide. The FITC-DFYNP peptide led to mis-localization of claudin-4 away from tight junctions (data not shown). These results suggest that the FITC-probe does not inhibit or change the activity of the peptide. To gain a better understanding of the timing of events, a time series experiment was performed to look at the localization of the FITC-DFYNP, claudin-4, and active caspase-8. Cells were first incubated with 400 μM FITC-DFYNP at 4° C. After 16 hours, cells were either fixed (“time 0”) or placed at 37° C. for 30 minutes, 2 hours, or 4 hours. After 30 mM, a significant amount of claudin-4 mis-localization was seen, with little caspase-8 activity (data not shown). However, by 4 hr there was significant claudin-4 mis-localization as well as significant caspase-8 activation. These results suggest that mis-localization of claudin-4 is upstream of caspase activation. The FITC-DFYNP appeared to form puncta within the cells, even at the earliest time point, and these puncta did not always correlate with claudin mis-localization.
To investigate the movement of the FITC-DFYNP peptide in the absence of fixation, cells were imaged live, in the presence of an active caspase-3 and -7 fluorescent indicator (Image-iT®LIVE Red Caspase-3 and -7 Detection Kit, FLICA reagent, described in methods). Both EPH4 epithelial cells (express claudin proteins) and 3T3L¹fibroblast cells (do not express claudin proteins) were treated with FITC-DFYNP. After treating cells with 400 μM FITC-DFYNP at 4° C., FLICA reagent was added to “time 0” cells for 1 hour (at 4° C.) before washing with cold PBS and imaging. All other cells were placed in 37° C. for 30 minutes, 2 hours, or 4 hours before adding FLICA reagent for 1 hour (at 37° C.), washing with room temperature PBS and imaging. It was observed that the FITC-DFYNP localized to the tight junctions of the epithelial cells after overnight incubation in the cold, with possible diffuse localization within the cells 30 min after warm-up n(data not shown). The FITC-DFYNP peptide did not, however, appear to interact with the fibroblast cells under the same conditions suggesting that it specifically interacts with the membrane or claudin proteins at the tight junctions of epithelia. In agreement with these results, caspase-3 and/or -7 activation was significantly increased at 4 hours in the epithelial EPH4 cells, but no caspase activation was seen in the fibroblast 3T3L¹cells, (data not shown).
A Glycine Scan Shows that 4 of the 4 Amino Acids are Necessary for Specificity
To confirm that the action of the DFYNP peptide is sequence specific, we performed a glycine scan of the peptide, substituting glycine for each amino acid, and tested each peptide's ability to induce apoptosis via caspase-3 activation. The D(G)YNP, DF(G)NP, and DFYN(G) peptides, after 16 hours incubation at 37° C., were unable to induce apoptosis significantly above control (1.46±0.53%, p=0.453437; 1.68±0.46%, p=0.21944; 1.32±0.17%, p=0.224133 respectively, FIG. 7). The DFY(G)P peptide induced apoptosis slightly, but significantly, above control (2.70±0.34, p=0.002614). The (G)FYNP peptide, however, induced apoptosis (13.13±1.28%) to a similar level as the linear D-form of the DFYNP peptide (12.27±0.72%, p=0.757506). This finding suggests that the aspartic acid is not required for the peptide interaction with the claudin protein, however, the remaining side-chains are required for this interaction.

Discussion

The present inventors have designed a peptide that mimics a highly conserved sequence in the second extracellular loop of claudin isoforms expressed in the mammary epithelium. This peptide disrupts normal claudin localization from tight junctions to the cytosol and induces apoptosis via the extrinsic apoptotic pathway as indicated by activation of caspase 8. This evidence shows involvement of claudins in cell death signaling, upstream of caspase activation. Similar results have been seen with mimic peptides that target the second extracellular loop of the tight junction protein occludin protein. In particular, others have shown that a mimic peptide to a small, highly conserved region within the second extracellular loop of occludin leads to the translocation of occludin away from the tight junctions and the induction of caspase activation and apoptosis. Those results along with the results from the present study suggest that interactions between the second extracellular loops of claudin and occludin are required for cell survival within an epithelial monolayer.
Although the potency of the D-amino acid form of the peptide was initially surprising, others have also reported D-amino acid peptides being as or more active than the L-amino acid form of the same peptide. If the interaction of the DFYNP peptide with the claudin protein were stereospecific, the mirror-image of the same peptide (D-amino acid form) would not be able to bind or interact with claudin. Therefore, the ability of the D-form of the DFYNP peptide to cause the mis-localization of claudin and induce apoptosis suggests that the interaction of the DFYNP to the second extracellular loop of claudin is not stereospecific. The effect of the mimic peptide on claudin localization suggests that claudin proteins interact with each other at this sequence to maintain claudin at the tight junction. Further, and perhaps more surprising, when this interaction is disrupted, cell death signaling is initiated. The ability of the D-form of the peptide to interact with endogenous claudin proteins to induce apoptosis also indicates that normal claudin-claudin trans-interactions at the second extracellular loop's conserved DFYNP sequence is not stereospecific.
The increased potency of the D-form of the DFYNP peptide can be explained by the fact that peptides in the D-form are more stable, as endogenous proteases recognize L-amino acid peptides. This contention is supported by the observation that the linear D-peptide was also more potent at inducing apoptosis than the cyclic L-form of the peptide.
The exact mechanism on how the D-form of the DFYNP peptide can influence endogenous claudin protein interactions is not apparent. One possibility is that the peptide is specifically competing with and binding to the endogenous claudin proteins to prevent normal claudin-claudin interactions at the second extracellular loops. Alternatively, the D-peptide could be interacting with the membrane of the epithelial cells at the site of integration of one claudin protein with the membrane of the neighboring cell. Results from the FITC-DFYNP treated 3T3L¹cells suggests that the D-peptide is not merely interacting with any membrane, but specifically with cells that express tight junctions and claudin proteins. The inhibition of apoptosis with glycine substitutions also suggests that the interaction of the peptide is specific to the DFYNP sequence within the claudin peptide. Therefore, it is likely that the DFYNP peptide is interacting with the claudin-3, -4, -7, and/or -8 proteins and preventing their interaction at the second extracellular loop.
Others have shown that mutations at amino acids F147, Y148, and P150 in the second extracellular loop of claudin-5 cause the mis-localization of claudin-5 away from the tight junctions and prevent trans-interactions of claudin-5 proteins and strand formation. The present inventors have shown similar results with mimic peptides disclosed herein. Disruption of F147, Y148, P150 and even N149 disrupted normal claudin-claudin interactions at the tight junctions, led to mis-localization of claudin-4 away from the tight junction, and initiated apoptosis. These results suggest these amino acids are significant for normal interactions of claudin proteins on opposing cells and when these interactions are disrupted, claudins no longer localize to the tight junction and a signaling cascade leading to cell death is activated.
The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

1. A method for treating a clinical condition associated with abnormal growth or development of cells in a subject, said method comprising administering to the subject in need of such a treatment a composition comprising a therapeutically effective amount of a compound comprising FYNP amino acid sequence, wherein F is phenylalanine, tyrosine, tryptophan, or leucine; Y is tyrosine, phenylalanine, tryptophan, or leucine; N is asparagine or glutamine; and P is proline.

2. The method of claim 1, wherein the compound stimulates apoptosis of cells.

3. The method of claim 1, wherein the cells comprise epithelial cells.

4. The method of claim 1, wherein the clinical condition associated with abnormal growth or development of cells comprises skin cancer, breast cancer, ovarian cancer, or metastases thereof.

5. The method of claim 1, wherein the compound comprises D-amino acids or retro-inverso D-amino acids.

6. A method for modulating apoptosis of cells comprising contacting the cells with an effective amount of a compound comprising FYNP amino acid sequence or a retro-inverso amino acid thereof, wherein F is phenylalanine, tyrosine, tryptophan, or leucine; Y is tyrosine, phenylalanine, tryptophan, or leucine; N is asparagine or glutamine; and P is proline.

7. The method of claim 6, wherein the cells comprises epithelial cells.

8. The method of claim 6, wherein the compound modulates an adhesion protein.

9. The method of claim 8, wherein the adhesion protein comprises occludin, claudin, junction adhesion molecule, integrins, or a combination thereof.

10. The method of claim 9, wherein the compound comprises at least 3 consecutive amino acid sequences of the second extracellular loop sequence of occludin or claudin, or a derivative thereof.

11. The method of claim 6, wherein the compound comprises D-amino acids or retro-inverso D-amino acids.

12. The method of claim 6, wherein the compound comprises about 20 amino acid sequences or less and is a cyclic peptide or a linear peptide, or a derivative thereof.

13. The method of claim 12, wherein the compound is a cyclic compound of the formula:

wherein

each of X¹and X⁴is independently cysteine, glutamic acid, aspartic acid, lysine, or ornithine;

each of L¹and L⁴is independently the corresponding functional group of the amino acid side chain of X¹and X⁴, respectively, or —(CH₂)_m—, wherein m is an integer from 1 to 4;

each of X²and X³is glycine;

each n is independently 0, 1 or 2;

Z is glycine, isoleucine, alanine, leucine, lysine, methionine, cysteine, phenylalanine, threonine, tryptophan, valine, proline, serine, tyrosine, arginine, or histidine;

F is phenylalanine, tyrosine, tryptophan, or leucine;

Y is tyrosine, phenylalanine, tryptophan, or leucine;

N is asparagine or glutamine; and

P is proline.

14. The method of claim 13, wherein F is phenylalanine, Y is tyrosine, N is asparagines, and Z is glycine.

15. The method of claim 13, wherein X¹and X⁴are cysteine.

16. The method of claim 15, wherein L¹and L²together form a moiety of the formula —S—S—.

17. A compound comprising 20 amino acid sequences or less and comprises D-amino acid sequences or retro-inverso D-amino acid sequences of FYNP, wherein F is phenylalanine, tyrosine, tryptophan, or leucine; Y is tyrosine, phenylalanine, tryptophan, or leucine; N is asparagine or glutamine; and P is proline.

18. The compound of claim 17, wherein said compound comprises a cyclic peptide or a linear peptide.

19. The compound of claim 18, wherein said cyclic peptide is of the formula:

wherein

each of X²and X³is glycine;

each n is independently 0, 1 or 2;

Z is glycine, isoleucine, alanine, leucine, lysine, methionine, cysteine, phenylalanine, threonine, tryptophan, valine, proline, serine, tyrosine, arginine, or histidine; and

F, Y, N, and P are those defined in claim 17.

20. The compound of claim 19, wherein Z is glycine, and L¹and L²together form a moiety of the formula —S—S—.