WO2000037653A1 - Structural basis for pterin function in nitric oxide synthase - Google Patents

Abstract

This invention describes methods and elucidates the three dimensional structure of nitric oxide synthase and its variants. Also described are methods of structural analysis to determine the binding of pterin to endothelial nitric oxide synthase and methods for screening and identifying small molecule modulators of endothelial nitric oxide synthase proteins and their variants. The invention also describes methods for identifying drugs that modulate nitric oxide synthase and its variants and are effective against diseased states in which NO signaling is defective or insufficient.

Description

3

STRUCTURAL BASIS FOR PTERIN FUNCTION IN NITRIC OXIDE SYNTHASE

The government owns rights in the present invention pursuant to grant numbers GM 32688, GM 52419 and HL 30050 from the National Institutes of Health.

1. Field of the Invention

The present invention relates generally to the fields of protein structure and crystallography. More particularly, it concerns the three dimensional structure of nitric oxide synthase.

2. Description of Related Art

Nitric oxide (NO^*), a free radical, is an ubiquitous signaling molecule which participates in diverse cellular processes, including regulation of blood pressure, neurotransmission, and the immune response (Dinerman et al, 1993).

NO, is well recognized as having various biologically relevant activities. For example, NO activates soluble guanylate cyclase in vascular smooth muscle cells which in turn increase cyclic guanosine monophosphate (cGMP) resulting in vasorelaxation, (Waldman et al, 1987) and ultimately leads to vasodilation and a reduction in blood pressure. It is well established that the NOS family of enzymes form nitric oxide from L- arginine, and the NO produced is responsible for the endothelium dependent relaxation and activation of soluble guanylate cyclase, neurotransmission in the central and peripheral nervous systems, and activated macrophage cytotoxicity (Sessa, William C, 1994).

NO^* production is tightly regulated by nitric oxide synthases (NOS), a family of enzymes of which three genetically encoded isoforms have been identified (Knowles and

Moncada, 1994; Marietta, 1993; Masters et al, 1996). The neuronal (nNOS) and endothelial NOS (eNOS) are constitutive with post-translational regulation of enzyme activity. The inducible isoform (iNOS) is regulated mostly at the level of transcriptioή

All three isoforms of NOS oxidize L-arginine to L-citrulline and NO.

Each NOS isoform consists of a heme domain linked via a calmodulin binding linker peptide to a P450 reductase-like diflavin domain giving a large polypeptide (130 kDa - 160 kDa). Only dimeric NOS is catalytically active. Upon Ca²⁺/calmodulin binding, the FAD of the reductase domain transfers reducing equivalents from NADPH to FMN, which in turn, reduces the heme iron. Reduction of the heme iron leads to O activation followed by oxidation of a L-Arg guanidino N atom to NO^* and L-citrulline. All three NOS isoforms exhibit an absolute requirement for tetrahydrobiopterin, (referred to as BH₄), as a cofactor to function (Tayeh and Marietta, 1989; Kwon et al., 1989), but the precise role it plays has remained elusive (Hemmens and Mayer, 1997).

Due to the linking of the heme and flavin domains, the various NOS isoforms are large and range in size from 130 to 160 kDa. The N-terminal domain of NOS contains the heme active center where L-arginine and BH₄ bind. While the flavin reductase domain is similar in sequence to the P450 reductase, the NOS heme domain bears little resemblance in sequence to P450s even though the NOS heme domain exhibits characteristics strikingly similar to those of cytochrome P450 monooxygenases.

Recently, the structures of mouse iNOS heme domains and cytochrome P450 reductase have both been determined (Crane and Trainer, 1997; Wang et al., 1997). In both of these studies, short versions of the full length iNOS were used. For iNOS, the heme domain consisting of residues 115-498 was used, whereas for P450 reductase the soluble fragment (amino acids 57-676) was used. These investigations revealed that the flavin domain of P450 reductase is structurally similar to flavodoxins while the FAD- NADPH domain is structurally homologous to ferrodoxin reductase. Additionally, a linker domain connects the FMN and FAD domains. The flavins are only 4A apart which blocks the edge of FMN that would normally be exposed in flavodoxins. Despite the extensive characterization of the heme domain of iNOS, little TS known about the structure of eNOS or the BH₄ binding domain therein. To understand the basis for the obligatory pterin cofactor requirement in catalysis and structure, there is a need to solve the structure of the eNOS heme domain, both in the presence and absence of BH₄ Such findings will have major implications in drug discovery and mechanisms of action of the proteins.

SUMMARY OF THE INVENTION

To overcome the limitations of the art the present inventors have developed methods for the structural analysis of endothelial nitric oxide synthase. In one embodiment the method comprises: a) subcloning a gene encoding the endothelial nitric oxide synthase in an expression vector; b) obtaining expression of the endothelial nitric oxide synthase protein from the vector; c) purifying the endothelial nitric oxide synthase protein; d) preparing an endothelial nitric oxide synthase protein sample amenable for crystallization; e) crystallizing the endothelial nitric oxide synthase protein sample; and f) performing a three-dimensional structural analysis of the endothelial nitric oxide synthase by x-ray crystallography.

In one embodiment, the endothelial nitric oxide synthase protein expressed is a variant endothelial nitric oxide synthase protein. In one aspect of the method, the crystallizing comprises practicing a sitting drop vapor-diffusion method. To compare the crystal structures in the presence and absence of tetrahydrobiopterin, in one embodiment, the crystallizing is performed in the presence of tetrahydrobiopterin while in another embodiment, the crystallizing is performed in the absence of tetrahydrobiopterin. In a further embodiment the crystal structures are determined by performing x-ray crystallography on the endothelial nitric oxide synthase protein crystallized both in the presence and in the absence of tetrahydrobiopterin.

The invention also describes a method of structural analysis to determine the binding of pterin to endothelial nitric oxide synthase that comprises: a) determining the crystal structure of a dimeric heme domain of the endothelial nitric oxide synthase in tne presence of the pterin; and b) determining the crystal structure of a dimeric heme domain of the endothelial nitric oxide synthase in the absence of the pterin; and comparing the crystal structures. In one embodiment the pterin is tetrahydrobiopterin. In a specific embodiment, the tetrahydobiopterin is (l'R,2'S,6R)-5,6,7,8-tetrahydrobiopterin. In one aspect, the crystal structure is determined at a resolution of between about 1.9 A to about

2.1 A.

The invention also describes methods for screening and identifying small molecule modulators of endothelial nitric oxide synthase proteins comprising: a) providing a pterin-free endothelial nitric oxide synthase structure; b) screening the small molecule modulators for their ability to bind to a pterin-binding site of the endothelial nitric oxide synthase ; and c) performing assays to determine the ability of the small molecule modulators to modulate the activity of endothelial nitric oxide synthase. A modulator is defined herein as a molecule that is capable of activating or inhibiting the activity of endothelial nitric oxide synthase. The molecule can be a small molecule.

In one embodiment, the small molecule modulator inhibits endothelial nitric oxide synthase. In another embodiment, the small molecule modulator activates endothelial nitric oxide synthase. In one embodiment, the endothelial nitric oxide synthase protein expressed is a variant endothelial nitric oxide synthase protein. In another embodiment, the pterin is tetrahydrobiopterin. In a specific embodiment, the tetrahydobiopterin is (l'R,2'S,6R)-5,6,7,8-tetrahydrobiopterin.

In one aspect of the method, the small molecule modulators are molecules and chemical-fragments from chemical-fragment libraries. In another aspect, the screening is performed by computerized methods. In yet another aspect of the method, the assays to determine the activity of endothelial nitric oxide synthase are performed in vitro or in vivo. In another embodiment, the invention describes methods for identifying drugs against diseased states in which nitric oxide signaling is defective or insufficient comprising: a) providing a tetrahydrobiopterin-free endothelial nitric oxide synthase structure; b) screening the drugs for their ability to bind the tetrahydrobiopterin binding site; and c) performing assays to determine the ability of the drugs to activate the endothelial nitric oxide synthase. The diseased states include but are not limited to conditions wherein defective or insufficient nitric oxide signaling leads to impaired neurotransmission; impaired insulin release; impaired penile erection; impaired vasorelaxation; and impaired oxygen detection.

The invention also provides endothelial nitric oxide synthase structure, obtained by the process comprising: a) subcloning a gene encoding the endothelial nitric oxide synthase in an expression vector; b) obtaining expression of the endothelial nitric oxide synthase protein from the vector; c) purifying the endothelial nitric oxide synthase protein; d) preparing an endothelial nitric oxide synthase protein sample amenable for crystallization; e) crystallizing the endothelial nitric oxide synthase protein sample; and f) performing a three-dimensional structural analysis of the endothelial nitric oxide synthase by x-ray crystallography. The structure of the endothelial nitric oxide synthase is described in the specification.

The invention also provides methods for screening and identifying a candidate substance with the ability to inhibit endothelial nitric oxide synthase comprising: a) obtaining a cell with endothelial nitric oxide synthase activity; b) admixing the candidate substance with the cell; and c) determining the ability of the candidate substance to inhibit the endothelial nitric oxide synthase activity of the cell.

The invention further provides a method for screening and identifying a candidate substance with the ability to inhibit endothelial nitric oxide synthase comprising: a) obtaining a purified endothelial nitric oxide synthase; b) admixing the endothelial nitric oxide synthase with the candidate substance; and c) performing X-ray crystallography analysis to determine the binding of the candidate substance. 3

A" or "an" is defined herein to mean "at least one" when used in combination with the term "comprising" in the specification and claims.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Panel A, schematic representation of the eNOS heme domain dimer viewed perpendicular to the dyed axis of symmetry. The two BH₄ molecules are shown as yellow space-filled models. The zinc located along the dyed axis of symmetry is highlighted as a red ball and labeled. Panel B, molecular surface map of the electrostatic potential of the eNOS heme domain dimer calculated using GRASP. The blue an99d red contours represent positive and negative potential, respectively. Fully saturated color indicates a potential of 5 kT. This view is rotated 90° from the orientation shown in Panel A such that the viewer is looking toward the ZnS center directly along the two-fold relating the monomers. The surface surrounding the ZnS₄ is the most extensive electropositive region on the dimer and could provide an electrostatic docking site for the FMN/FAD reductase. This could enable the electron donors (FMN domains of the reductase) to approach close to the ZnS₄ center thereby providing a conduit to either/both pterin and heme groups. Interestingly, both Mss4 and LIM proteins utilise

ZnS₄ containing surfaces to mediate protein-protein interaction (Yu and Schreiber, 1995;

Rabbits and Boehm, 1990; Schmeichel and Beckerle, 1994).

FIG. 2. Stereo view of the 2Fo-Fc 1.9A omit electron density map around the ZnS center. The map was obtained from model phases after a round of simulated annealing with the atoms shown excluded from the refinement. The map is contoured at lσ (blue) and lOσ (black). The zinc ion was identified using anomalous dispersion effects characteristics for the metal. X-ray wavelengths of 1.280 A and 1.286 A (zinc absorption edge, λ = 1.283 A) were chosen using a tunable synchrotron X-ray source. Zinc exhibits significant anomalous scattering effects at 1.280 A with little anomalous scattering contribution at 1.286 A. Heme Fe exhibits some anomalous scattering at both wavelengths (iron absorption edge, λ = 1.739 A). Direct methods (Sheldrick, 1997) were also used to independently confirm the location of the metal center. The cysteine residues of eNOS involved in zinc coordination are strictly conserved in all NOS sequences known to date indicating that the metal center is a common feature in all NOS isoforms. The ligands, Cys 96 and Cys 101, are part of a small 3-stranded antiparallel β-sheet (2 strands from one monomer and 1 strand from the other) that orients Cys 96 and Cys 101 in the same direction directly across antiparallel strands. In addition to β-strand main chain H-bonds, Sγ (96) and Sγ (101) form H-bonds with the peptide NH of residues 102 and 103, respectively. Crane et al. (1998) interpreted this region in iNOS as an inter-subunit disulfide bond between symmetry related Cys 109 residues which corresponds to one of the ligands in eNOS, Cys 101. The reason for the discrepancy is not clear. However, the iNOS heme domain dimer structure was solved at medium resolution (2.6 A) and Crane et al. (1998) noted a disordering in residues 101-107 immediately preceding Cys 109. Considering the strongly reducing conditions in the cytosol, the formation of a disulfide would be both kinetically and thermodynamically disfavored (Braakman et al, 1994). Hence, the inventors conclude that the loss of zinc in the iNOS structure led to the disordering of the polypeptide chain. ZnS₄ centers have been observed in four other enzymes where they play a structural role (Lipscomb and Strater, 1996; Tsukihara et al, 1995; Vallee and Auld, 1993). In E. coli Ada protein a catalytic function of ZnS₄ has been demonstrated (Lipscomb and Strater, 1996; Tsukihara et ai, 1995; Vallee and Auld, 1993).

FIG. 3. Comparison of the pterin-free and -bound structures at BH binding site. L-Arg was found in the BH₄ binding pocket in one subunit whereas only glycerol and water molecules could be modeled in the other subunit. Panels A and B are the 2Fo-Fc omit electron density maps contoured at lσ with arginine or glycerol excluded in the calculation, respectively. Side chains of the same color belong to the same monomer. Panel C shows BH being sandwiched between Trp 449 of the same subunit (green) and Phe 462 from the other (cyan). Among the extensive H-bonding interactions of BH₄ to protein, two crucial ones are between N at position 3 of the pterin ring and a heme propionate, and between the OH in dihydroxypropyl side chain of BH and Serl04 carbonyl oxygen. These interactions are closely mimicked by L-arginine with its guanidino nitrogen and primary amino group, respectively, as depicted in pang D. In addition, a new water molecule in panel D satisfies the H-bonding interactions of the BH₄ amino group. Therefore, L-Arg in the BH₄ site is able to closely mimic the H-bonding and aromatic stacking interactions in the BH complex. The structure of the BH complex in panel C has L-Arg bound in the heme pocket while in panel D, the- inhibitor, SE1TU, is in the heme pocket. The protein structure of the inhibitor and substrate complexes are essentially identical. In addition, the structure of the substrate or inhibitor complex with BH₄ bound exhibit no differences at the BH₄ site.

FIG. 4. The ZnS₄ center and its relation to BH₄. The metal ion (white ball) is equidistant from each BH , a distance of 12 A. The peptide carbonyl oxygen of Ser 104 H-bonds with one BH₄ OH group. ZnS₄ plays an effector role in helping to form and stabilize the pterin binding pocket which, in turn, promotes substrate binding. Therefore, the ZnS₄ center, BH₄, and substrate are all structurally linked at the dimer interface. Both the zinc atom and its cysteine ligands are accessible to solvent.

FIG. 5. The proposed mechanism for pterin radical formation in NOS catalysis. I-BH₄ showing the ring numbering scheme. The pKa of N3 is near 10.6. II- BH₄ mono cation form and III the BH₄ ^+* radical, respectively. The inventors are proposing that NOS is designed to stabilize III, the radical cation. Stabilization of aromatic cation radicals is reminiscent of cytochrome c peroxidase which is designed to stabilize a cationic Trp radical essential for catalysis (Sivaraja et al, 1989; Houseman et al, 1993).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

NO is a key intracellular signal and defensive cytotoxin in the nervous, muscular, cardiovascular, and immune systems (Moncada and Higgs, 1993; Schmidt and Walter, 1994; Nathan, 1997; Christopherson and Bredt, 1997; Marietta, 1993; Mayer and Werner, 1995; Masters et al, 1996; Steuhr et al, 1997). eNOS produces low NO concentrations for neurotransmission, insulin release, penile erection, vasorelaxation, oxygen detection, and the like. Since NO was voted the molecule of the year by Science in 1992, there has been a tremendous amount of work on the pharmacological properties of this molecule. Despite all these studies, little is known about the structure of eNOS. Once the structural determination of eNOS has been made, designing agents to potentiate or inhibit the action of NO becomes a realizable goal. The present invention is directed to addressing these needs.

A. The Present Invention

The present invention describes the crystal structure of the dimeric heme domain of endothelial nitric oxide synthase (eNOS). This structure was determined both in the presence and absence of (l'R,2'S,6R)-5,6,7,8-tetrahydrobiopterin (BH₄) to understand its role as an indispensable cofactor in nitric oxide biosynthesis. The BH₄ bound structure at 1.9 A reveals a novel zinc tetrathiolate (ZnS₄) sandwiched at the dimer interface which functions by maintaining the integrity of the pterin and substrate binding sites. The pterin-free structure at 2.1 A unambiguously establishes an obligatory function for BH₄ in catalysis and rules out a role in the dimerization process. These structures suggest a reaction mechanism that involves a pterin radical. The unusual finding that an L-arginine

(L-Arg) is bound at the BH₄ site in the pterin-free structure suggests evolution of cofactor recognition from a common L-Arg-binding ancestor in the primordial NOS catalytic machinery.

Here the inventors report the 1.9 A crystal structure of the dimeric heme domain of constitutive eNOS (Table 1), which maintains the catalytic site for NO" synthesis. The overall fold of the bovine eNOS heme domain (FIG. 1) is similar to that reported for the 2.6 A structure of the iNOS heme domain dimer (Crane et al, 1998). eNOS belongs to the α/β protein class and the quaternary structure is characterized by a tightly packed dimer interface which buries 3000 A² per subunit of solvent accessible surface (55% hydrophobic and 45% polar).

To understand the basis for the obligatory pterin cofactor requirement in catalysis and structure, the inventors have solved the structure of eNOS heme domain both in the presence and absence of BH₄ resulting in three major structural findings. First, the inventors have found a novel ZnS center (in both pterin-bound and -free structures) located at the bottom of the dimer interface (FIG. 1) with the metal tetrahedrally coordinated by two pairs of symmetry-related cysteine residues (FIG. 2) from each subunit. BH H-bonds directly with a heme propionate which also entertains H-bonds with the substrate, L-Arg (FIG. 3C). The zinc is positioned equidistant from each heme (21.6 A) with one of its ligands, Cys 101, separated by only two residues from Ser 104 which H-bonds directly to BH (FIG. 4). In addition, Val 105 forms a direct nonbonded contact with BH₄. Therefore, disruption of the metal center either by demetallation or ligand removal via mutagenesis will distort this region of the polypeptide chain resulting in greatly diminished affinity for BH . Since BH₄ couples directly to the heme, alterations at the pterin site will in turn affect the heme pocket and L-Arg binding. A number of studies (Chen et al, 1995; Rodrfguez-Crespo et al, 1997; Ghosh et al, 1997; Venema et al, 1997; Miller et al, 1997) confirm the dramatic loss in protein stability, catalytic activity, and BH₄ binding upon removal of Cys 96 and/or Cys 101 in eNOS or their counterparts in the inducible and neuronal isoforms. The identification of the new ZnS center in NOS unambiguously establishes me structural role played by these cysteine residues in forming and maintaining the integrity of the pterin site. The inventors' finding of zinc in eNOS has pathophysiological implications as well. Inherited vascular dysfunction may arise from mutations that specifically weaken zinc affinity resulting in a dysfunctional eNOS. For example, in familial amyotropic lateral sclerosis (ALS), over 50 independent mutations in Cu/Zn-superoxide dismutase give rise to a common toxic phenotype invariably characterized by decreased zinc affinity up to 100,000 fold (Lyons et al, 1996; Crow et al, 1997).

Second, in the absence of BH , the overall eNOS heme domain structure remains unchanged both in the tertiary topology and quaternary structure. These results sharply contrast with the conclusions of Crane et al, (1998) who proposed that large conformational changes concomitant with BH binding were necessary for inducible NOS dimerization. By comparison of the pterin-bound and pterin-free structures, it is clear that the site preexists (FIG. 3) and does not form via an induced fit process. The pterin-free structure also does not show any major changes at the active site either in the substrate- or inhibitor (S-ethylisothiourea, SEITU)-bound conformations.

Third, in addition to the new ZnS₄ center, the most striking and novel finding is the identification of an L-Arg bound to the pterin site in the absence of BH (FIG. 3A and FIG. 3D). The mode of L-Arg interaction at the pterin site surprisingly mimics that entertained by BH₄ itself. Two crucial H-bonds, one between a guanidino N with the heme propionate and the other between the primary amino group and Ser 104 are strictly conserved (FIG. 3D). Moreover, the planar guanidinium group is sandwiched between two aromatic groups, one from each monomer, exactly as in the BH₄ complex. Solvent interactions also are similar to the BH₄ complex. The affinity for L-Arg must be great since no exogenous L-Arg was added during crystallization. The specificity of L-Arg recognition at the pterin site appears to require both the guanidino function and the primary amino group since SEITU was unable to displace L-Arg from the pterin site. The inventors' observation involving L-Arg mimicry of BH₄ has not been previously observed and has broad evolutionary implications. It is possible that the pterin site was originally an L-Arg binding site and later evolved into a BH site. The "arginine paradox" (McDonald et al, 1997), which refers to the ability of extracellular L-Arg to drive NO^* biosynthesis, amidst the large intracellular substrate pool, suggests limited substrate availability in specialized cellular compartments and may have favored the evolution of a second L-Arg site as a substrate reserve in a primordial setting. The inventors propose that the higher affinity of NOS for BH (K_d ~ 20 nM; Werner- Felmayer and Gross, 1996) evolved to repel competition from L-Arg which is abundant in cells (0.2 - 0.8 mM; Hecker et al, 1990). A striking corollary can be established between the inventors' finding and the ability of Tetrahymena group I catalytic RNA to specifically recognize L-Arg as a mimic for guanosine binding (Yarus, 1988).

Given this elegant mimicry of the obligatory cofactor by L-Arg, the question arises as to why NOS chose a pterin in place of L-Arg for sustaining function. The strict requirement for reduced pterin cannot apparently be explained by a purely structural role since L-Arg can serve this same function. BH likely plays a direct functional role. A well-known function of pterin is to cycle between quinonoid BH and BH₄ in metal-dependent aromatic amino acid hydroxylases (Kaufman, 1997). To date, direct evidence for both pterin cycling and pterin function in NOS is lacking (Hemmens and

Mayer, 1997). The inventors' finding that the pterin site in NOS recruits L-Arg, provides structural insights on why BH₄ can serve as a single electron donor. The guanidino group of L-Arg is one of the strongest organic bases and the ability of the pterin site to bind

L-Arg argues in favor of preferential binding of a fully protonated species of BH₄. Owing to the essentially identical nature of the pterin site in both the L-Arg and BH₄ complexes, it appears that bound BH₄ experiences the same electrostatic environment as

L-Arg.

The inventors propose a mechanism (FIG. 5) in which the NOS pterin site modulates the pKa of N5 of the pyrazine ring and also provides an "acidic" milieu known to stabilize pterin radicals (Pfleiderer, 1985; Kappock and Caradonna, 1996; Eberlein et al, 1984) that can serve as one electron donors (Bee et al, 1998) in NO^* biosynthesis.

Cycling from the pterin radical back to BH₄ may be achieved via electron transfer from the reductase domain while the pterin remains bound to NOS. Another important structural feature that will substantially contribute to pterin radical stabilization is the π stacking interaction with Trp 449. Such aromatic stacking is not found in binding sites of other pterin utilizing enzymes (Bourne et al, 1991; Auerbach et al, 1997), but resonance stabilization of flavin semiquinone radical found in flavoproteins are mediated through stacking interactions (Massey, 1994; Wang et al, 1997). NOS provides an interesting scenario in which an enzyme has evolved to produce a pterin function that may mimic flavoprotein systems in structure and function.

Finally, the pterin-free eNOS structure also has relevance to human medicine. There is strong evidence for superoxide generation by the heme domain of eNOS in the absence of BH thereby leading to potential pathophysiology. Endothelial dysfunction is reversed in hypercholesterolemic patients treated with BH₄ and has been shown to be a NOS related action (Stroes et al, 1997; Piper, 1997; Kinoshita et al, 1997; Cosentino et al, 1998). It is conceivable that BH₄ deficient eNOS may be a reality in vascular pathologies in which L-Arg could substitute at the BH₄ site in pterin-depleted states. The availability of pterin-free eNOS structure paves the way for rational design of both pterin-dependent and independent activators that can restore endothelial function. These and other implications of the findings present herein are discussed in further detail herein below.

B. Crystallization Techniques The techniques used for the crystallization of a protein for crystallographic resolution of protein are well known to those of skill in the art. One technique for crystallization is referred to as the microbatch technique. The microbatch technique is ideal for the rapid determination of the phase diagram of a protein. If the concentration of crystallizable protein is plotted against the concentration of a precipitant, microbatch results can be used to divide the space represented into several areas. Microbatching has been extensively described in the literature, see for example, Chayen et al, 1990; ChayJn et al, 1992; Chayen et al, 1994.

In microbatching, at high concentrations of both protein and precipitant, the protein precipitates as an amorphous material. At lower concentrations, crystal nuclei may form, which may grow to form diffracting crystals. At still lower concentrations, nuclei will not form, so generally no crystals appear. However, if a nucleus or crystal is placed in such a solution, it will grow to form a large crystal. This area, where crystal growth but not nucleation takes place, is sometimes referred to as "the metastable zone". At the lowest concentrations, the protein is completely soluble. It is often found that crystals grown in the metastable zone are better ordered and diffract better than crystals grown at higher concentrations. The microseeding approach described herein below includes a simple method of finding the metastable zone and introducing crystal seeds to it.

Single crystals of a given protein are generally obtained by microseeding, a valuable measurement in this step is the precipitation point of a protein at a single protein concentration which is described by Stewart and Khimasia (1994). A well with a well- formed crystal is selected and the crystal is transferred to a glass depression plate containing 40 μl of harvesting buffer with a high PEG concentration. The initial crystal is ground up with a needle or a glass fibre with a ball at the tip. The resulting suspension is added to an Eppendorf tube containing 100 μl of the harvesting buffer the tube was centrifuged for a five minutes at around 100 g. The supernatant from this step contains the seeds; this supernatant may then be diluted to yield varying concentrations of seeding solution. The seeding solutions are used to seed sitting drop crystallization trials in appropriate crystallization plates (e.g., CrystalClear plates Douglas Instruments). 100 μl of solution was used in the reservoirs. Next, buffer, protein and PEG are dispensed automatically into the sample wells of the CrystalClear plates. By dispensing droplets marginally below the reservoir concentration, the need for equilibration before seeding may be avoided - the concentration was not so low that the nuclei dissolved. Finally 0.3 μl of each of the seeding solutions produced is added by hand to each sample well with a 5 μl Hamilton syringe. The plates were then sealed and crystals allowed to form. Small single crystals will appear after several days.

Large crystals are obtained by macroseeding. Using a rayon loop, a small single crystal is transferred into reservoir solution, allowed to wash for several minutes, and then transferred into another drop that has been equilibrated for 3-5 days. The same reservoir and drop condition used to obtain the initial aggregates also are used for the subsequent micro and macroseeding. The crystals attain their maximum size in 5-10 days following macroseeding. Typical crystal dimensions are 0.3 mm x 0.3 mm x 0.6 mm.

C. Specific eNOS Active Site Modifications

Given that the present invention has determined the crystal structure of eNOS, it is now possible to modify various specific residues within the protein to determine the roles of particular residues within the active site. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table, Table 1 :

TABLE 1

Amino Acid Names and abbreviations Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC uuu

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp w UGG

Tyrosine Tyr Y UAC UAU

It is known that certain amino acids may be substituted for other amino acids in a protein structure in order to modify or improve its antigenicity or activity (see, e.g., Kyte and Doolittle, 1982; Hopp, U.S. Patent 4,554,101, incorporated herein by reference). For example, through the substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide which result in increased activity or stability. Alternatively, amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide- molecule conjugates which retain enough antigenicity of the starting peptide to be useful for other purposes. For example, a selected eNOS peptide bound to a solid support might be constructed which would have particular advantages in diagnostic embodiments. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein has been discussed generally by Kyte and Doolittle (1982), wherein it is found that certain amino acids may be substituted for other amino acids having a similar hydropathic index or core and still retain a similar biological activity. As displayed in Table 2 below, amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with substrate molecules. Preferred substitutions which result in an antigenically equivalent peptide or protein will generally involve amino acids having index scores within ±2 units of one another, and more preferably within ±1 unit, and even more preferably, within ±0.5 units.

TABLE 2 3

Amino Acid Hydropathic Index

Isoleucine 4.5

Valine 4.2

Leucine 3.8

Phenylalanine 2.8

Cysteine/cystine 2.5

Methionine 1.9

Alanine 1.8

Glycine -0.4

Threonine -0.7

Tryptophan -0.9

Serine -0.8

Tyrosine -1.3

Proline -1.6

Histidine -3.2

Glutamic Acid -3.5

Glutamine -3.5

Aspartic Acid -3.5

Asparagine -3.5

Lysine -3.9

Arginine -4.5

Thus, for example, isoleucine, which has a hydropathic index of +4.5, will preferably be exchanged with an amino acid such as valine (+ 4.2) or leucine (+ 3.8). Alternatively, at the other end of the scale, lysine (- 3.9) will preferably be substituted for arginine (-4.5), and so on.

Substitution of like amino acids may also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with an important biological property of tr?e protein.

As detailed in U.S. Patent 4,554,101, each amino acid has also been assigned a hydrophilicity value. These values are detailed below in Table 3.

TABLE 3

Amino Acid Hydrophilic Index arginine +3.0 lysine +3.0 aspartate +3.0 ± 1 glutamate +3.0 ± 1 serine +0.3 asparagine +0.2 glutamine +0.2 glycine 0 threonine -0.4 alanine -0.5 histidine -0.5 proline -0.5 ± 1 cysteine -1.0 methionine -1.3 valine -1.5 leucine -1.8 isoleucine -1.8 tyrosine -2.3 phenylalanine -2.5 tryptophan -3.4

It is understood that one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Accordingly, these amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like. In general, preferred substitutions which take various of the foregoing characteristics into consideration will be known to those of skill in the art and include, for example, the following combinations: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine (See Table 4, below). The present invention thus contemplates functional or biological equivalents of an eNOS or variant eNOS polypeptide as set forth above.

TABLE 4

Original Residue Exemplary Substitutions

Ala Gly; Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala

His Asn; Gin

He Leu; Val

Leu He; Val

Lys Arg

Met Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu

Biological or functional equivalents of a polypeptide can also be prepared using site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of second generation polypeptides, or biologically functional equivalent polypeptides or peptides, derived from the sequences thereof, through specific mutagenesis of the underlying DNA. As noted above, such changes can be desirable where amino acid substitutions are desirable. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 2 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by Adelman, et al. (1983). As will be appreciated, the technique typically employs a phage vector which can exist in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing, et al, 1981). These phage are commercially available and their use is generally known to those of skill in the art.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes all or a portion of the eNOS or variant eNOS enzyme polypeptide sequence selected. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al, (1978). This primer is then annealed to the singled-stranded vector, and extended by the use of enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as E. coli cells and clones are selected which include recombinant vectors bearing the mutation. Commercially available kits come with all the reagents necessary, except the oligonucleotide primers.

In addition, peptides derived from these polypeptides, including peptides of at least about 6 consecutive amino acids from these sequences, are contemplated. Alternatively, such peptides may comprise about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 consecutive residues. For example, a peptide that comprises 6 consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3 to 8 and so on of the eNOS protein. Such peptides may be represented by the formula

x to (x + n) = 5' to 3' the positions of the first and last consecutive residues

where x is equal to any number from 1 to the full length of the eNOS protein and n is equal to the length of the peptide minus 1. Where the peptide is 10 residues long (n = 10- 1), the formula represents every 10-mer possible for each antigen. For example, where x is equal to 1 the peptide would comprise residues 1 to (1 + [10-1]), or 1 to 10. Where x is equal to 2, the peptide would comprise residues 2 to (2 + [10-2]), or 2 to 11, and so on.

Syntheses of peptides are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptide(s) are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled, deionized) or buffer prior to use. Of particular interest are peptides that represent antigenic epitopes that lie within the eNOS polypeptides of the present invention. An "epitope" is a region of a molecule that stimulates a response from a T-cell or B-cell, and hence, elicits an immune response from these cells. An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is structurally "complementary" to, and therefore will bind to, binding sites on antibodies or T-cell receptors. It will be understood that, in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitopic core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the corresponding eNOS antigen to the corresponding eNOS-directed antisera.

The identification of epitopic core sequences is known to those of skill in the art. For example U.S. Patent 4,554,101 teaches identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity, and by Chou-Fasman analyses. Numerous computer programs are available for use in predicting antigenic portions of proteins, examples of which include those programs based upon Jameson-Wolf analyses (Jameson and Wolf, 1988; Wolf et al., 1988), the program PepPlot® (Brutlag et al, 1990; Weinberger et al, 1985), and other new programs for protein tertiary structure prediction (Fetrow and Bryant, 1993) that can be used in conjunction with computerized peptide sequence analysis programs.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would be on the order of about 6 amino acids in length. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence. D. Small Molecule Modulators of eNOS

The present invention provides methods for screening and identifying small molecule modulators of eNOS proteins and identifies such compounds. One rationale behind the design of the small molecule eNOS protein modulators is that in the absence of BH₄ it is seen that the heme domain of eNOS generates superoxide radicals thereby leading to pathophysiology. For example, further endothelial dysfunction in hypercholesteremia is reversed as a result of BH₄ treatment. The activated eNOS protein is thus able to produce NO and restore endothelial function. The present invention provides a pterin-free eNOS structure that can be used to model drugs (i.e., ligands) that will ameliorate the effect of BH₄ depletion. Such ligands will be useful in any diseased state in which NO signaling is defective or insufficient.

The findings of the present invention will be exploited to design chemical ligands that bind to the active site of the different variant proteins to yield complexes with sufficient thermodynamic stability to effectively modulate the functional activity of the protein. To obtain appropriate ligands that bind to the active sites of different eNOS variant proteins, the inventors may utilize the technique of force-field docking of chemical fragments from both commercially available chemical fragment libraries, as well as in-house generated libraries, into the active electrophile-binding (H-) site in the derived crystal structure of each variant protein. The docked fragments will be energy- minimized and the binding energies computed and used to select candidate ligands.

Generation of eNOS modulators: Generation of modulators is accomplished by a rational drug development strategy involving force field docking and energy- minimization of chemical fragments and compounds into the active site of the eNOS protein(s). The. Additional chemical libraries also may be generated as necessary. The active compounds and chemical fragments can be drawn from chemical fragment libraries, such as that available in the Leapfrog database site and other structural components of the eNOS proteins will be derived from the crystal structure of the eNOS described by the present invention. One potential substitution that confers a functional change to the eNOS protein is to replace Cys 101 and/or Ser 104 which bond to BH₄ (FIG. 4). In addition, Val 105, which forms a direct nonbonded contact with BH₄ may be altered. Therefore, disruption of the metal center either by demetallation or ligand removal via mutagenesis will distort this region of the polypeptide chain resulting in greatly diminished affinity for BEL. Additional mutations are contemplated which may result in increased stability. For example, increased protein stability results from the addition of disulfide bonds and the creation of more hydrophobic interactions within the protein structure.

Based on the resultant DDH values obtained after energy minimization of chemical fragments/compounds, candidate modulators are selected and/or newly constructed from chemical fragments for synthesis and further analyses for their inhibitory or other action on the eNOS proteins. Selection criteria for such modulators for synthesis and further analysis includes lipophilicity, chemical stability, and availability or ease of synthesis.

If the identified and/or newly constructed potential inhibitors are not commercially available, then they will be synthesized using standard organic synthetic methodology, including heterocyclic ring construction and functionalization, and electrophilic and nucleophilic substitution reactions. Reaction mixtures will be separated by thin layer, flash silica gel column, and high performance liquid chromatography (TLC, CC and HPLC). The compounds will be purified using standard techniques modified as necessary. Characterization of synthetic products will be done by melting point determination, Fourier transform infrared (FT-1R), ultraviolet (UV), and high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Compounds for biological testing will be purified by preparative HPLC. The purity of compounds will be determined by elemental analysis and HPLC.

Candidate modulators of the present invention will be useful in the treatment of nitric oxide synthase mediated diseases and disorders, including neurodegenerative disorders, disorders of gastrointestinal motility and inflammation. These disease and disorders include hypotension, septic shock, toxic shock syndrome, hemodialysis, IL* therapy such as in cancer patients, cachexia, immnunosuppression such as in transplant therapy, autoimmune and/or inflammatory indications including sunburn or psoriasis and respiratory conditions such as bronchitis, asthma, and acute respiratory distress (ARDS), myocarditis, heart failure, atherosclerosis, arthritis, rheumatoid arthritis, chronic or inflammatory bowel disease, ulcerative colitis, Crohn's disease, systemic lupus erythematosis (SLE), ocular conditions such as ocular hypertension and uveitis, type 1 diabetes, insulin-dependent diabetes mellitus, and cystic fibrosis. These compounds will be similar to those already described in the art in for example, U.S. Patent 5,821,261; U.S. Patent 5,821,267; U.S. Patent 5,807,886; U.S. Patent 5,776,979; U.S. Patent 5,767,160; U.S. Patent 5,728,728; U.S. Patent 5,723,451; U.S. Patent 5,710,181; U.S. Patent 5,688,499; U.S. Patent 5,684,008; U.S. Patent 5,674,907; U.S. Patent 5,645,839; U.S. Patent 5,629,322; U.S. Patent 5,585,402; U.S. Patent 5,543,430; U.S. Patent 5,480,999; U.S. Patent 5,436,271; U.S. Patent 5,380,945; U.S. Patent 5,362,747; U.S. Patent 5,296,466 and U.S. Patent 5,266,594 (each incorporated herein by reference). The compositions disclosed in these patents may be used as starting materials for rational drug design to yield modulators that best fit the crystal structure of NOS described herein.

The different compounds may have varying substituents which result in significant changes in binding energies of the compounds in the active site pocket of the eNOS protein. An individual skilled in the art of organic synthesis in light of the present disclosure will be able to prepare or identify a large variety of candidate molecules which would be expected to have eNOS modulatory effects in the light of the present disclosure.

The modulators identified may be inhibitors or stimulators of eNOS activity. Inhibitors will be used in treating various conditions where there is an advantage in inhibiting nitric oxide biosynthesis, as described in e.g., U.S. Patent 5,821,261; U.S. Patent 5,821,267; 5,807,886; U.S. Patent 5,789,442; U.S. Patent 5,789,395; U.S. Patent 5,776,979; U.S. Patent 5,756,540; 5,741,815; U.S. Patent 5,723,451; U.S. Patent 5,721,278; U.S. Patent 5,710; U.S. Patent 5,710,181; U.S. Patent 5,695,761; U.S. Patent 5,684,008; U.S. Patent 5,674,907; 5,645,839; U.S. Patent 5,629,322; U.S. Patent

5,585,402. Inhibition of NOS activity will be useful in treating conditions such as hypotension, inhibition of ovulation, inflammatory bowel disease, inflammation, autoimmune diseases and septic shock variety of cardiovascular fibrotic pathologies, such as that associated with left ventricular hypertrophy secondary to hypertension, myocardial infarction, myocarditis, and the like. Stimulators will be useful in treating conditions where there is an advantage to stimulating nitric oxide biosynthesis. Such conditions include diseases related to vasoconstriction, wherein the vasoconstriction is relieved by stimulating the NOS to produce native nitric oxide, e.g., as described in U.S. Patent 5,767,160; 5,543,430; stimulation of ovulation as described in U.S. Patent 5,721,278. NOS stimulators also are used to slow and reverse the process of fibrosis in the body, useful in the treatment of a variety of cardiovascular fibrotic pathologies, such as that associated with left ventricular hypertrophy secondary to hypertension, myocardial infarction, and myocarditis as described in U.S. Patent 5,645,839.

Screening for modulators of eNOS: Within certain embodiments of the invention, methods are provided for screening for modulators of eNOS protein activity. Such methods may use labeled eNOS proteins or analogs, anti-eNOS proteins or anti-eNOS antibodies and the like as reagents to screen small molecule and peptide libraries to identify modulators of eNOS protein activity. Within one example, a modulator screening assay is performed in which cells expressing eNOS proteins are exposed to a test substance under suitable conditions and for a time sufficient to permit the agent to effect activity of eNOS proteins.

Generally the test substance is added in the form of a purified agent. However, it is also contemplated that test substances useful within the invention may include substances present throughout the handling of test sample components. For example, host cell factors that are present in a cell lysate may be used for generating the test sample. Such endogenous factors may be segregated between the test and control samples, for example, by using different cell types for preparing lysates. In such preparations, the cell type used for preparing the test sample expresses a putative test substance that is not expressed by the cell type used in preparing the control sample.

The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it may be necessary to test a variety of candidates to determine which have potential.

Accordingly, in screening assays to identify agents which alter the activity of eNOS proteins, it is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.

In these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to decrease the eNOS activity of cells, the method including generally the steps of:

(a) obtaining a cell with eNOS activity;

(b) admixing a candidate substance with the cell; and

(c) determining the ability of the candidate substance to inhibit the eNOS activity of the cell.

To identify a candidate substance as being capable of decreasing eNOS activity, one would measure or determine the basal eNOS status of the cell prior to any additions or manipulation. One would then add the candidate substance to the cell and re- determine the eNOS activity in the presence of the candidate substance. A candidate substance which decreases the eNOS activity relative to the composition in its absence is indicative of a candidate substance being an inhibitor of eNOS. A similar assay may 1?e set up to determine whether the candidate substance is a stimulator of eNOS activity.

"Effective amounts", in certain circumstances, are those amounts effective to reproducibly alter eNOS activity in an assay in comparison to their normal levels.

Compounds that achieve significant appropriate changes in activity will be used. If desired, a battery of compounds may be screened in vitro to identify other agents for use in the present invention.

A significant change in eNOS activity is represented by a change in eNOS protein activity levels of at least about 30%-40%, and most preferably, by a change of at least about 50%, with higher values of course being possible. Assays that measure eNOS activity in cells are well known in the art and may be conducted in vitro or in vivo, and have been described elsewhere in the specification.

Quantitative in vitro testing of the eNOS modulators is not a requirement of the invention as it is generally envisioned that the agents will often be selected on the basis of their known properties or by structural and/or functional comparison to those agents already demonstrated to be effective. Therefore, the effective amounts often will be those amounts proposed to be safe for administration to animals in another context.

E. Antibodies to eNOS

Within certain embodiments of the present invention, antibodies raised against eNOS may be useful in aiding the identification of drugs. An antibody that recognizes the active site of an enzyme will act as a mimic of the drug that fits that active site. Using this information, drugs may be designed that mimic the shape of such an antibody.

Antibodies to eNOS variant peptides or polypeptides may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent

4,196,265. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., purified or partially purified protein, synthetic protein or fragments thereof, as discussed in the section on polypeptides. Animals to Se immunized are mammals such as cats, dogs, and horses, although there is no limitation other than that the subject be capable of mounting an immune response of some kind. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals. However, the use of rabbits, sheep or frogs is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.

For generation of monoclonal antibodies (MAbs), following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10⁷ to 2 x 10⁸ lymphocytes.

The antibody-producing B cells from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas."

Any one of a number of myeloma cells may be used and these are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use

P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 41, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-A^g 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell line is the NS-1 myeloma cell line (also termed P3-NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies, from about

1 x 10^~6 to 1 x 10^~8. This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium. The selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate. and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography.

Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art. Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, i.e., hybrids of two or more antibody molecules. In another embodiment, monoclonal antibodies according to the invention are chimeric monoclonal antibodies. In one approach, the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene. The antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.

In another embodiment, monoclonal antibodies according to the present invention is a "humanized" monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. "Humanized" monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods.

As stated above, the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art. Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.

Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogenous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture. Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain, and/or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.

The monoclonal conjugates of the present invention are prepared by methods known in the art, e.g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fiuorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as ³H, ¹²⁵I, ¹³¹1 ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶C1, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, and ^99mTc, are other useful labels which can be conjugated to antibodies. Radioactively labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium-⁹⁹"¹ by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC1 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.

F. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Determination of the Crystal Structure of eNOS

Crystals were grown as described. Briefly, bovine eNOS heme domain (39-482; Mr 49,000) was obtained via trypsinolysis of a modified version of the holo eNOS construct (expressed in E. coli) missing 75% of the calmodulin binding region. Crystals suitable for diffraction were grown by the sitting drop vapor-diffusion method from 15% PEG 3350, 200 mM magnesium acetate, 100 mM sodium cacodylate, pH 6.5, and 2 mM S-ethylisothiourea (or 10 mM L-Arg) in the presence of 75 μM sodium dodecyl sulfate (SDS) as an additive and 10 mM tris(2-carboxyethyl)phosphine (TCEP) or 5 mM glutathione sulfonate as reducing agent. No BH was added during crystallization. Pterin-free protein expressed in E. coli was purified either in the presence (50 μM) or absence of BH₄ towards obtaining crystals with cofactor-bound and -free forms. Crystals grown under these conditions belong to the orthorhombic space group, 2 22_\, with cell constants =58.00 A, b=106.55 A, and c=156.22 A. There is one dimer in the asymmetric unit (50% solvent content). All native and derivative crystals were flash frozen in liquid nitrogen for both storage and data collection at cryogenic temperatures (100 K). A protein stabilization cocktail containing 15% glycerol, 11% trehalose, 8% mannitol and 8% sucrose was used as cryoprotectant.

Data were collected with a charge coupled device (CCD) detector at CHESS, NSLS and SSRL (beamlines FI and F2, X12B and 1-5, respectively) and with a Mar Research image plate scanner at SSRL (beamlines 7-1 and 9-1). Multiwavelength anomalous diffraction (MAD) data were collected using the inverse beam mode after aligning the crystal with a major axis coincident with the rotation axis so that Bijvoet pairs could be measured simultaneously. Image plate data were reduced using the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997) and CCD data were processed with the DPS, MOSFLM and CCP4 suite of programs. Mercury and osmium positions (three sites each) were readily identified by SHELXS (Sheldrick, 1997) and could later be confirmed in the isomorphous and anomalous difference Patterson maps. Iterative rounds of rejections performed with ENDHKL (Louis Sanchez, Cal. Tech.) in conjunction with SCALEPACK and local scaling were both critical for the identification of the heavy atoms. For MAD phasing an inhibitor, S-(2- (5((amidinothio)methyl)-2-thienyl)ethyl)isothiourea (Garvey et al, 1994), was prepared with the sulfur atoms replaced with selenium. After 15 min, a solution of 2.401 g (0.011 mol) of ethyl 2-selenophenacetate in 5 ml of dichlormethane was added dropwise over several minutes. The mixture was poured into water and ice after 1 h and stirred for 30 min. The dichlormethane layer was washed with water, dried over sodium sulfate, and concentrated. The crude product was purified by silica gel chromatography with 10% ethyl acetate in petrolether to yield 1.77 g ( 65%) of 5-formyl-2-selenophenacetic acid ethyl esther intermediate. H NMR (300 MHz, CDC 13) 9.83 (formyl, IH), 7.63-7.04 (aromatic, 2H), 4.19-4.17 (methyl, 5H), 3.86 (ethyl, IH), 1.28-1.24 (methyl, 5H). To a 0 oC stirred suspension of 0.77 g (20.29 mmol) of lithium aluminium hydride (Aldrich) in 200 ml of tetrahydrofuran was added a solution of 2.0 g (8.65 mmol) of the intermediate prepared above in tetrahydrofuran. The suspension was stirred at 20 oC for 16 h, cooled to 0 oC, and the exess hydride was quenched by the carefull addition of 0.8 ml of water, 0.8 ml of IN sodium hydroxyde solution, and 2.4 ml of water. The suspension was stirred with magnesium sulfate, filtered, concentrated, and purified by silica gel chromatigraphy with 50% ethyl acetate in petrolether. There was isolated 1.0 g (66%) >f diol intermediate (yellow oil). H NMR (300 MHz, CDC 13) 797- 7.13 (aromatic, 2H), 4.83 (hydroxyl, 2H), 3.63 (ethyl, 5H), 1.63-1.42 (ethyl, IH). A solution of this diol (1 g, 5.6 mmol) in dichlormethane (20 ml) at 0 oC was treated with 3.1 g (9.7 mmol) of carbon tribromide and 2.5 g (9.7 mmol) triphenylphosphine. The mixture was stirred at 20 oC for 4 h before 50 ml of petrolether was added. After 15 h, the solution was decanted from brown-colored solid, concentrated, and purified by silica gel chromatography (ethyl acetate/petrolether, 80:20) to yield 0.7 g (63%) of dibromide as an oil. A solution of 0.7 g (2.3 mmol) of dibromide and 1 g (8.12 mmol) of selenourea in 20 ml of absolute ethanol was refluxed for 2 h, cooled and concentrated to dryness. The crude solid was recristallized from ethanol to yield 0.1 g (9.4%) of the bis-ISU as a yellow crystalline solid. H NMR (300 MHz, CDCL3) 9.25 (NH, 6H), 6.2 (aromatic, 2H), 4.22-4.15 (ethyl, 4H), 1.23-1.13 (ethyl, 4H).

The preparation of 2-selenophenacetate:

To a solution of 3.18 g ethylbromoacetate and 0.362 g of tin (IV) chloride in 10 ml of carbon disulfide at 0 oC was added dropwise over several minutes 2.5 g of selenophene in 1 ml CS2. The mixture was stirred at room temperature overnight, poured into water and ice, extracted by dichlommethane, washed by saturated solution of sodium hydrocarbonate, dried over sodium sulfate and concentrated. The crude product was purified by silica gel chromatography in dichlormethane to yield 0.7 g ethyl 2-sdenophenacetate (18.3%). IH NMR (300 MHz, DMSO) 7.23-7.2 (IH), 6.97-6.94(2H), 4.22-4.15 (ethyl 5H), 3.83 (methylen, 2H), 1.29-1.25 (methyl, 5H).

Once the structure was refined, it was evident that the inhibitor had not bound.

One of the six sites initially assigned to selenium was the new zinc site and two others are most likely adducts of (CH )₂As to both Cys384 residues (one per monomer) from the cacodylate buffer used in crystallization. Since the crystallization solution contains excess reducing agents, the inventors attribute this chemistry to the reduction of dimethylarsenate (V) to dimethyl arsenite (III) followed by reaction with Cys384 (Barber, 1932; Tsao and Maki, 1991). The three remaining sites initially thought to be selenium, two near both Cys214 sulfurs and one near one Cys87, were much weaker and could not be confidently modeled as (CH₃) As sites in the final refined electron density map. Despite the incorrect assignment of the arsenic and zinc sites as selenium, their inclusion was essential for obtaining an interpretable electron density map. To the best of the inventors' knowledge this is the first study where arsenic atoms have been used successfully in phasing. Availability of heme iron positions, identified independently via anomalous scattering at the Fe edge, greatly facilitated the location of heavy atom sites. Heavy atom derivative screening and preliminary phase refinements were carried out with PHASES and visualized using XTALVIEW.

The final combined MAD and heavy atom refinement was done with SHARP (de La Fortelle and Bricogne, 1997) followed by density modification with either SOLOMON (Abrahams and Leslie, 1996) or DM. The latter calculation includes non- crystallographic symmetry (TICS) averaging. The resulting experimental MAD-heavy atom map at 2.35 A was of sufficient quality to allow nearly all main chain atoms and 80% of the side chain atoms to be built into the model before the first round of refinement. Phase refinement with heavy atom derivatives alone did not produce an interpretable map. Heme iron positions, SHELXS, MAD phasing, and SHARP were a sine qua non for success in the structure solution.

Structural refinement was performed with XPLOR (Brunger, 1992) and SHELXL (Sheldrick and Schneider, 1997). Five percent of the data were set aside for free-R cross validation prior to any structural refinement. The protein model was built using TOM and improved with SigmaA- weighted 2|Fobs | - |Fcalc | and |Fobs | - |Fcalc | maps iteratively with X-PLOR refinement. A bulk solvent correction was used in the final stages of the X-PLOR but not in the SHELXL refinement. The current model at 1.9 A resolution includes 830 residues (residues 67 - 482 in molecule A; 69 - 482 in molecule B) and 591 waters. Residues 39-66 and 108-121 are disordered primarily due to the proline-rich nature of this region. Ramachandran plots generated with PROCHECK showed that 88.9% of the residues were in the most favored regions, 11% in additional allowed regions and 0. 1% in disallowed regions. Solvent accessible surface area calculations were done with MSP.

Table 5

Data collection Native H4 free Se edge Se-peak Se- remote Se-EMP* Se- Os0₃(PY)₂*

Wavelength (A) 0.9798 1.08 0.9801 0.9794 0.9252 1.08 1.08 Resolution limits (A) 1.9 2.1 2.3 2.3 2.3 2.3 3.0 Total observations 217,080 204,608 131 ,275 127,968 144,570 131 ,287 74,034 Unique observations 74,337 57,723 40, 100 36,351 40,965 40,508 16,204

0.044 0.084 0.048 0.054 0.044 0.059 0.085

R_sy_m (outer shell) 0.175 0.563 0.121 0.219 0.185 0.325 0.336

<I/σ> 19.3 7.7 16.9 15.3 17.1 9.8 8.9

<I/σ> (outer shell) 3.5 2.4 3.2 2.2 3.1 2.2 4.0

Completeness 0.965 0.996 0.962 0.988 0.985 0.977 0.808

Completeness (outer shell) 0.800 0.996 0.795 0.802 0.983 0.801 0.829 MAD phasing (20.0 - 2.35 A)

Number of sites 6

Phasing power Iso/Ano 0.0/0.76 0.0/0.78 1.1/0.75

Rcuiiis Iso/Ano 0.0/0.96 0.97/0.96 0.41/0.95 MIRAS phasing

Number of sites 5

* so 0.157 0.173 K-cullis 0.89 0.92

Phasing power Iso/Ano 1.15/0.87 0.71/0.85 Overall FOM (MAD+MIRAS) 0.29 (2.35 A)

Refinement Resolution (A) Protein Waters R-factor R-free Reflections R.m.s. deviation⁹

Data collection Native H4 free Se edge Se-peak Se- remote Se-EMP* Se- Os0₃(PY)₂* atoms

X-PLOR (native) 30.0 - 1.9 6593 591 0.225 0.262 73,483

(F>2σF) (F>2σF)

SHELXL (native) 10.0 - 1.9 6593 591 0.207 0.278 70,029 0.007A 0.021 A

X-PLOR (BH₄ free) 30.0 - 2.1 6593 336 0.187 0.247 48,347 0.007A

(F>2σF) (F>2σF) 1.408°

Rs m = ∑| - < >/∑, where / is the observed intensity and <I> the average intensity of multiple symmetry-related observations of that reflection. R_1S0 = ∑IEPH - E_P|/∑ E_P. Rcuihs ISO (acentric) = r.m.s. lack of closure / r.m.s. isomorphous difference (statistics from SHARP). R_Cuiiι_s Ano = r.m.s. lack of closure / r.m.s. anomalous difference (statistics from SHARP). Phasing power = r.m.s. heavy atom structure factor / phase integrated lack of closure (statistics from SHARP). Overall FOM = overall figure of merit (from SHARP). R-free = R-factor calculated using 5% (3685 reflections) of the reflection data chosen randomly and set aside from the start of the refinement. ΕMP = ethyl mercuric phosphate. OsO₃Py₂ = osmium bis-pyridine. ^s r.m.s. deviations are defined as bond length and angular distances in SHELXL (Sheldrick and Schneider, 1997) and bond length and bond angle, respectively, in X-PLOR (Brϋnger, 1992).

3

The inventors report the 1.9 A crystal structure of the dimeric heme domain of constitutive eNOS (Table 5), which maintains the catalytic site for NO^* synthesis. The overall fold of the bovine eNOS heme domain (FIG. 1) is similar to that reported for the 2.6 A structure of the iNOS heme domain dimer (Crane et al, 1998). The quaternary structure of eNOS is characterized by a tightly packed dimer interface which buries 3000 A² per subunit of solvent accessible surface (55% hydrophobic and 45% polar).

The inventors solved the structure of eNOS heme domain both in the presence and absence of BH₄ resulting in three major structural findings. First, the inventors found a novel ZnS₄ center (in both pterin-bound and -free structures) located at the bottom of the dimer interface (FIG. 1) with the metal tetrahedrally coordinated by two pairs of symmetry-related cysteine residues (FIG. 2) from each subunit.

Second, in the absence of BH₄, the overall eNOS heme domain structure remains unchanged both in the tertiary topology and quaternary structure. These results sharply contrast with the conclusions of Crane et al, (1998) who proposed that large conformational changes concomitant with BH₄ binding were necessary for inducible NOS dimerization. By comparison of the pterin-bound and -free structures, it is clear that the site preexists (FIG. 3) and does not form via an induced fit process. The pterin-free structure also does not show any major changes at the active site either in the substrate- or inhibitor (S-ethylisothiourea, SEITU)-bound conformations.

Third, in addition to the new ZnS center, the most striking and novel finding is the identification of an L-Arg bound to the pterin site in the absence of BH₄ (FIG. 3A and FIG. 3D). The mode of L-Arg interaction at the pterin site surprisingly mimics that entertained by BH₄ itself. Two crucial H-bonds, one between a guanidino N with the heme propionate and that between the primary amino group and Ser 104 are strictly conserved (FIG. 3D). Moreover, the planar guanidinium group is sandwiched between two aromatic groups, one from each monomer, exactly as in the BH₄ complex. Solvent interactions also are similar to the BH₄ complex. The affinity L-Arg must be great since no exogenous L-Arg was added during crystallization. The specificity of L-Arg recognition at the pterin site appears to require both the guanidino function and the primary amino group since SEITU was unable to displace L-Arg from the pterin site. The relevance of these findings is discussed in greater detail herein above.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS: #

1. A method for the structural analysis of endothelial nitric oxide synthase comprising: a) subcloning a gene encoding the endothelial nitric oxide synthase in an expression vector; b) obtaining expression of the endothelial nitric oxide synthase protein from the vector; c) purifying the endothelial nitric oxide synthase protein; d) preparing an endothelial nitric oxide synthase protein sample amenable for crystallization; e) crystallizing the endothelial nitric oxide synthase protein sample; and f) performing a three-dimensional structural analysis of the endothelial nitric oxide synthase by x-ray crystallography.

2. The method of claim 1, wherein the endothelial nitric oxide synthase protein expressed is a variant endothelial nitric oxide synthase protein.

3. The method of claim 1, wherein the crystallizing comprises practicing a sitting drop vapor-diffusion method.

4. The method of claim 1 , wherein the crystallizing is performed in the presence of tetrahydrobiopterin .

5. The method of claim 1, wherein the crystallizing is performed in the absence of tetrahydrobiopterin.

6. The method of claim 1, wherein the x-ray crystallography is performed in the presence of tetrahydrobiopterin.

7. The method of claim 1, wherein the x-ray crystallography is performed in tne absence of tetrahydrobiopterin.

8. A method of structural analysis to determine the binding of pterin to endothelial nitric oxide synthase comprising: a) determining the crystal structure of a dimeric heme domain of the endothelial nitric oxide synthase in the presence of the pterin; and b) determining the crystal structure of a dimeric heme domain of the endothelial nitric oxide synthase in the absence of the pterin; and comparing the crystal structures.

9. The method of claim 8, wherein the pterin is tetrahydrobiopterin.

10. The method of claim 9, wherein the tetrahydobiopterin is (l'R,2'S,6R)-5,6,7,8- tetrahydrobiopterin .

11. The method of claim 8, wherein the crystal structure is determined at a resolution of between about 1.9 A to about 2.1 A.

12. A method for screening and identifying small molecule modulators of endothelial nitric oxide synthase proteins comprising: a) providing a pterin-free endothelial nitric oxide synthase structure; b) screening the small molecule modulators for their ability to bind to a pterin-binding site of the endothelial nitric oxide synthase ; and c) performing assays to determine the ability of the small molecule modulators to modulate the activity of endothelial nitric oxide synthase.

13. The method of claim 12, wherein the small molecule modulator inhibits endothelial nitric oxide synthase.

14. The method of claim 12, wherein the small molecule modulator activates endothelial nitric oxide synthase.

15. The method of claim 12, wherein the endothelial nitric oxide synthase protein expressed is a variant endothelial nitric oxide synthase protein.

16. The method of claim 12, wherein the pterin is tetrahydrobiopterin.

17. The method of claim 16, wherein the tetrahydobiopterin is (l'R,2'S,6R)-5,6,7,8- tetrahydrobiopterin .

18. The method of claim 12, wherein the small molecule modulators are molecules and chemical-fragments from chemical-fragment libraries.

19. The method of claim 12, wherein the screening is performed by computerized methods.

20. The method of claim 12, wherein the assays are performed in vitro or in vivo.

21. A method for identifying drugs against diseased states in which nitric oxide signaling is defective or insufficient comprising: a) providing a tetrahydrobiopterin-free endothelial nitric oxide synthase structure; b) screening the drugs for their ability to bind the tetrahydrobiopterin binding site; and c) performing assays to determine the ability of the drugs to activate the endothelial nitric oxide synthase.

22. The method of claim 21, wherein the diseased states include impaired neurotransmission; impaired insulin release; impaired penile erection; impaired vasorelaxation; and impaired oxygen detection. 3

23. Endothelial nitric oxide synthase structure, obtained by the process comprising: a) subcloning a gene encoding the endothelial nitric oxide synthase in an expression vector; b) obtaining expression of the endothelial nitric oxide synthase protein from the vector; c) purifying the endothelial nitric oxide synthase protein; d) preparing an endothelial nitric oxide synthase protein sample amenable for crystallization; e) crystallizing the endothelial nitric oxide synthase protein sample. f) performing a three-dimensional structural analysis of the endothelial nitric oxide synthase by x-ray crystallography.

24. A nitric oxide synthase obtained according to the method of claim 1.

25. A method for screening and identifying a candidate substance with the ability to inhibit endothelial nitric oxide synthase comprising: a) obtaining a cell with endothelial nitric oxide synthase activity; b) admixing the candidate substance with the cell; and c) determining the ability of the candidate substance to inhibit the endothelial nitric oxide synthase activity of the cell.

26. The method of claim 25, wherein the candidate substance is a small molecule modulator of endothelial nitric oxide synthase.

27. A method for screening and identifying a candidate substance with the ability to inhibit endothelial nitric oxide synthase comprising: a) obtaining a purified endothelial nitric oxide synthase; b) admixing the endothelial nitric oxide synthase with the candidate substance; and performing X-ray crystallography analysis to determine the binding of the candidate substance.