2. Description of the Bar Related Art Selectins are a class of highly homologous proteins that are responsible for the recognition of animal cell surface oligosaccharides. The role of selectins in mediating cellular adhesion, and thereby initiating the inflammatory and cell-mediated immune responses, and cell-cell interaction has attracted much attention. (1,2,3,4,5) Even though the 3-dimensional crystal structure of a fragment of human E-selectin (157 residues), is available, it was determined in the absence of ligands and thus certain aspects of the molecular mechanism of selectin: ligand recognition remain unknown. (6) E-selectin binds to sialyl LewisX (SLeX) (branched tetrasaccharide ligand), relatively weakly in vitro (Kd on the order of 1 mM) (7,8) This interaction gives rise to a characteristic rolling of lymphocytes along the endothelial surface that is observed prior to the onset of the neutrophil migration or extravasation into injured tissue. (9) The tighter adhesion of lymphocytes at the onset of the inflammatory response is triggered by an inadequately characterized process. (10) Controlling or regulating this triggering process is required for the development of materials suitable for artificial implants and for

the development of anti-inflammatory, anti-thrombotic, anti-cancer, and immunoregulatory agents.

It is known that cyclic nucleotides, could be involved in cell-cell signaling and cell-cell adhesion.

Early experiments implicated that cGMP is present at sites or tight cell-cell adhesion, and that cAMP is present at sites lacking cell-cell adhesion. These initial studies took place in the early 1970's, before adhesion molecules had been well characterized. It was not known whether the correlation between the presence of cyclic nucleotides and adhesion represented a direct causal relationship. Adhesion molecules have since been identified, but previously observed cyclic nucleotide effects were, before the present invention, attributed entirely to the action of other established pathways for control by cyclic nucleotides.

The present invention provides bifunctional compounds to regulate cellular adhesion and extravasation and a method to determine same. These were determined by analyzing the crystal structure of E-selectin using recently described computer software. (11) This computer software defines the shapes and positions of ligands on the surfaces of proteins of known structure.

The present invention provides compounds which are useful in the prevention of thrombosis, cancer and inflammatory responses. Further it provides compounds useful in enhancing the acceptance of artificial implants.

SUMMARY OF THE INVENTION An object of the present invention is a bifunctional compound for regulating adhesion and cellular proliferation.

A further object of the present invention is a method to control cellular adhesion and extravasation.

An additional object of the present invention is a method of detecting test compounds for agonistic and antagonistic activity to cell proliferation.

Thus, in accomplishing the foregoing objects there is provided in accordance with one aspect of the present invention a bifunctional compound for regulating cellular proliferation comprising a cell- adhesion oligosaccharide, a linker group attached to said cell-adhesion oligosaccharide by the reducing end of the oligosaccharide or by a primary hydroxyl group and a ribonucleotide cyclic-3'-5'monophosphate attached to the linker group through a heterocyclic base.

In specific embodiments of the present invention the linker group is attached to the C8 of the purine ring or C, or Ce of pyrimidine rings.

In another specific embodiment of the bifunctional compound, the nucleotide cyclic-3'5'monophosphate is selected from the group consisting of the compounds in Fig. 10.

In another specific embodiment of the bifunctional compound, the linker is attached to C8 of the purine ring or the C5 or C6 of the pyrimidine ring.

In another specific embodiment of the bifunctional compound, the linker is selected from the group consisting of the compounds in Fig. 9A, 9B, 9C, 9D.

In another specific embodiment of the bifunctional compound, the oligosaccharide is selected from the group consisting of compounds in Fig. 8.

In a specific embodiment, the bifunctional compound is selected from the group consisting of compounds in Fig. 11.

An alternative embodiment includes, a method of regulating cell proliferation comprising the step of contacting a therapeutic amount of bifunctional compound with a selectin.

In a specific embodiment, the method includes a selectin selected from the group consisting of E-selectin, P-selectin and L-selectin.

Another specific embodiment includes a method of regulating cell-cell adhesion comprising the step of contacting an effective amount of bifunctional compound with a selectin.

A further method includes detecting a test compound for agonistic or antagonistic activity to cell proliferation comprising the steps of contacting said test compound with a selectin in cell culture, measuring the growth of the cells in the cell culture wherein increased cell growth over normal cell growth indicates agonistic activity and decreased cell growth over normal cell growth indicates antagonistic activity.

In another embodiment of the present invention there is a method of detecting a test compound for agonistic or antagonistic activity to cell adhesion or cell migration comprising the steps of contacting the test compound with a selectin in a cell culture, measuring the cell-cell adhesion of cells in the cell culture; wherein increased cell-cell adhesion compared to normal cell-cell adhesion indicates agnostic activity and decreased cell-cell adhesion compared to normal cell-cell adhesion indicates antagonistic activity.

Another specific embodiment includes a method of treating the inflammatory response comprising the step of contacting a therapeutic amount of bifunctional compound with a selectin.

Other and further objects features and advantages will be apparent from the following description of the presently preferred embodiments of the invention, which are given for the purpose of disclosure when taken in conjunction with the accompanying drawings.

DESCRIPTION OF THE DRAWINGS Figures 1A and 1B are computer-derived sites of interest on the surface of E-selectin. Figure 1A shows Putative ligand binding sites (transparent density) for oligosaccharides (upper left) and for cyclic nucleotides (lower right) superimposed upon the structure of E-selectin. Figure 1B is a rotated view with the putative peptide/glycoconjugate site included.

Figures 2A, 2B, 2C and 2D are close-up stereoviews of the unexpected cyclic purine nucleoside monophosphate site as viewed from opposite sites of the protein.

Figure 3 shows the amino acid resides in the vicinity of the predicted cyclic nucleotide (dark shading) and trisaccharide (light shading) sites on human E-selectin superimposed on a PILEUP diagram of sequence similarities among selectins. The N-and C-termini of the crystallographically examined fragment are denoted by arrows. The commercially available soluble fragment contains 535 out of 611 residues present in full length human E-selectin.

Figure 4 demonstrates E-selectin (soluble fragment, R&D Biosystems) binding to a cAMP Sephrarose affinity column.

Figure 5 is a reference chart of electrophoretic bands that result from the V8 proteolytic cleavage of E-selectin as analyzed on 15% of polyacrylamide gels under denaturing conditions.

Figures 6A, 6B, 6C and 6D show Titration of E-selectin with a cAMP or cGMP in the absence of exogenous metals; and Titration of E-selectin with SLeX at fixed concentrations of Mg++ (0.8 mM), Ca++ (0.8 mM), and either cAMP or cGMP (100 jus).

Figure 7 is a schematic model for the onset of inflammation.

Figure 8 is a schematic representation of some examples of cell-adhesion oligosaccharides that are useful in the present invention.

Figure 9A, 9B, 9C, 9D is a schematic representation of some examples of linkers that are useful in the present invention.

Figure 10 is a schematic representation of some examples of ribonucleotide cyclic-3'-5'monophosphates that are useful in the present invention. In Fig. 10: X1, X2, X3 can independently be any of : N, CH, C-Halogen.

X4, X5, X7, X8, X11, X13 can independently be any of: H, F, Cl, Br, I, NH2, OH, OR, NHR, NR RÆ wherein R' and R' can independently be any of: alkyl, acyl or aryl group. <BR> <BR> <BR> <P> X5, X9, X10, X12 can independently be any of:<BR> <BR> <BR> <BR> <BR> O, S, NH, CH2, CHOH, CH-Halogen, C (Halogen),.<BR> <BR> <BR> <BR> <BR> <P> X14 can be any nitrogen-containing heterocycle to which linker groups are attached.

For the case of co-administration, Ri, R2 linker groups can independently be any of: H, F, Cl, Br, I, NH2, OH, OR, NHR', NR Rt wherein R'and R'can independently be any aklyl, alkenyl, acyl or aryl group.

In addition, but independently, R, and R, can be linker groups that connect to a cell surface oligosaccharide. For example, the R1 and R, linker groups can independently be: G-X16[(CH2)10X16]n(CH2)pX17-,G-X15(CH2)nX16-,or wherein G is site of attachment of the cell surface oligosaccharide and: Xi5, , X16 and X17 can independently be any of: O, NH, S, CH2, -CO-NH- or -NH-CO- (amides), -CO-O-of-O-CO- (esters), or carbonates or urethanes.

The numbers"m","n"and"p"may be any number from 0-20.

Figure 11 is a schematic representation of some bifunctional compounds useful in the present invention.

The drawings are not necessarily to scale. Certain features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness.

DETAILED DESCRIPTION OF THE INVENTION It is readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The term"agonist"refers to any compound or molecule which interacts with or binds to a selectin or interacts with the function of selectin to promote or enhance the selectin's oligosaccharide binding activity. As described in the examples, cGMP shows agonistic activity for E-selectin.

The term"antagonist"as used herein refers to a compound or molecule which interacts with or binds to a selectin or interacts with selectin's function to inhibit or block selectin's activity. As described in the examples, cAMP shows antagonistic activity for E-selectin.

The term"bifunctional compound"as used herein refers to a compound of the present invention which has molecules of two types linked by a linker group, each molecule showing a different function, thus, the term bifunctional. For instance, there would be a cell adhesion oligosaccharide with one type of activity and a nucleotide cyclic-3'5'monophosphate showing a different type of activity. Some examples of bifunctional compounds are shown in Fig. 11.

A specific example is shown by the distinct cyclic nucleotide binding site on E-selectin which suggests some small organic molecules that can moderate the specific cell-cell interactions promoted by selectin.

The general class of compounds that can have such a moderating influence are shown in Fig. 11. These compounds are bifunctional ligands composed of three key portions: (1) a cell-adhesion oligosaccharide linked via its reducing end or via a primary hydroxyl group through (2) a linker group, and (3) a nucleoside cyclic-3'-5'monophosphate, which attaches to the linker via a heterocyclic base. The proposed class of ligands possess two independent types of biomolecular fragments, each recognized by a spatially independent binding site on the target protein and a linker that may be designed for optimal specificity and binding affinity.

The term"cyclic nucleotide monophosphate"refers to the cyclic purine and pyrimidine monophosphate nucleotides. The most common Examples include guanosine-3'-5'cyclic monophosphate (cGMP) and adenosine-3'-5'cyclic monophosphate (cAMP). Some examples of other cyclic nucleotide monophosphates that are useful in the present invention are shown in Fig 10.

Although only one tautomer, ionized form, conformation and protonated state is drawn for each compound in Fig. 10, one skilled in the art recognizes that, some of the depicted structures correspond to minor species in solution and are listed as such for economy in description. The invention relates to all tautomers, conformers, ionized forms and protonated states, and especially is implied to hold for the major tautomeric, ionic, and protonated forms of these compounds that exist when they are in aqueous solution or in sera, or in the presence of solubilizing agents

under conditions of administration. Any counter-cation or counter-anion may be present with the major ionized form of the compound, under conditions of administration. Also, the compound need not be administered in solution, but it may be administered as a solid or liquid that forms such ionized or neutral species upon dissolution. In Fig 10. the depicted linker groups correspond to straight monomeric or polymeric compounds with a site of attachment at each end. The invention also includes the depicted straight linker or, alternatively for analogous branched linker groups or linker group containing internal carbocyclic or heterocyclic groups. Such branched linker groups may allow connection from one or multiple cell surface oligosaccharide units or mono-or oligosaccharide mimetic units (i. e. glycomimetic units) or glycoconjugate units to a chemical unit consisting of one or more cyclic nucleotides or cyclic nucleotide analogues. The invention includes all formulations in which a cyclic nucleotide or analogue is co-administered with a cell surface oligosaccharide or glycomimetic analogue. In the case of co-administration, the linker groups may be of the first type described above for Ri and R. For co-administration, the cyclic nucleotide or analogue need not be attached covalently to the cell surface oligosaccharides, as it is in some of the prior examples. Co-administration includes for example, cyclic nucleotides encapsulated within vesicles of a glycolipid containing a cell surface oligosaccharide as the sugar component of the glycolipid.

The term"oligosaccharide"as used herein refers to a sugar composed of two or more monosaccharide units joined by glycosidic bonds or analogs of same, or a glycomimetic compound that mimics such a cell surface mono-or oligosaccharide, or glycoconjugate. Examples

of some oligosaccharides that have been found useful in the present invention are shown in Fig. 8.

As used herein the term"regulation of cell proliferation"includes the regeneration or growth of tissue or the inhibition of growth of tissue and generally encompasses all of the treatments discussed herein whether or not they are providing an inhibitory effect (antagonistic) or a enhanced effect (agonist).

The regulation of cell proliferation can be by affecting cell-cell adhesion or migration of cells.

Thus, the inhibitory effect (antagonistic) or enhanced effect (agonist) of the present invention includes regulation of cell-cell adhesion or cell migration.

As used herein"selectin"refers to a family of molecules which are cell-surface carbohydrate binding proteins. They mediate cell-cell adhesions. Examples include E-selectin, P-selectin and L-selectin.

The expected effect of the length and hydrophobicity or hydrophilicity of the linker is illustrated below. If the linker between the oligosaccharide and the nucleotide functionalities is long enough, it will be possible for both of the biomolecular functional groups to bind to a single protein molecule. This would result in the temporary knockout of that particular target protein molecule from the pool of protein molecules participating in adhesion. If the linker is too short, oligomerization of the target proteins is expected. The oligosaccharide group would bind to one molecule of the target protein, and the cyclic nucleotide would bind to its characteristic binding site on a second molecule of the target protein.

Preferably a hydrophobic linker could interconnect the two protein-bound biomolecular functional groups via the channel corresponding to the lipid/peptide binding site, but either hydrophilic or hydrophobic or

amphipathic linkers are effective at inducing the effects and at promoting binding by the bifunctional ligand.

One aspect of the present invention is a bifunctional compound for the regulation of cellular proliferation, cell-cell adhesion or cell migration comprising a cell-adhesion oligosaccharide; a linker group attached to said cell-adhesion oligosaccharide by the reducing end of the cell adhesion oligosaccharide or by a primary hydroxyl group and a nucleotide cyclic- 3'-5'monophosphate attached to a linker group through a heterocyclic base.

In specific embodiments of the present invention the linker is attached to C8 of the purine ring of the nucleotide-3'-5'monophosphate.

A further specific embodiment includes a nucleotide-3'-5'monophosphate selected from the group in Fig. 10.

In additional specific embodiments the bifunctional compound includes a linker selected from the group consisting of the structures shown on Fig.

9A, 9B, 9C, 9D.

Another specific embodiment includes oligosaccharide selected from the group consisting of the compounds shown on Fig. 8.

Specific bifunctional compounds which are useful in the present invention include the compounds shown on Fig. 11.

Another aspect of the present invention is the regulation of cell-cell adhesion, cell migration and cellular proliferation. This method comprises contacting a therapeutic amount of the bifunctional compound with a selectin. The contacting of the bifunctional compound with selectin is done under conditions where the bifunctional compound either binds

or attaches to selectin or interferes with selectin's function in cell-cell adhesion or cell migration.

A further embodiment of the present invention is a method of detecting a test compound for agonistic or antagonistic activity to cell adhesion, migration or proliferation comprising the steps of contacting said test compound with selectin in cell culture; measuring the adhesion or growth of the cells in the cell culture, wherein compounds with agonistic activity to selectin will increase cell adhesion or proliferation or regeneration in growth of tissue as compared to normal cell adhesion or growth while those with antagonistic activity to E-selectin will inhibit or slow down the cell adhesion, proliferation or regeneration of growth in tissue as compared to normal cell adhesion or growth.

One skilled in the art readily recognizes the advantages of this procedure. It is apparent that depending on the linkers, oligosaccharide and nucleotide-3'-5'monophosphate used and the mode of administration the bifunctional compounds can control excessive cell proliferation as seen in cancer. Thus, the bifunctional compounds can be used as anti-cancer drugs for the treatment of cancer. They can also be used to serve as immunosuppressants in certain applications such as organ or tissue transplantation.

They also can be used as immunostimulators and providing immunological activity for disease such as AIDS. They can also have important uses in the control and generation and growth for new tissue. This can be useful in the repair and regeneration of tissue as well as in the transplantation or introduction of artificial tissue into the body. This can have beneficial effects in the treatment of burns or other replacement therapies requiring increased cell-cell adhesion, cell migration and/or cellular growth.

It is important to recognize that the bifunctional compounds can assert their effect by contact with the selectin and/or effecting selectin binding. The bifunctional compounds will be used in a pharmaceutically acceptable mode of delivery to the source of the tissue. This can include in vitro, in vivo or ex vivo administration.

THERAPEUTIC EFFECTIVE AMOUNT As used in an immunosuppressive application a compound will be considered therapeutically effective if it decreases or delays the onset of an immunoreactive or an inflammatory response to rejection of tissues or to some other disease state. When used as an immunostimulator it will be therapeutically effective if it increases the immune response or provides enhanced or increased immunological protection to the mammal receiving the bifunctional compound.

Further, in the case of cell growth or cell-cell adhesion or cell migration or cell regeneration, if it enhances the adhesion, migration or growth of cells or increases the rate of adhesion, migration or growth of cells, it is considered to be therapeuticly effective.

A skilled artisan readily recognizes that in many of these cases the bifunctional compound may not provide a cure but may only provide partial benefit. A physiological change having some benefit is considered therapeutically beneficial. Thus, an amount of bifunctional compound which provides a physiological change is considered an"effective amount"or a "therapeutic effective amount".

When discussing controlling or regulating cell proliferation herein, it includes the ability to either increase cellular proliferation as in the case of regeneration or growth of tissue or the ability to decrease the excessive proliferation or metastases seen

in the inflammation or cancer. The proliferative effect may be brought about by contact between other cell surface proteins that are brought together upon cellular adhesion A compound, molecule or composition is said to be "pharmacologically acceptable"if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a"therapeutically effective amount"if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in technical change in the physiology of a recipient mammal. For example, in the treatment of cancer or neoplastic disease, a compound which inhibits the tumor growth or decreases the size of the tumor would be therapeutically effective.

Dosage and Formulation The bifunctional compounds (active ingredients) of this invention can be formulated and administered to inhibit a variety of disease and nondisease states (including tumors, neoplasty, cancer, inflammatory disease, transplantation, tissue regeneration, tissue replacement) by any means that produces contact of the active ingredient (agonist or antagonist) with the agent (selectin) or its site of action in the body of a mammal. The bifunctional compounds can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dosages given as examples herein are the dosage ranges usually used in treating tumors, neoplasty and cancer. Dosages for other uses will vary depending on the physical effect desired. These relationships are generally known in the art for compounds having similar effects and can be readily determined by the skilled artisan.

The dosage administered will be a therapeutically effective amount of active ingredient and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired. Usually a daily dosage (therapeutic effective amount) of active ingredient can be about 5 to 400 milligrams per kilogram of body weight.

Ordinarily, 10 to 200, and preferably 10 to 50, milligram per kilogram per day given in divided doses 2 to 4 times a day or in sustained release form is effective to obtain desired results.

Dosage forms (compositions) suitable for internal administration contain from about 1.0 to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.05-95% by weight based on the total weight of the composition.

The active ingredient can be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups, emulsions and suspensions. The active ingredient can also be formulated for administration parenterally by injection, rapid infusion, nasopharyngeal absorption or dermoabsorption. The agent may be administered intramuscularly,

intravenously, or as a suppository. Additionally, gene therapy modes of introduction can be used to target the introduction of the compound. The skilled artisan readily recognizes that the dosage for this method must be adjusted depending on the efficiency of delivery.

Gelatin capsules contain the active ingredient and powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.

Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid either alone or combined are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl-or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's

Pharmaceutical Sciences, a standard reference text in this field.

Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyaminoacids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release.

Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows.

Capsules: Capsules are prepared by filling standard two-piece hard gelatin capsulates each with 100 milligram of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient. 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275

milligrams of microcrystalline cellulose, 11 milligrams of cornstarch and 98.8 milligrams of lactose.

Appropriate coatings may be applied to increase palatability or to delay absorption.

Injectable: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

Suspension: An aqueous suspension is prepared for oral administration so that each 5 millimeters contain 100 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution U. S. P. and 0.025 millimeters of vanillin.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner. The synthetic bifunctional compounds described in the examples can include any of the substitutions discussed earlier. Modifying groups can be added to the oligosaccharide, nucleotide cyclic-3'-5'and linker. The substitutions can enhance individually or collectively the affinity, the chemical stability and the cellular uptake properties of the specific bifunctional compound.

Example 1 Detection of Binding Sites Computational protein structural analysis, when applied to the crystal structure of E-selectin, identifies three significant sites of interest (Figs.

1A, 1B, 2A, 2B, 2C and 2D). The first of these sites (Fig. 1A), is a ligand binding cavity that is chemically complementary to and has a shape consistent with a branched trisaccharide portion of the earlier identified oligosaccharide ligand, Salez (12) This

binding site agrees with earlier models that were based upon analogy with similar sugar binding proteins. (13) The site is also consistent with recent crystallographic observations of SLeX binding to a related, modified mannose-binding protein. (14,15) If protein side chains are held fixed, interatomic clashes are difficult to avoid and it is difficult to distinguish a unique best binding mode from several alternative, nearly equivalent binding modes by rudimentary inspection. The depicted orientation of the tetrasaccharide is based upon the interpretation of the second site.

The second site (Figs. 2A-2D) is bound adjacent to the branched trisaccharide binding site. It is an elongated hydrophobic groove that corresponds to a recognition site for the peptide or lipid component of the glycoconjugates that comprise the predominant forms of the SLeX ligand on cell surfaces. Since this analysis is based on a crystal structure that represents a small fragment (157 residues) of a larger protein (619 residues), part of the second site may correspond to sites of contact between the analyzed fragment and the remainder of the protein. (11) The third binding site (Fig. 2B) is spatially removed from the oligosaccharide and peptide sites. It is located near the junction of the sugar binding protein (SBP) and human growth factor (HGF) subdomains of E-selectin. The size and shape of this binding site are consistent with a cyclic purine monophosphate in a syn conformation. This was confirmed by model building using the molecular graphics display program 0. (16) Upon manually positioning a cyclic nucleotide based solely upon the shape of the computer-generated binding site. It was found that (i) complementary hydrogen bonding arises between the nucleotide and the protein,

and (ii) face to edge stacking between a tryptophan residue (W1) and the heterocyclic base is observed. (17) Example 2 Experimental Confirmation of Cyclic Nucleotide Binding The specificity of E-selectin: nucleotide interaction was verified by examining the elution behavior of nanogram quantities of the soluble fragment of human E-selectin (R&D Systems, 535 residues) on three distinct types of cyclic-AMP affinity columns in the presence and absence of soluble, competitively binding cyclic and acyclic purine nucleotides. ELISA analyses were used to quantify the eluted E-selectin selectively. Commercially available E-selectin bound tightly to two of the affinity columns in a phosphate buffered saline solution lacking added cyclic nucleotides. (Fig. 3) The two cAMP-Sepharose columns to which E-selectin binds are those for which cAMP is covalently attached to the solid support via C8 of the purine base rather than via N-6. Thus, the C8 linkage appears to be critical for E-selectin adhesion and the N-6 linkage prevents binding. The molecular modeling suggests that C8 is directed away from the protein and towards solution in the syn conformer of the bound nucleotide.

E-selectin leaches slowly from the C8-linked affinity column with a 4-atom spacer group and gives rise to two broad, overlapping chromatographic peaks.

On the other hand, it binds tightly to the C8 linker cAMP-Sepharose with a 9-atom spacer group. Of the large portion (> 95%) of E-selectin that is initially bound to the longer affinity column, only about 15% can be eluted from the column with a buffer containing cAMP or cGMP (10 mM). The remaining E-selectin was eluted from this longer cAMP Sepharose column only after the column was digested by incubation (2h, room

temperature) with buffer containing cyclic nucleotide phosphodiesterase, divalent calcium cations, and phosphodiesterase activator (calmodulin). This digestive treatment of the cAMP Sepharose column was able to promote the displacement of E-selectin from its tight binding sites on this column.

The acyclic nucleotides AMP, GMP, ATP, and GTP do not elute E-selectin from undigested affinity columns under conditions similar to those examined for the cyclic nucleotides. If these acyclic nucleotides bind, they bind weakly, or at an alternative site on E-selectin. All affinity chromatographic studies were undertaken in the presence of a relatively high ionic strength phosphate buffered saline solution ( [PJ = 340 mM), to minimize any possible"ion-exchange"effects.

Non-specific binding to negatively charged polymers was observed for P-selectin but was not observed for E-selectin. (18,19) Furthermore, the marked increase in the chromatographic mobility of E-selectin, after treatment of the higher affinity column with cyclic nucleotide phosphodiesterase, implies that binding is weak to the resulting polyanionic Sepharose linked <BR> <BR> <BR> <BR> 5'-AMP column. Thus, as predicted by the computational analysis, the cyclic purine nucleotides bind tightly and selectively to E-selectin, whereas the acyclic nucleotides bind weakly or at an alternative site on E-selectin.

The results of affinity chromatography are consistent with binding between E-selectin and cyclic nucleotides. The small quantity of E-selectin used in these studies (sub-stoichiometric), along with the observation that E-selectin does not bind upon repeated reuse of the intact, undigested affinity column, suggests that E-selectin might bind to a small number of high affinity, cyclic nucleotide sites on the affinity column. These sites might correspond to small

contaminating amounts of cGMP in the commercially available cAMP-Sepharose affinity resin or to unique oligosaccharide structures present in the Sepharose matrix.

Assuming that binding of E-selectin to cAMP-Sepharose is exclusively due to nucleotide recognition, and if the rates of binding to and dissociation from the column are fast, then the observed adhesion to the column, in the presence of 10 mM cyclic nucleotides (a 3: 1 molar ratio between free and immobilized cyclic nucleotide) suggests at least one cyclic nucleotide site on E-selectin for which the value of KD is smaller than 1 ßM. This value for the dissociation constant represents binding that is comparable, if not tighter than, the values observed for the specific binding of cyclic nucleotides to other proteins. (20) Example 3 Ligand Dependent Protease Cleavage Patterns To demonstrate the cooperativity between the binding of E-selectin to cyclic nucleotides and to oligosaccharides, the proteolytic behavior of E-selectin in the presence of varying concentrations of cyclic nucleotides, SLeX, and divalent metal cations was analyzed. V8 protease digestion of E-selectin results in the formation of several distinct peptides (fragments f0 through fll) (20, 21) The V8 protease digestion products were analyzed by polyacrylamide gel electrophoresis under denaturing conditions (Fig. 4).

The binding in Fig. 4 is performed in phosphate-buffered saline (PBS, pH 7.0 w = 200 mM).

Approximately 15% of the bound E-selectin can be eluted from the column if cGMP (lOmM) is added to the eluent buffer, but most of the protein remains bound to the column. This protein also remains bound if cGMP is

removed from the eluent buffer and if the column is washed with PBS containing divalent cations. Upon treatment of the affinity column with cyclic nucleotide phosphodiesterase (1. OU, 2h, 23°C) and the enzyme activator calmodulin in the presence of divalent Ca (II) and Mg (II), the remaining E-selectin is quantitatively eluted by PBS, suggesting either that E-selectin is displaced from the column by the phosphodiesterase or, more likely, that E-selectin binds weakly to the immobilized 5'-AMP that is formed by the diesterase. A separate experiment showed that most of the bound E-selectin is not eluted by calmodulin alone.

Varying the concentration of ligands present during protease digestion results in distinct, characteristic differences in the quantity of each peptide fragment that is produced. Ligand dependent differences in the protease cleavage pattern result from relative differences in the rates and affinities (relative vmax/Km values) of the V8 protease for the different cleavage sites in ligand-bound forms of the protein. The cleavage results depend critically upon the presence of contaminating cleavage fragments in the different preparations of the protease and upon gel development procedures. However certain consistent features are found that can be used as markers of selectin-ligand interactions. Proteolysis of bovine serum albumin by V8 protease is unaffected by the presence of either nucleotides or oligosaccharides.

Thus, ligand dependent effects on cleavage were from the binding of ligands to E-selectin.

The pattern depicted in Fig. 5 is similar to the pattern observed in the presence of cGMP at high concentrations. The band FO is strong, however, only in the presence of high concentrations of SLeX.

High concentrations of free SLeX ligand and Ca+= markedly protect certain sites on E-selectin from

cleavage by V8 protease. When the concentration of free SLeX is greater than 1 mM, loss of bands for peptide fragments f7 through f9 become weak and flO and fll become markedly weaker (Fig. 5a through 5d). The decrease in the intensities of bands flO and fll is accompanied by an increase on the intensity of band fo.

Titration with SLeX shows that the transition of proteolysis pattern occurs at concentration of 1mM for SLeX. This dependence of protection from protease cleavage upon SLeX concentration is consistent with previously measured dissociation constants for SLe. (9,10) The presence of Ca+ and Mgit cations in the proteolysis mixture results in a marked decrease in the intensities of bands f7 through f9 relative to bands flO and fll, with differences being most pronounced in the absence of other ligands.

In the presence of cyclic purine nucleotides, but in the absence of both exogenous Ca"cation and free SLeX ligand, very slight yet discernible changes occur in the protease cleavage pattern. In the presence of cGMP, for example, very slight increase in the intensities of proteolysis bands f9 and flO occurs relative to the intensities of bands f7, f8 and fll.

Also bands f4 and f5, that are initially absent, begin to appear as very faint bands. When bands f4 and f5 are initially present, they become slightly darker.

There are also differences in the appearance of higher molecular weight bands fO, and fl. Although the differences in the intensities of the bands are slight and variable, the concentration range at which such transitions occur remains relatively consistent from experiment to experiment. In several experiments, the midpoint of transition in cleavage pattern occurs when the concentration of cGMP is in the range of 0.1-1.0 yM or above. The analogous transition point for cAMP,

when it is seen on the gels, occurs at a concentration that is about 100 fold greater than the concentration required for cGMP, under similar conditions. E-selectin quantitatively binds more tightly to cGMP than to cAMP when binding of the two is monitored at identical conditions.

The cleavage patterns obtained upon titrating E-selectin with SLeX at a fixed concentration of Mut+, Cl++, and one of the cyclic purine nucleotides ( [cNMP] = 100 ßM) indicates that transition in the cleavage pattern due to the low-affinity SLeX site is still present, but that an additional transition in the cleavage pattern, occurs at lower concentrations of SLeX. The appearance of this transition depends upon whether cAMP or cGMP is present. This lower concentration transition is signaled by an initial increase in the intensity of band fO and by the initial faint appearance of bands f4 and f5.

The decreased susceptibility to protease cleavage in the presence of ligands is consistent with a conformational change that is induced upon the binding of cGMP or divalent cations to E-selectin. The dependence of the effect upon ligand concentration provides an indication of the dissociation constant for a given ligand.

Example 4 Cyclic Nucleotide Binding Effects Cyclic nucleotides and SLeX ligand both appear to bind to E-selectin in the presence or absence of divalent cations and induce differences in the protease cleavage pattern. These differences are more pronounced for SLeX ligand. In the presence of cyclic nucleotides (100 M), an altered cleavage pattern is seen. The pattern shows increased intensity of bands fO, f4 and f5 when the SLeX ligand concentration

exceeds 100 nM for cGMP or 10 ßM for cAMP. This effect is most pronounced at higher concentrations of the SLeX ligand. At higher SLeX concentrations, the characteristic loss of peptide fragments flO-fll, along with bands f8 and f9 is observed. This observation is consistent with two binding sites, a high affinity site that forms in the presence of cyclic nucleotides, and a second, lower-affinity (rolling state) binding site (KD approximately 1 mM) that is unaffected by the presence of cyclic nucleotides. The apparent value of KD for the higher affinity E-selectin: SLeX complex is thus approximately 100 nM to 10 UM as ascertained by the approximate midpoint of the appearance of altered cleavage upon titration with free Solex. Thus the presence of bound cyclic nucleotides on E-selectin appears to uncover a site with enhanced binding affinity toward the SLeX ligand.

These conclusions about the interdependence of ligand affinities for E-selectin are consistent with earlier physiological studies on the contributions of externally added cyclic nucleotides to the inflammatory response. (23,24) Similar relative affinities for cAMP and cGMP were observed earlier in investigations on the effect of externally added cyclic nucleotides on inflammation and on lymphocyte proliferation. (22) Inhibitors of cyclic nucleotide phosphodiesterase diminish the inflammatory response and are among the more useful anti-inflammatory agents. Thus cGMP or higher concentrations of cAMP can serve as a trigger for E-selectin to assume the conformation required for the tight binding to oligosaccharides. It appears that cGMP can uncover a site for SLeX for which binding to SLeX is enhanced by 2 to 3 orders of magnitude. If cAMP binds competitively and does not induce tight oligosaccharide binding, then cAMP at high concentrations could attenuate the effect of cGMP.

Cyclic-GMP can be a selective trigger (agonist) for adhesion and cAMP can be an antagonist.

Example 5 Selective action by cGMP The role of cAMP and cGMP as chemical messengers of signal transduction is well established in organisms from prokaryotes to mammals. Furthermore, results prior to the biochemical and genetic characterization of selectins, implicated cyclic purine nucleotides as modulators of the inflammatory and immune responses. (22,23,24) In the selectin literature, the necessity for a third"chemoattractant"signal has been recognized, but the precise nature of this signal is uncertain. (10) In the absence of chemoattractants, lymphocytes undergo their characteristic rolling motion on the surface of blood vessel endothelium, whereas in the presence of chemoattractants, the lymphocytes adhere to this surface, undergo extravasation, and elicit an inflammatory response. The cyclic nucleotides are part of the ultimate signals that are generated by the presence of the chemoattractants. The selectins represent important sites of direct molecular action by these cyclic nucleotides in the inflammatory response. The earlier investigations that linked cyclic nucleotides with the inflammatory response, suggested that increased exogenous cGMP levels enhance inflammation and that increased cAMP levels diminish inflammation. (23) Results of immunostaining from over 20 years ago provides additional compelling evidence that cGMP is involved in cellular adhesion. (25,26) Cyclic-GMP is present at the sites of tight cell-cell contacts, where intercellular adhesion is enhanced, and cAMP is present at sinusoidal sites where direct cell- cell contacts are lacking. (See especially Fig. 2 and Fig. 3 of reference 25.) These data in combination

with that presented herein suggest a consistent model for lymphocyte targeting during tissue disruption (Fig.

7).

In the model shown in Fig. 7, the surfaces of cells lacking tight cell-cell contacts appear to have moderate surface concentrations of cAMP (amber). Those surfaces with tight cell-cell contacts are coated with cGMP (Green) (Steiner, et al. 1976). Normally (left) binding of selectin-bearing cells to cell-surface oligosaccharides is weak if the oligosaccharides are presented from surfaces that are uncoated with cGMP.

Based upon the findings presented here, tissue disruption (by either mechanical or biopathogenic means) is expected to expose cGMP-coated surface patches to the lymph or blood and to promote the enhanced adhesion of selectin-bearing cells to cell surface oligosaccharides (red). Although it seems likely that cAMP is normally present on the surfaces lacking tight contacts, it is possible that the observed cAMP arises from the response of cells to the procedures for microscopic sample preparation, perhaps as a further signal for the induction of inflammation.

According to the model tissue disruption or chemokine induced cytoskeletal motions, causes the cGMP coated surfaces of cells, which are normally involved in close cell-cell contacts, to become exposed to the blood or lymph. The combined, localized presence of cGMP and cell surface SLeX results in immediate adhesion mediated by selectins on the surfaces of lymphocytes and endothelial cells. Chemokine effectors of cellular adhesion can also exert their action on non-disrupted cells by locally activating nucleotide cyclases, by modulating the local concentrations of divalent cations, or by modulating the transport of cyclic nucleotides. (27) Thus cGMP, membrane bound selectins, and the cognate oligosaccharides can

comprise a ternary intercellular glue in which all three components must be present locally for the tightest binding to occur between cells. Cyclic-AMP at higher concentrations could substitute for cGMP. The presence of an exposed, cGMP-rich cell surface patch can serve as a signal to the immune system that tissue disruption has occurred, perhaps due to the presence of an invading organism. Formation of a selectin-cGMP-SLeX complex represents an immediate molecular response to this signal.

Complete mechanisms for the spatial localization of cyclic nucleotides at selected cell surfaces remain to be elucidated. The skilled artisan is aware of several possible mechanisms including directional export of cyclic nucleotides, directed deposition of adenylate cyclases, guanylate cyclases, cAMP or cGMP specific phosphodiesterases, or the directed deposition of alternative effectors that modify the nucleotide base preference of the cyclases and phosphodiesterases. (28) Anchored macromolecules are likely to be required for spatial localization because the relatively rapid physical diffusion of low molecular weight cyclic nucleotides must be overcome in vivo. The present invention provides bifunctional compounds for spatial localization.

The requirement of polar presentation of cyclic nucleotides and other effectors in the selective formation of intercellular contacts during development in multicellular organisms is implied by the observed stasis seen in figure 6 (see also reference 25). This stasis condition is recognized in the present invention as a means by which multicellular organisms detect and control of aberrant cellular growth in tissues. Polar presentation of cyclic nucleotides by cell surfaces can also serve as a signal to the immune system that cellular disruption has occurred. The local

concentration of cyclic nucleotide can serve as a marker that multicellular organisms use to detect whether a cell is living or is dead. Because polar cyclic nucleotide presentation is necessary for the static growth of tissues, a delicate balance is required for the position dependent production and removal of cyclic nucleotides on the cell surface.

Example 6 Cyclic Nucleotide Induction The E-selectin: cyclic nucleotide complex, provides an explanation for enhancement oligosaccharide binding in the presence of cyclic nucleotides. Such enhanced oligosaccharide binding can lead either to enhanced or diminished cell-cell adhesion, depending on the presence of the additional factors. One skilled in the art readily recognizes that the bifunctional compounds of the present invention are well suited to affect this process. Further it is recognized that the methods of identifying additional compounds are useful to deter antagonists and agonists of this process.

The cyclic nucleotide binding site lies at the junction between two covalently connected protein subdomains with few close, non-bonded interdomain interactions. The structural analysis software also reveals the presence of further unfilled space in the nucleotide cavity near this junction (Figs. 1A and 1B).

The location and shape of the extra unfilled space of the nucleotide cavity are consistent with those of similar cavities that become filled upon ligand-induced protein conformational changes. For example, a similar disposition of a familiar, ligand-shaped cavity bearing extra unfilled space was observed for both the open form of yeast hexokinase and for the Klenow fragment of DNA polymerase I, each of which has been observed to undergo a protein conformational change in the presence

of physiological ligands. (31,32) This extra space is consistent with a conformational change in E-selectin that is induced by the selective presence cyclic nucleotides. The observed alteration in the proteolytic cleavage pattern when SLeX is added, in the presence of cGMP, can arise from such a conformational change Example 7 Generality of the Cyclic Nucleotide Site Several residues in the general vicinity of the proposed cyclic nucleotide site are conserved identically or are conservatively replaced across several mammalian species and across selectin classes E, P, and L. (Fig. 2) Thus, the recognition that cyclic nucleotides represent a general mechanism for triggering throughout the three known classes. No sequence similarity to other proteins for which interactions with cyclic nucleotides had been established was seen. Likewise, no striking structural similarity could be observed when the experimental structure of a complex between cAMP and the catabolite gene activator protein (CAP) of E. coli.

To search for other proteins with possibly unidentified cyclic nucleotide binding sites, an amino sequence"profile"was generated from the residues 101 through 112 in the alignment in Fig. 2. This portion of the sequence corresponds to the longest contiguous segment where a large proportion of the residues are proximal to the cyclic nucleotide of the present invention. A search of the protein sequence database with this profile revealed few notably strong"hits" (scores greater than 5 o from the mean). Notable among these"hits"were plant derived superoxide dismutases.

These plant derived superoxide dismutases are known to respond to the presence of ethylene as a ripening

hormone. (36) Intriguingly, the homologous peptide segment in human superoxide dismutase (SOD residue numbers 146 through 157), a protein of known structure, is adjacent to a protein surface cavity and is opposed in space by a peptide strand with the amino acid sequence IQN. (37) This is similar to that observed at the proposed cyclic nucleotide site in E-selectin (residues 71 through 73). However, SOD only shows weak inhibition by cyclic nucleotides (KI >lmM).

Example 8 Cyclic Nucleotide Signaling Cyclic nucleotides exert an influence on intracellular processes by modulating the activity of cyclic nucleotide dependent protein kinases. (34) Extracellular action of cyclic nucleotides has been suggested since the discovery of extracellular cyclic nucleotide phosphodiesterases. (28) Inhibitors of cyclic nucleotide phosphodiesterases are presently used as anti-inflammatory drugs. For consistency with the present invention, such an anti-inflammatory effect would be expected if the phosphodiesterases that are inhibited by these drugs were selective for cAMP over cGMP. The results presented here suggest a new means by which cyclic nucleotides exert their influence on the onset inflammation.

Example 9 Summary To summarize the present invention, (i) cyclic purine nucleotides bind to E-selectin orders of magnitude more tightly than the originally recognized cognate oligosaccharide, Salez; (ii) prior association of cGMP with E-selectin produces, a form of the protein with heightened affinity for the previously recognized SLeX related oligosaccharide; (iii) the presence of

cyclic nucleotides is a signal for high affinity cell-cell contact; (iv) cAMP is a competitive ligand that binds to E-selectin more weakly than cGMP; (v) cAMP does not enhance the affinity between E-selectin and SLeX; (vi) cAMP and its analogues and derivatives can serve to inhibit the formation of cell-cell contacts competitively; (vii) cGMP and its analogues and derivatives can serve to enhance formation of selectin-oligosaccharide cell-cell interaction that can lead to enhanced contacts and cellular proliferation; (viii) this molecular mechanism of cyclic nucleotide action in cell adhesion was first suggested by the computational analysis of the structure of a fragment of human E-selectin first described herein; and (ix) the present invention was unexpected and unnoticeable prior to this computer-based prediction; and (x) the amino acid sequence similarity of all selectins in the region close to the predicted cyclic nucleotide site, reveals that cyclic nucleotides are ligands for all P-, L-and E-selectins.

REFERENCES All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains.

All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

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One skilled in the art readily appreciates that the invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned as well as those inherent therein. Bifunctional compounds, pharmaceutical compositions, treatments, methods, procedures and techniques described herein are presently representative of the preferred embodiments and are intended to be exemplary and are not intended as limitations of the scope. Changes therein and other uses will occur those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the pending claims.