EP1141320A1 - Base structurelle de la fonction pterine dans l'oxyde nitrique synthase - Google Patents

Base structurelle de la fonction pterine dans l'oxyde nitrique synthase

Info

Publication number
EP1141320A1
EP1141320A1 EP99967546A EP99967546A EP1141320A1 EP 1141320 A1 EP1141320 A1 EP 1141320A1 EP 99967546 A EP99967546 A EP 99967546A EP 99967546 A EP99967546 A EP 99967546A EP 1141320 A1 EP1141320 A1 EP 1141320A1
Authority
EP
European Patent Office
Prior art keywords
nitric oxide
oxide synthase
endothelial nitric
pterin
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99967546A
Other languages
German (de)
English (en)
Inventor
C.S Raman
Bettie Sue S. Masters
Pavel Martasek
Linda J. Roman
Vladimir Kral
Thomas L. Poulos
Huiying Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
Original Assignee
University of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP1141320A1 publication Critical patent/EP1141320A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)

Definitions

  • 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.
  • 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.
  • 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.
  • cGMP cyclic guanosine monophosphate
  • 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
  • nNOS neuronal
  • eNOS endothelial NOS
  • iNOS inducible isoform
  • 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.
  • 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.
  • NOS isoforms exhibit an absolute requirement for tetrahydrobiopterin, (referred to as BH 4 ), 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).
  • BH 4 tetrahydrobiopterin
  • 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 BH 4 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.
  • 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.
  • the endothelial nitric oxide synthase protein expressed is a variant endothelial nitric oxide synthase protein.
  • 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.
  • the pterin is tetrahydrobiopterin.
  • the tetrahydobiopterin is (l'R,2'S,6R)-5,6,7,8-tetrahydrobiopterin.
  • the crystal structure is determined at a resolution of between about 1.9 A to about
  • 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.
  • 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.
  • the small molecule modulators are molecules and chemical-fragments from chemical-fragment libraries.
  • the screening is performed by computerized methods.
  • the assays to determine the activity of endothelial nitric oxide synthase are performed in vitro or in vivo.
  • 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.
  • FIG. 1 Panel A, schematic representation of the eNOS heme domain dimer viewed perpendicular to the dyed axis of symmetry. The two BH 4 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 ZnS 4 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 ZnS 4 center thereby providing a conduit to either/both pterin and heme groups.
  • both Mss4 and LIM proteins utilise
  • 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 l ⁇ (blue) and lO ⁇ (black).
  • the zinc ion was identified using anomalous dispersion effects characteristics for the metal.
  • Heme Fe exhibits some anomalous scattering at both wavelengths (iron absorption edge, ⁇ 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 ⁇ -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.
  • FIG. 3 Comparison of the pterin-free and -bound structures at BH binding site.
  • L-Arg was found in the BH 4 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 l ⁇ 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).
  • 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.
  • the structure of the substrate or inhibitor complex with BH 4 bound exhibit no differences at the BH 4 site.
  • FIG. 4 The ZnS 4 center and its relation to BH 4 .
  • the metal ion (white ball) is equidistant from each BH , a distance of 12 A.
  • ZnS 4 plays an effector role in helping to form and stabilize the pterin binding pocket which, in turn, promotes substrate binding. Therefore, the ZnS 4 center, BH 4 , 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-BH 4 showing the ring numbering scheme.
  • the pKa of N3 is near 10.6.
  • the inventors are proposing that NOS is designed to stabilize III, the radical cation. Stabilization of aromatic cation radicals is pronounced of cytochrome c peroxidase which is designed to stabilize a cationic Trp radical essential for catalysis (Sivaraja et al, 1989; Houseman et al, 1993).
  • 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.
  • 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 (BH 4 ) to understand its role as an indispensable cofactor in nitric oxide biosynthesis.
  • the BH 4 bound structure at 1.9 A reveals a novel zinc tetrathiolate (ZnS 4 ) sandwiched at the dimer interface which functions by maintaining the integrity of the pterin and substrate binding sites.
  • L-Arg is bound at the BH 4 site in the pterin-free structure suggests evolution of cofactor recognition from a common L-Arg-binding ancestor in the primordial NOS catalytic machinery.
  • eNOS dimeric heme domain of constitutive eNOS
  • Table 1 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 ⁇ / ⁇ protein class and the quaternary structure is characterized by a tightly packed dimer interface which buries 3000 A 2 per subunit of solvent accessible surface (55% hydrophobic and 45% polar).
  • the inventors have solved the structure of eNOS heme domain both in the presence and absence of BH 4 resulting in three major structural findings.
  • 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).
  • Val 105 forms a direct nonbonded contact with BH 4 . 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 BH 4 couples directly to the heme, alterations at the pterin site will in turn affect the heme pocket and L-Arg binding.
  • Inherited vascular dysfunction may arise from mutations that specifically weaken zinc affinity resulting in a dysfunctional eNOS.
  • ALS familial amyotropic lateral sclerosis
  • 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).
  • L-Arg 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 BH 4 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.
  • L-Arg argues in favor of preferential binding of a fully protonated species of BH 4 . Owing to the essentially identical nature of the pterin site in both the L-Arg and BH 4 complexes, it appears that bound BH 4 experiences the same electrostatic environment as
  • 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 BH 4 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 ⁇ 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.
  • 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 BH 4 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 BH 4 deficient eNOS may be a reality in vascular pathologies in which L-Arg could substitute at the BH 4 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.
  • 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.
  • the protein precipitates as an amorphous material.
  • crystal nuclei may form, which may grow to form diffracting crystals.
  • nuclei will not form, so generally no crystals appear.
  • the metastable zone This area, where crystal growth but not nucleation takes place, is sometimes referred to as "the metastable zone".
  • 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 ⁇ l 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 ⁇ l 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 ⁇ l 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 ⁇ l of each of the seeding solutions produced is added by hand to each sample well with a 5 ⁇ l Hamilton syringe. The plates were then sealed and crystals allowed to form. Small single crystals will appear after several days.
  • appropriate crystallization plates e.g., CrystalClear plates Douglas Instruments.
  • 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.
  • 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).
  • substitution of alternative amino acids small conformational changes may be conferred upon a polypeptide which result in increased activity or stability.
  • 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.
  • 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.
  • 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.
  • isoleucine which has a hydropathic index of +4.5
  • an amino acid such as valine (+ 4.2) or leucine (+ 3.8).
  • lysine (- 3.9) will preferably be substituted for arginine (-4.5), and so on.
  • each amino acid has also been assigned a hydrophilicity value. These values are detailed below in Table 3.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by Adelman, et al. (1983).
  • 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.
  • 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.
  • 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.
  • peptides derived from these polypeptides are contemplated.
  • 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.
  • 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 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.
  • 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.
  • peptides 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.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of 7.0 to 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • 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.
  • a predetermined amount of water preferably distilled, deionized
  • 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 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 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.
  • this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • 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 BH 4 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 BH 4 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 BH 4 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.
  • 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.
  • 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 BH 4 (FIG. 4). In addition, Val 105, which forms a direct nonbonded contact with BH 4 may be altered.
  • 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.
  • 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).
  • TLC, CC and HPLC high performance liquid chromatography
  • 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.
  • ARDS acute respiratory distress
  • SLE systemic lup
  • 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
  • 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.
  • 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.
  • 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.
  • test substance is added in the form of a purified agent.
  • test substances useful within the invention may include substances present throughout the handling of test sample components.
  • host cell factors that are present in a cell lysate may be used for generating the test sample.
  • endogenous factors may be segregated between the test and control samples, for example, by using different cell types for preparing lysates.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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
  • 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.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells) are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • 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.
  • 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.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 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.”
  • myeloma cells 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
  • NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
  • P3-NS-l-Ag4-l NS-1 myeloma cell line
  • 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).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate.
  • Fusion procedures usually produce viable hybrids at low frequencies, from about
  • the selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate. and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine 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.
  • HPRT hypoxanthine phosphoribosyl transferase
  • 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.
  • monoclonal antibodies according to the invention are chimeric monoclonal antibodies.
  • 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.
  • 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.
  • CDRs mouse complementary determining regions
  • Humanized monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods.
  • 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.
  • suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • feeder cells such as normal mouse peritoneal exudate cells, spleen
  • Techniques for large scale hybridoma cultivation under tissue culture conditions 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.
  • the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
  • 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.
  • 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.
  • 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.
  • a reducing agent such as SNC1
  • a buffer solution such as sodium-potassium phthalate solution
  • 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 ⁇ M 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.
  • 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).
  • CCD charge coupled device
  • NSLS and SSRL beamlines FI and F2, X12B and 1-5, respectively
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 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 A 2 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 BH 4 resulting in three major structural findings.
  • the inventors found a novel ZnS 4 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.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne des méthodes permettant d'élucider la structure tridimensionnelle de l'oxyde nitrique synthase et de ses variantes. Elle porte également sur des techniques d'analyse structurelle visant à déterminer la liaison de la ptérine à l'oxyde nitrique synthase endothéliale et à identifier de petites molécules modulatrices de protéines d'oxyde nitrique synthase endothéliale et de leurs variantes. Sont également décrites des méthodes permettant d'identifier des médicaments qui modulent l'oxyde nitrique synthase et ses variantes et sont efficaces contre des états pathologiques caractérisés par une signalisation NO défectueuse ou insuffisante.
EP99967546A 1998-12-22 1999-12-22 Base structurelle de la fonction pterine dans l'oxyde nitrique synthase Withdrawn EP1141320A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11320498P 1998-12-22 1998-12-22
US113204P 1998-12-22
PCT/US1999/030707 WO2000037653A1 (fr) 1998-12-22 1999-12-22 Base structurelle de la fonction pterine dans l'oxyde nitrique synthase

Publications (1)

Publication Number Publication Date
EP1141320A1 true EP1141320A1 (fr) 2001-10-10

Family

ID=22348141

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99967546A Withdrawn EP1141320A1 (fr) 1998-12-22 1999-12-22 Base structurelle de la fonction pterine dans l'oxyde nitrique synthase

Country Status (4)

Country Link
EP (1) EP1141320A1 (fr)
AU (1) AU2380800A (fr)
CA (1) CA2361196A1 (fr)
WO (1) WO2000037653A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2543315A1 (fr) * 2003-11-13 2005-06-02 The General Hospital Corporation Methodes pour traiter la douleur
ES2596827T3 (es) 2003-11-17 2017-01-12 Biomarin Pharmaceutical Inc. Tratamiento de la fenilcetonuria con BH4
US9216178B2 (en) 2011-11-02 2015-12-22 Biomarin Pharmaceutical Inc. Dry blend formulation of tetrahydrobiopterin

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993018156A1 (fr) * 1992-03-05 1993-09-16 The General Hospital Corporation Synthase de l'oxyde d'azote endothelial
US5498539A (en) * 1992-07-02 1996-03-12 Daiichi Pharmaceutical Co., Ltd. Bovine endothelial nitric oxide synthase nucleic acids
US5744340A (en) * 1995-06-09 1998-04-28 Schering Corporation Expression of human inducible nitric oxide synthase
US5919682A (en) * 1995-08-24 1999-07-06 Board Of Regents, University Of Texas System Overproduction of neuronal nitric oxide synthase
US6150500A (en) * 1996-07-12 2000-11-21 Salerno; John C. Activators of endothelial nitric oxide synthase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0037653A1 *

Also Published As

Publication number Publication date
CA2361196A1 (fr) 2000-06-29
AU2380800A (en) 2000-07-12
WO2000037653A1 (fr) 2000-06-29

Similar Documents

Publication Publication Date Title
Schiffer et al. Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site
Mirwaldt et al. The Crystal Structure of Dihydrodipicolinate Synthase fromEscherichia coliat 2.5 Å Resolution
US20060188959A1 (en) Crystal structure of worm NitFhit reveals that a Nit tetramer binds tow Fhit dimers
Karplus et al. Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family
Raman et al. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center
Sirinupong et al. Structural insights into the autoinhibition and posttranslational activation of histone methyltransferase SmyD3
Hurley et al. Structure of human beta 1 beta 1 alcohol dehydrogenase: catalytic effects of non-active-site substitutions.
US7858745B2 (en) Use of specifically engineered enzymes to enhance the efficacy of prodrugs
Vihinen et al. Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease.
Blaszczyk et al. Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes
Hermoso et al. Mechanism of coenzyme recognition and binding revealed by crystal structure analysis of ferredoxin–NADP+ reductase complexed with NADP+
Xu et al. Internally deleted human tRNA synthetase suggests evolutionary pressure for repurposing
JP2003501038A (ja) 蛋白質キナーゼ
Yang et al. Molecular mechanism for the regulation of human mitochondrial NAD (P)+-dependent malic enzyme by ATP and fumarate
JP2000511884A (ja) Ｉｍｐｄｈ様結合ポケットを含む分子およびそれらをグラフィック表示することが可能なコード化データ記憶媒体
Frey et al. Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: A basis for the design of specific inhibitors
Feng et al. Fusion surface structure, function, and dynamics of gamete fusogen HAP2
JP2007528731A (ja) ３’，５’−環状ヌクレオチドホスホジエステラーゼ（ｐｄｅ１０ａ）の結晶構造およびその使用
Jelakovic et al. Structure and catalytic mechanism of the cytosolic D-ribulose-5-phosphate 3-epimerase from rice
Milani et al. Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis
US20040171019A1 (en) PIN1 peptidyl-prolyl isomerase polypeptides, their crystal structures, and use thereof for drug design
ES2306802T3 (es) Proteina de pliegue con nudo de cistina.
WO2000037653A1 (fr) Base structurelle de la fonction pterine dans l'oxyde nitrique synthase
JP2001161387A (ja) ヒトエフェクタ細胞サイクルチェックポイントタンパクキナ−ゼＣｈｋ１の触媒ドメイン、そのインヒビタの同定のための物質および同定方法
JP2003523175A (ja) スタフィロコッカスアウレウスＵＤＰ−Ｎ−アセチルエノールピルビルグルコサミン＝リダクターゼ（Ｓ．ａｕｒｅｕｓＭｕｒＢ）の結晶化および構造決定

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010723

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20020719

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040630