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
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

• the present invention relates to crystal structures of a phosphodiesterase 5 (PDE5) and ligand complexes of PDE5 and to their uses in identifying PDE5 ligands, including PDE5 inhibitor compounds.
• PDE5 phosphodiesterase 5
• the present invention also relates to methods of identifying such PDE5 inhibitor compounds and their medical use.
• the present invention additionally relates to crystals of PDE5 into which ligands may be soaked and to crystals of PDE5 comprising ligands that have been soaked into the crystal. Also contemplated by the present invention is the use of crystals of PDE5 into which ligands may be soaked in identifying ligands of PDE5, including PDE5 inhibitor compounds.
• PDE cyclic nucleotide dependent protein kinases
• PDEs class I phosphodiesterases
• PDE4 The family of cyclic nucleotide phosphodiesterases catalyse the hydrolysis of 3', 5 '-cyclic nucleotides to the corresponding 5' monophosphates.
• Current literature shows that there are eleven related, but biochemically distinct, human phosphodiesterase gene groups and that many of these groups include more than one gene subtype giving a total of twenty genes.
• Some PDEs are highly specific for hydrolysis of cAMP (PDE4, PDE7, PDE8), some are highly cGMP specific (PDE5, PDE6, PDE9), and some have mixed specificity (PDE1, PDE2, PDE3, PDE10, PDE11).
• PDEs are multi-domain proteins; each PDE has a -270 amino acid domain located towards the C-terminus, which has a high degree of amino acid sequence conservation between families (Charbonneau 1986). This domain has been extensively studied and shown to be responsible for the common catalytic function (Francis, S. H. et al. 1994). Non-homologous segments in the remainder of the protein have regulatory function or confer specific binding properties.
• PDE2, PDE5, PDE6 and PDE10 are all reported to contain putative GAF domains within their regulatory amino terminal portion (Aravind & Ponting 1997 and Soderling & Beavo 2000). These GAF domains have been shown to bind cGMP but their function is not yet fully understood.
• PDE5 a cGMP specific PDE, has been recognised in recent years as an important therapeutic target. It is composed of the conserved C-terminal, zinc containing, catalytic domain, which catalyses the cleavage of cGMP, and an N-terminal regulatory portion, which contains two GAF domain repeats. Each GAF domain contains a cGMP-binding site, one of high affinity and the other of lower affinity. PDE5 activity is regulated through binding of cGMP to the high and low affinity cGMP binding sites followed by phosphorylation, which occurs only when both sites are occupied (Thomas et al. 1990).
• PDE5 is found in varying concentrations in a number of tissues including platelets, vascular and visceral smooth muscle, and skeletal muscle.
• the protein is a key regulator of cGMP levels in the smooth muscle of the erectile corpus cavernosal tissue.
• the physiological mechanism of erection involves release of nitric oxide (NO) in the corpus cavernosum during sexual stimulation. NO then activates the enzyme guanylate cyclase, which results in increased levels of cGMP, producing smooth muscle relaxation in the corpus cavernosum and allowing in flow of blood.
• Inhibition of PDE5 inhibits the breakdown of cGMP allowing the levels of cGMP, and hence smooth muscle relaxation, to be maintained (Corbin & Francis 1999).
• Sildenafil (UK-092,480), the active ingredient of Viagra® and a potent inhibitor of PDE5, has attracted widespread attention for the effective treatment of male erectile dysfunction.
• Structural information has recently been shown for the catalytic domain of PDE4b a cAMP-specific PDE (Xu et al. 2000). This structure provides information about the overall fold of the catalytic domains of the PDE family, but, to date, no structural information is known about the way in which potential inhibitors bind to the enzyme.
• the most desirable crystal form of a macromolecule of interest is one which is amenable to having potential ligands soaked into the preformed crystal of the apo macromolecule.
• This has distinct advantages over conventionally produced co-crystals.
• the lattice symmetry and cell dimensions for each new ligand complex will be substantially the same as for the apo macromolecule such that the data collection parameters for the apo and all complex crystals will essentially be identical.
• the structure solution can then occur using rapid difference fourier methods, thus avoiding more involved time and labour intensive phasing methods. Consequently, soakable crystals of macromolecules allow for particularly rapid screening and structural determination of new macromolecule-ligand complexes.
• PDE5* The engineered form of PDE5, PDE5*, allows the production of soakable crystals of ligand complexes, and the structure of such a soaked PDE5*-ligand complex has been determined.
• PDE5 can be crystallised. It has also been found that manipulating the wild-type PDE5 amino acid sequence can facilitate the crystallisation of PDE5. Specifically, it has been found that manipulations of certain portions of the PDE5 amino acid sequence can facilitate the crystallisation of PDE5. More specifically it has been shown that particular manipulations of the PDE5 amino acid sequence can provide soakable crystal forms of PDE5 into which potential PDE5 ligands may be introduced by the process of crystal soaldng. Manipulating the wild- type PDE5 amino acid sequence results in a sequence modified PDE5 protein.
• This manipulation can be achieved by deletion, addition or substitution of one or more amino acid residues of the- PDE5 loop region or it can be achieved by complete replacement of the PDE5 loop region with a loop region (or other amino acid sequence) from another protein, preferably another PDE, more preferably PDE4, most preferably PDE4b.
• Crystals of PDE5 have been found to be useful for screening for PDE5 ligands, especially PDE5 inhibitors by (i) co-crystallising PDE5 with the PDE5 ligand (e.g. PDE5 inhibitor), as shown in Application Number PCT/TB 02/04426 (Example 9, "crystallisation of wild type PDE5 catalytic domain with Sildenafil", page 44, line 29 to page 45, line 17; Eample 11, "crystallisation of PDE5* with Sildenafil", page 46, lines 8 to 25; Example 13, “Data collection, structure determination and refinement of wild type PDE5 with Sildenafil", page 48, line 1 to page 49, line 9; Example 15, “data collection, structure determination and refinement of PDE5* with Sildenafil", page 50, line 9 to page 51, line 6; Table 4 "atomic co-ordinates for wild type PDE5 complexed with Sildenafil", pages 91 to 240; and Table 6, "atomic co-ordinates for baculovirus-
• PDE5 ligands especially PDE5 inhibitors, as identified by the methods of the present invention are useful in curative, palliative or prophylactic treatments.
• a crystal of phosphodiesterase 5 (PDE5), wherein the crystal is soakable.
• SEQ ID NO: 1 is the so-called "loop region" of PDE5.
• This loop region or a homologue, fragment, variant, analogue or derivative thereof includes additions, deletions or substitutions of amino acid residues comprised within the loop region.
• a variant in relation to the amino acid sequence of the crystal of the PDE5 of the present invention includes the deletion or substitution of the histidine (Fiis/H) residue as shown emboldened and underlined in SEQ ID NO: 1 (HRGVNNSYIQRSEHPLAQLYCHSLME).
• Replacement of said histidine (H) residue is preferably by way of incorporating one or more amino acid residues (other than histidine), preferably wherein said amino acid residues are neutral or non-polar.
• a variant in relation to the amino acid sequence of the crystal of the PDE5 of the present invention includes the complete replacement of the loop region with a loop region (or other amino acid sequence) from another protein, preferably a PDE, more preferably PDE4, most preferably PDE4b (see hereinafter).
• the crystal of PDE5 according to any of the preceding aspects which is grown using polyethylene glycol as a precipitant.
• Polyethylene glycol of molecular weights 2000 to 8000 are commonly used.
• the molecular weight of the polyethylene glycol is 4000.
• the polyethylene glycol may be at a concentration of between 10% and 30%, but is most preferably 20%.
• the PDE5 protein used to grow the crystals is desired to be of a concentration between 1 and
• the crystal of PDE5 according to any of the preceding aspects which is grown in a buffer in the pH range of 6.5 to 8.0.
• the pH is in the range of 7.0 to 7.8; most preferably the pH is 7.4.
• the buffer should be one capable of providing buffer capacity over the required pH range, preferably HEPES.
• the crystal of PDE5 according to any of the preceding aspects which is grown in the presence of an alcohol.
• the alcohol is isopropanol.
• the crystal of PDE5 according to any of the preceding aspects which is grown in a solution containing HEPES buffer, polyethylene glycol 4000 and iso-propanol.
• the solution contains 0.1M sodium HEPES pH 7.4, 20% polyethylene glycol 4000 and 10% iso-propanol.
• the crystal is grown from solution inclubated at or below room temperature (20-25 °C).
• the crystal is grown from a solution inclubated at a temperature within the range of 2-6 °C. More preferably, the crystal is grown from a solution inclubated at about 4°C.
• Soakable crystals may be grown using a variety of methods such as dialysis, sitting drop vapour diffusion or batch methods, microcrystallisation methods, micro or macro seeding methods or gel crystallisation methods but are preferably grown using hanging drop vapour diffusion. 13.
• (d) comprises a PDE5 of a molecular weight of approximately 40kDa ⁇ 2 kDa;
• UK-088,800 is illustrated in Figure 4 and is a structural representation of the compound with the chemical name 5-(2-ethoxyphenyl)-l-methyl-3-propyl-l,6- dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one.
• PDE5 has an active site within the third sub-domain of the protein and comprises Leu 765, Ala 767 and He 768 and one or more of Phe 820, Val 782, Phe 786, Tyr 612, Leu 804, Ala 779, Ala 783, He 813, Met 816 and Gin 817.
• the crystal of PDE5 according to any of the preceding aspects, wherein a PDE5 ligand has been soaked in.
• the process of soaking may involve transferring the crystal to be soaked from the solution in which it is grown into a stabilising solution containing the ligand or to which the ligand is to be added once the crystal is present in it.
• the stabilising solution has the physical properties such that the crystal, when transferred, retains its structural integrity and does not crack or dissolve or alter significantly in its crystal parameters, symmetry or cell dimensions.
• the stabilising solution comprises some or all of the chemical constituents from which the crystal was grown.
• the stabilising solution is of similar or identical composition to the solutions used for crystal growth as set out in aspects 9 to 12 above, although the concentration of precipitant used may be slightly increased. Most preferably the stabilising solution has the same pH as the crystal growth solution.
• the ligand may be added to the solution containing, or to contain, the crystal for soaking as a solid or in a liquid form (i.e. the ligand already being in solution).
• the ligand is in solution and the solvent maybe aqueous, organic or non-organic.
• the ligand is in DMSO.
• the ligand is commonly added to the soalcing solution to yield a final ligand concentration above the expected binding constant for the ligand by PDE5; preferably this is greater than or equal to 10 times the binding constant.
• the final concentration of ligand present in the crystal soalcing solution is preferably between 0.1 and 20 mg/ml, more preferably between 0.5 and 10 mg/ml, and most preferably between 1 and 5 mg/ml.
• PDE5 inhibitor is a pyrazolo- pyrimidinone, preferably UK-088,800.
• a method of selecting a compound capable of associating with PDE5 from a group of potential PDE5 ligand compounds comprising the following steps: i. soaking the crystal of PDE5 according to any one of aspects 1 to 19 in a solution containing a potential PDE5 ligand compound; ii. determining the three-dimensional structure from the soaked crystal; and iii. assessing whether the compound is bound to PDE5.
• a method of selecting a PDE5 ligand from a group of potential PDE5 ligands comprising the following steps:
• ligands are built using molecular graphics tools, more preferably the designed ligand is energy minimised prior to co-display and analysis.
• the ligand is a PDE5 inhibitor.
• a PDE5 ligand according to aspect 38 is a pharmaceutical.
• the ligand is a PDE5 inhibitor.
• a pharmaceutical composition comprising one or more PDE5 ligands according to aspect 38 and one or more pharmaceutically acceptable excipients.
• PDE5 ligand according to aspect 38 in the manufacture of a medicament for the prophylaxis or treatment of a condition, disease, disorder or dysfunction where the inhibition of PDE5 is prophylactically or therapeutically beneficial.
• the curative, palliative or prophylactic treatments contemplated by the present invention include the curative, palliative or prophylactic treatment of mammalian sexual disorders, in particular the treatment of mammalian sexual dysfunctions such as male erectile dysfunction (MED), impotence, female sexual dysfunction (FSD), clitoral dysfunction, female hypoactive sexual desire disorder, female sexual arousal disorder (FSAD), female sexual pain disorder or female sexual orgasmic dysfunction (FSOD) as well as sexual dysfunction due to spinal cord injury or selective serotonin re-uptake inhibitor (SSRI) induced sexual dysfunction but, clearly, will also be useful for treating other medical conditions for which PDE5 inhibitor is indicated.
• mammalian sexual dysfunctions such as male erectile dysfunction (MED), impotence, female sexual dysfunction (FSD), clitoral dysfunction, female hypoactive sexual desire disorder, female sexual arousal disorder (FSAD), female sexual pain disorder or female sexual orgasmic dysfunction (FSOD)
• SSRI selective serotonin re-uptake inhibitor
• Such conditions include premature labour, dysmenorrhoea, benign prostatic hyperplasia (BPH), bladder outlet obstruction, incontinence, stable, unstable and variant (Prinzmetal) angina, hypertension, pulmonary hypertension, chronic obstructive pulmonary disease, coronary artery disease, congestive heart failure, atherosclerosis, conditions of reduced blood vessel patency, e.g.
• post-PTCA post-percutaneous transluminal coronary angioplasty
• peripheral vascular disease stroke, nitrate induced tolerance, bronchitis, allergic asthma, chronic asthma, allergic rhinitis, diseases and conditions of the eye such as glaucoma, optic neuropathy, macular degeneration, elevated intra-occular pressure, retinal or arterial occulsion and diseases characterised by disorders of gut motility, e.g. irritable bowel syndrome (LBS).
• LBS irritable bowel syndrome
• pre- eclampsia Kawasaki's syndrome, nitrate tolerance
• multiple sclerosis diabetic nephropathy
• neuropathy including autonomic and peripheral neuropathy and in particular diabetic neuropathy and symptoms thereof
• Particularly preferred conditions include MED and FSD (preferably FSAD).
• a method of soaking a chemical compound into a crystal comprising the following steps: a) incubating the crystal in an aqueous stabilising solution comprising buffer and polyethylene glycol; b) combining the chemical compound with the stabilising solution preferably wherein the chemical compound is in solid or liquid form, more preferably in liquid form, most preferably solublised in DMSO; and c) optionally adding a cryo-protectant to the stabilising solution.
• the stabilising solution comprises polyethylene glycol which is of the molecular weight 4000.
• the polyethylene glycol may be at a concentration of between 10% and 30%, but is most preferably
• the pH is preferably in the range of 7.0 to 7.8; most preferably the pH is 7.4.
• the buffer should be one capable of providing buffer capacity over the required pH range; preferably HEPES.
• the stabilising solution contains an alcohol; most preferably this is isopropanol.
• the stabilising solution contains 0.1M sodium HEPES pH 7.4, 20% polyethylene glycol 4000 and
• cryoprotectants include 2-methyl-2,4- pentanediol (MPD) and organic polymers e.g. lower molecular weight PEG.
• Carbohydrates such as sorbitol or xylitol, and alcohols may also be used.
• cryoprotectant is glycerol.
• a PDE5 ligand (also known as a PDE5 inhibitor compound) according to aspect 38 (hereinafter referred to as "the compound”) can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• the pharmaceutical compositions, pharmaceuticals and medicaments contemplated by the present invention may be formulated in various ways well-known to one of skill in the art and administered by similarly well-known methods.
• the compound of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, or controlled-release such as sustained-, dual-, or pulsatile delivery applications.
• the compound may also be administered via intracavernosal injection.
• the compound may also be administered via fast dispersing or fast dissolving dosages forms or in the form of a high-energy dispersion or as coated particles.
• Suitable pharmaceutical formulations of the compound may be in coated or un-coated form as desired.
• Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably com, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably com, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and
• Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
• Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
• the compound may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
• Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device.
• Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof.
• Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients.
• Release rate-modifying excipients maybe present both within the dosage form i.e. within the matrix, and/or on the dosage form i.e. upon the surface or coating.
• Fast dispersing or dissolving dosage formulations may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.
• dispersing or dissolving as used herein to describe FDDFs are dependent upon the solubility of the drug substance used i.e. where the drug substance is insoluble a fast dispersing dosage form can be prepared and where the drug substance is soluble a fast dissolving dosage form can be prepared.
• the compound can also be administered parenterally, for example, intracavernosally, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion or needleless injection techniques.
• parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
• the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
• the daily dosage level of the compound will usually be from 10 to 500 mg (in single or divided doses).
• tablets or capsules of the compound may contain from 5mg to 250mg of active compound for administration singly or two or more at a time, as appropriate.
• the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
• the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
• the compound may be taken as a single dose on an "as required" basis (i.e. as needed or desired).
• the compound can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EATM), carbon dioxide or other suitable gas.
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM or 1,1,1,2,3,3,3
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
• a lubricant e.g. sorbitan trioleate.
• Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
• Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff contains from 1 to 50 mg of a compound of the invention for delivery to the patient.
• the overall daily dose with an aerosol will be in the range of from 1 to 50 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
• the compound may also be formulated for delivery via an atomiser.
• Formulations for atomiser devices may contain the following ingredients as solubilisers, emulsifiers or suspending agents: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycols, sodium chloride, fluorocarbons, polyethylene glycol ethers, sorbitan trioleate, oleic acid.
• the compound can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
• the compound may also be dermally administered.
• the compound may also be transdermally administered, for example, by the use of a skin patch.
• the compound may also be administered by the ocular, pulmonary or rectal routes.
• the compound can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
• the compound may be formulated in an ointment such as petrolatum.
• the compound of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
• it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• the compound may also be used in combination with a cyclodextrin.
• Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes.
• the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser.
• Alpha-, beta- and garnma- cyclodextrins are most commonly used and suitable examples are described in WO-A- 91/11172, WO-A-94/02518 and WO-A-98/55148.
• oral administration the compound is the preferred route, being the most convenient and, for example in MED, avoiding the well-known disadvantages associated with intracavernosal (i.e.) administration.
• a preferred oral dosing regimen in MED for a typical man is from 25 to 250 mg of compound when required.
• the drug may be administered parenterally, sublingually or buccally.
• the compound, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate or pro-drug thereof is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
• the present invention provides the following numbered preferred aspects:
• a crystal of sequence-modified PDE5 protein comprising SEQ ID NO: 4 into which a compound of the formula represented in Figure 4 is capable of being soaked such that the compound is bound to the active site of the protein.
• sequence modified PDE5 protein includes the wild- type PDE5 protein amino acid sequence which has been manipulated to facilitate the crystallisation of the protein, and in particular provide soakable crystal forms of the protein into which a PDE5 ligand or potential ligand may be introduced by soalcing the crystal in a solution comprising the ligand so that the ligand can enter the crystal and bind to the active site of the protein in the process of crystal soalcing.
• the term includes a PDE5 protein that has been subjected to manipulations of the amino acid sequence of the catalytic domain of PDE5, specifically the 657-682 region of PDE5 (the "loop region") or SEQ ID No:l, and that facilitate the crystallisation of the protein and particularly provide soakable crystal forms of the protein.
• the relevant manipulations include manipulation by deletion, addition or substitution of one or more amino acid residues of the PDE5 loop region.
• Such manipulations for example include the deletion or substitution of the histidine (His/H) residue as shown emboldened and underlined in SEQ ID NO: 1 (HRGVNNSYIQRSEHPLAQLYCHSLME) to incorporate one or more amino acid residues (other than histidine), particularly amino acid residues that are neutral or non-polar.
• the relevant manipulations also include manipulation by complete replacement of the PDE5 loop region with a loop region (or other amino acid sequence) from another protein or protein sequence, for example another PDE such as PDE4, preferably PDE4b.
• the sequence modified PDE5 protein may comprise a manipulated sequence of the full PDE5 sequence or alternatively a manipulated sequence of a sub-domain or fragment of the PDE5 sequence, and is preferably a manipulated sequence of the PDE5 catalytic domain.
• a crystal according to preferred aspect 1 comprising SEQ ID NO:5.
• a crystal according to either preferred aspect 1 or preferred aspect 2 which is grown using polyethylene glycol as a precipitant Polyethylene glycol of molecular weights 2000 to 8000 are commonly used. Preferably the molecular weight of the polyethylene glycol is 4000.
• the polyethylene glycol may be at a concentration of between 10% and 30%, but is most preferably 20%.
• the protein used to grow the crystals is desired to be of a concentration between 1 and 20 mg/ml, preferably between 5 and 15 mg/ml and most preferably is at 10 mg/ml concentration.
• the pH is in the range of 7.0 to 7.8; most preferably the pH is 7.4.
• the buffer should be one capable of providing buffer capacity over the required pH range, preferably HEPES.
• the alcohol is isopropanol.
• a crystal according to any of the preceding preferred aspects which is grown in a solution containing HEPES buffer, polyethylene glycol 4000 and iso-propanol.
• the solution contains 0.1M sodium HEPES pH 7.4, 20% polyethylene glycol 4000 and 10% iso-propanol.
• the crystal is grown from a solution incubated at or below room temperature (20-25 °C).
• the crystal is grown from a solution incubated at a temperature within the range of 2-6 °C. More preferably, the crystal is grown from a solution incubated at about 4°C.
• (j) comprises a protein of a molecular weight of approximately 40kDa ⁇ 2 kDa; (lc) a calculated solvent content of approximately 44 + 5%; and (1) a monoclinic crystal system.
• the protein has an active site within the third sub-domain of the protein and comprises Leu 765, Ala 767 and He 768 and one or more of Phe 820, Val 782, Phe 786, Tyr 612, Leu 804, Ala 779, Ala 783, He 813, Met 816 and Gin 817.
• the process of soalcing may involve transferring the crystal to be soaked from the solution in which it is grown into a stabilising solution containing the ligand or to which the ligand is to be added once the crystal is present in it so that the ligand can enter the crystal and preferably bind to the active site of the protein.
• the stabilising solution has the physical properties such that the crystal, when transferred, retains its structural integrity and does not crack or dissolve or alter significantly in its crystal parameters, symmetry or cell dimensions.
• the stabilising solution comprises some or all of the chemical constituents from which the crystal was grown.
• the stabilising solution is of similar or identical composition to the solutions used for crystal growth as set out in aspects 3 to 6 above, although the concentration of precipitant used may be slightly increased by between 1 to 10%.
• the stabilising solution has the same pH as the crystal growth solution.
• the ligand may be added to the solution containing, or to contain, the crystal for soaking as a solid or in a liquid form (i.e. the ligand already being in solution).
• the ligand is in solution and the solvent maybe aqueous, organic or non-organic.
• the ligand is in DMSO.
• the ligand is commonly added to the soaking solution to yield a final ligand concentration above the expected binding constant for the ligand by PDE5; preferably this is greater than or equal to 10 times the binding constant.
• the final concentration of ligand present in the crystal soaking solution is preferably between 0.1 and 20 mg/ml, more preferably between 0.5 and 10 mg/ml, and most preferably between 1 and 5 mg/ml.
• a cryoprotectant such as 2-methyl-2,4-pentanediol (MPD), alcohols and organic polymers e.g. lower molecular weight PEG or carbohydrates such as sorbitol or xylitol, may also added to the soalcing solution if the crystal is to be frozen prior to data collection.
• the co-ordinate set or a part thereof can be used according to known methods to provide a full or partial phasing model for molecular replacement procedures to determine the X-ray crystal structure of a structurally similar or related protein or protein sub-domains or fragments, additionally the coordinate set can provide a structural model from which NMR assignments for NMR structure solution of structurally similar or related proteins or protein sub-domains or fragments can be determined.
• a method of identifying a compound which is a ligand of PDE5 comprising the following steps: a) providing a crystal according any one of preferred aspects 1 to 14, b) contacting the crystal with the compound under conditions conducive to soalcing the compound into the crystal, c) determining, by X-ray diffraction and structure solution, whether or not the compound is bound to the active site of the protein in the resultant soaked crystal, d) optionally assaying the ligand to determine whether it is an inhibitor of PDE5.
• the crystal may be soaked under the conditions as set out in preferred aspect 12 and the X-ray crystal structure may be determined by X-ray diffraction of the crystal and application of any suitable known method such as direct methods, heavy atom phasing methods, single or multiple anomalous dispersion, molecular replacement or difference Fourier methods.
• the presence or absence of the ligand at the active site of the protein can be determined from the electron density maps, for example from difference Fourier maps or omit maps, calculated from the X-ray diffraction data.
• a method of selecting a PDE5 ligand from a group of potential PDE5 ligands comprising the following steps:
• the three- dimensional representation of the structure of the protein may be directly derived from the atomic coordinates determined for the crystal and may be suitably displayed to identify the active site of the protein as defined in preferred aspect 8 and 9.
• the active site of the protein may be computationally and graphically rendered to display the atomic radii of the atoms comprising the active site such that a similarly graphically rendered compound molecule may be docked into the active site to look for the goodness of fit between the protein active site and the compound, preferably by examination for steric clashes and/or favourable packing and/or bonding interactions between the groups and atoms comprising the active site of the protein and those of the compound.
• the resulting docked compound-protein model may be analysed computationally to assess the fit or may be adjusted computationally, for example by molecular dynamics and energy minimisation of the model, to improve the fit of the compound in the active site prior to fit analysis.
• a site directed mutant is preferably designed by computationally creating a three-dimensional representation of the structure of the protein present in the crystal according to any of preferred aspects 1 to 14, or a protein fragment or protein subdomain as may be directly derived from the atomic coordinates determined for the crystal.
• the model may be overlayed with, or least squares fitted against, a model of a similar or related protein or protein sequence fragment in order to allow substitution of amino acids or whole peptide regions between the model on a knowledge based basis to alter the physical characteristics of the protein, for example to substitute a known unstable loop or region with a known stable loop or region between structures (the stability of a region of protein sequence can be judged by the B factors for the protein region in the coordinate set).
• One or more side chain replacements, additions or deletions may be made in the model of the protein or whole fragments of polypeptide may be replaced, added or deleted to potentially affect the physical property of the protein, for example, ability to crystallise by alteration of unstable regions, solubility by substitution of hydrophobic groups with hydrophilic groups.
• the site directed mutant of the protein may be made and tested for the improvement in the physical parameter for example by assessing ability to crystallise the mutated protein or the solubility of the mutated protein in comparison to the native protein.
• site directed mutation of the active site residues may be designed in order to assess side chains involved in key bond donor or acceptor functions with a PDE5 ligand or potential ligand compound, or to design active site mutations to better accommodate a compound.
• This may be achieved by co-displaying the protein and compound models as detailed in preferred aspects 19 and/or 20 and making replacements of protein side chains at the active site of the protein model in order to assess, either by graphics visualisation of the fit and bonding interactions between the compound and the protein model or by computational analysis of the model complex for steric fit or clashing and/or bonding interactions, whether a compound is better accommodated in the active site by such changes; this process may optionally involve model energy minimisation and/or molecular dynamics steps as described in the preref erred embodiments in preferred aspects 19 and/or 20.
• the site directed mutant of the protein may be made and assayed in a PDE5 ligand binding assay.
• a method of soaking a compound into a crystal comprising the following steps: a) incubating the crystal in an aqueous stabilising solution comprising buffer and polyethylene glycol; b) combining the compound with the stabilising solution; and c) optionally adding a cryo-protectant to the stabilising solution.
• the stabilising solution is of similar or identical composition to the solutions used for crystal growth as set out in aspects 3 to 6 above, although the concentration of precipitant used may be slightly increased by between 1 to 10%.
• the stabilising solution has the same pH as the crystal growth solution. More preferably the stabilising solution comprises 0.1M sodium hepes pH 7.4, 20% PEG 4000, 10% isopropanol.
• the compound is in solution and the solvent maybe aqueous, organic or non-organic. Most preferably the compound is in DMSO.
• the ligand is commonly added to the soaking solution to yield a final ligand concentration above the expected binding constant for the ligand by PDE5; preferably this is greater than or equal to 10 times the binding constant.
• the final concentration of ligand present in the crystal soaking solution is preferably between 0.1 and 20 mg/ml, more preferably between 0.5 and 10 mg/ml, and most preferably between 1 and 5 mg/ml.
• the cryoprotectant is selected from one or more of 2-methyl-2,4-pentanediol (MPD), alcohols, organic polymers e.g. lower molecular weight PEG, carbohydrates such as sorbitol or xylitol, most preferably the cryoprotectant is glycerol.
• apo as used herein is taken to mean macromolecule and inparticular any protein (or named protein) that is detached from a/its ligand(s) and/or prosthetic group(s).
• buffer as used herein is taken to include any solution containing a weak acid and a conjugate base of this acid (or, less commonly, a weak base and its conjugate acid).
• a “buffer” as used herein resists change in its pH level when an acid or a base is added to it, because the acid neutralises an added base (or, less commonly, the base neutralises an added acid).
• an "activity assay” as referred to herein with reference to PDE5 is taken to mean an in vitro assay of PDE inhibitory activities against cyclic guanosine 3',5'-monophosphate (cGMP) and cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterases which can be determined by measurement of their IC 50 values (the concentration of compound required for 50% inhibition of enzyme activity).
• cGMP cyclic guanosine 3',5'-monophosphate
• cAMP cyclic adenosine 3',5'-monophosphate
• the required PDE enzymes can be isolated from a variety of sources, including human corpus cavernosum, human and rabbit platelets, human cardiac ventricle, human skeletal muscle and bovine retina, essentially by a modification of the method of Thompson WJ and Appleman MM; Biochemistry 10(2),311-316, 1971, as described by Ballard SA et al; J. Urology 159(6), 2164-2171, 1998.
• cGMP-specific PDE5 and cGMP-inhibited cAMP PDE3 can be obtained from human corpus cavernosum tissue, human platelets or rabbit platelets.
• Assays can be performed either using a modification of the "batch” method of Thompson, WJ et al; Biochemistry 18(23), 5228-5237, 1979, essentially as described by Ballard SA et al.; J. Urology 159(6), 2164-2171, 1998, or using a scintillation proximity assay for the direct detection of [ 3 H] -labelled AMP/GMP using a modification of the protocol described by Amersham pic under product code TRKQ7090/7100.
• the final assay volume is made up to lOO ⁇ l with assay buffer [20mM Tris-HCl pH 7.4, 5mM MgCl 2 , lmg/ml bovine serum albumin].
• Reactions are initiated with enzyme, incubated for 30-60min at 30°C to give ⁇ 30% substrate turnover and terminated with 50 ⁇ l yttrium silicate SPA beads (containing 3mM of the respective unlabelled cyclic nucleotide for PDE5). Plates are re-sealed and shaken for 20min, after which the beads are allowed to settle for 30min in the dark and then counted on a TopCount plate reader (Packard, Meriden, CT). Radioactivity units are converted to % activity of an uninhibited control (100%), plotted against inhibitor concentration, and inhibitor IC 50 values obtained using the 'Fit Curve' Microsoft Excel extension.
• precipitant is taken to include any substance that, when added to solution comprising a biological molecule, causes the biological molecule to precipitate or crystallise from the solution.
• complex as used herein is taken to mean a biological macromolecule, preferably a protein, with ligand(s) bound and may be formed before, during or after protein crystallisation.
• soaking or “capable of being soaked” as used herein is taken to mean a process of placing a crystal in an aqueous solution containing a chemical compound, (usually) small molecule (e.g. inhibitor), or adding a chemical compound to an aqueous solution containing a crystal such that the compound may be able to enter the crystal lattice by diffusion and may interact (e.g. contact and bond with) with the molecules comprising the lattice preferably, where the molecules comprising the lattice are protein molecules, to form a protein-ligand complex, most preferably where the molecules comprising the lattice are protein molecules and the ligand or compound become bound to the active site of the protein molecule.
• the compound may be added to the aqueous solution in solid form, or it may be dissolved in a suitable solvent, preferably di-methyl-sulphoxide (DMSO).
• DMSO di-methyl-sulphoxide
• a soakable crystal is taken to mean a crystal into which a small molecule or ligand may be soaked without significant disruption of the lattice such that the ligand can enter and pass through the crystal lattice and have access to, and may associate with, the molecules comprising the lattice.
• a soakable crystal is a crystal which is ameanable to the soaking process in which a small molecule or ligand either in solution or in solid form is added to the solution containing the crystal. The small molecule may then enter the crystal lattice by diffusion and associate with the molecules comprising the crystal lattice.
• the soakable crystal binds the ligand without disruption of the lattice or cracking of the crystal and that the lattice symmetry and crystal parameters are not significantly altered by the soalcing and ligand binding process. It is preferable that the soakable crystal diffracts X-rays to atomic resolution, ' preferably to beyond 3.5 A, more preferably beyond 2.5A, most preferably 1.5 A, after undergoing the soalcing process.
• stabilising solution as used herein is taken to mean a solution into which a crystal may be transferred for the purposes of further manipulation, for example for soaking, and in which the crystal retains its structural integrity and does not crack or dissolve or alter significantly in its crystal parameters, symmetry or cell dimensions.
• the stabilising solution comprises some or all of the chemical constituents from which the crystal was grown and is of the approximately the same, and more preferably identical, pH.
• cryoprotectant as used herein is taken to mean a chemical compound which, when added to a solution, allows the solution to be rapidly frozen without the formation of ice crystals.
• a cryoprotectant is preferably an alcohol such as ethanol or a carbohydrate such as xylitol or sorbitol, but may also be 2-methyl-2,4- pentanediol (MPD) or other organic polymers e.g. lower molecular weight PEG.
• MPD 2-methyl-2,4- pentanediol
• Most preferably the cryoprotectant is glycerol.
• co-crystallisation is taken to mean crystallisation of a pre-formed comple of a macromolecule with it's ligand i.e. a protein/small molecule complex.
• mutant are in relation to the amino acid sequence of the PDE5 protein or polypeptide sequence which is used to produce the crystal of the present invention.
• the terms include any substitution of, variation of, modification of, replacement of, deletion of, or addition of one (or more) amino acids from (or to) the sequence providing the resultant PDE5 is capable of being crystallised.
• amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions, provided that the modified PDE5 retains the ability to be crystallised in accordance with present invention. Amino acid substitutions may include the use of non-naturally occurring analogues.
• variant refers to additions, deletions or substitutions of amino acid residues comprised within the wild- type amino acid sequence or fragment thereof.
• a variant in relation to the amino acid sequence of the crystal of the PDE5 of the present invention could include the deletion or substitution of the histidine (His/H) residue as shown emboldened and underlined in SEQ ID NO: 1 (HRGVNNSYIQRSEHPLAQLYCHSIME), which sequence is comprised in the protein or polypeptide of PDE5 of the crystal of the present invention.
• Replacement of said histidine (H) residue is preferably by way of incorporating one or more amino acid residues (other than histidine), preferably wherein said amino acid residues are neutral or non-polar.
• mutants in relation to the nucleotide sequence coding for the PDE5 of the crystal of the present invention include any substitution of, variation of, modification of, replacement of, deletion of, or addition of, one (or more) nucleotide from (or to) the sequence providing the resultant nucleotide sequence codes for, or is capable of coding for, a PDE5 which is capable of being crystallised.
• variant refers to additions, deletions or substitutions of nucleotides of the wild-type nucleotide sequence or fragment thereof.
• fragment refers to any portion of the PDE5 amino acid sequence as defined in the present invention provided the resultant PDE5 comprising said PDE5 portion is capable of being crystallised.
• fragment also includes PDE5, which comprises any portion of SEQ ID NOS: 1, 2, 3, 4, 5, or 6.
• SEQ JO NO: 6 full-length "loop-swapped" PDE5 sequence
• SEQ ID NO: 5 full-length "loop-swapped" PDE5 catalytic domain
• SEQ ID NO: 4 PDE4 "loop region"; HPGVSNQFLTNTNSELALMYNDESVLE
• analogue as used herein means a sequence similar to the amino acid sequence of the PDE5 polypeptide of the crystal of the present invention or of any one of SEQ JJD NOS: 1, 2, 3, 4, 5 or 6, but wherein non-detrimental (i.e. not detrimental to the PDE5's capability of being crystallised) amino acid substitutions or deletions have been made.
• derivative as used herein in relation to the amino acid sequence of the PDE5 of the crystal of the present invention, or of any one of SEQ ID NOS: 1, 2, 3, 4, 5 or 6, includes chemical modification of PDE5. Hlustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.
• heavy atom derivative refers to a crystal comprising macromolecules in the crystal lattice which are modified by the inclusion of a heavy atoms (i.e. of significantly greater atomic mass than the atoms common to organic macromolecules, e.g. carbon, nitrogen , oxygen, phosphorous, etc.) into their structure.
• a heavy atoms i.e. of significantly greater atomic mass than the atoms common to organic macromolecules, e.g. carbon, nitrogen , oxygen, phosphorous, etc.
• ionic compounds such as salts of mercury, platinum, gold or silver which may covalently bond to certain groups on the macromolecule either during co-crystalisation with the ionic compound or by soaking the compound into the crystal.
• Other processes may also be used such as derivitisation with seleno-methionine or with noble gases such as Xenon.
• the purpose of such heavy atom derivatives of crystals is to provide phasing information for structure solution.
• deletion is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
• an "insertion” or “addition” is a change in a nucleotide or amino acid sequence, which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the sequences of the naturally occurring PDE5.
• substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
• homologue covers homology specifically with respect to protein structure, primary secondary, tertiary and quarternary, and covers any structural PDE5 homologue that is capable of being crystallised.
• homology of the amino acid sequences detailed herein preferably there is at least 70%, more preferably at least 75%, more preferably at least 80%, yet more preferably at least 85%, even more preferably at least 90% homology to SEQ ID NOS: 1, 2, 3, 4, 5 or 6. More preferably there is at least 95%, and most preferably at least 98%, homology to SEQ ID NOS: 1, 2, 3, 4, 5 or 6.
• homology of the nucleotide sequences coding for the amino acid sequences detailed herein preferably there is at least 70%, more preferably at least 75%, more preferably at least 80%, yet more preferably at least 85%, even more preferably at least 90% homology to the nucleotide sequences which code for SEQ ID NOS: 1, 2, 3, 4, 5 or 6. More preferably there is at least 95%, and most preferably at least 98%, homology to the nucleotide sequences which code for SEQ ID NOS: 1, 2, 3, 4, 5 or 6.
• homologue with respect to the nucleotide sequence of the PDE5 as defined in the present invention and the amino acid sequence of the PDE5 as defined in the present invention may be synonymous with allelic variations of the sequences.
• sequence homology with respect to, for example, the amino acid sequence of PDE5 of the crystal of the present invention can be determined by a strict comparison of any one or more of the sequences with another sequence to see if that other sequence has at least 70% identity to the sequence(s).
• Relative sequence homology i.e. sequence identity
• sequence identity can also be determined by commercially available computer programs that can calculate percentage (%) homology between two or more sequences.
• a typical example of such a computer program is CLUSTAL.
• Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
• the alignment process itself is typically not based on an all-or-nothing pair comparison.
• a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
• An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
• GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
• sequence homology may be determined using any suitable homology algorithm, using for example default parameters.
• BLAST algorithm is employed, with parameters set to default values.
• the BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html.
• substantially homology when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more.
• the default threshold for EXPECT in BLAST searching is usually 10.
• amino acid sequence of the PDE5 of the present invention present invention may be produced by expression of a nucleotide sequence coding for the same in a suitable expression system.
• the protein itself could be produced using chemical methods to synthesize a PDE5 amino acid sequence, in whole or in part.
• peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g. Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman and Co., New York, NY, USA). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g. the Edman degradation procedure).
• Direct peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al, Science, Vol 269, 1995, pp. 202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer, Boston, MA, USA) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequence of PDE5, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant polypeptide.
• This engineered protein has been shown to be stable to degradation by mass spectrometry and SDS-PAGE (data not shown).
• the protein shows improved biophysical properties allowing an alternative purification protocol to be developed.
• the new protocol utilises binding to a Blue sepharose column and specific elution with cGMP.
• the wild-type protein had been shown not to bind to this column probably due to the disorder of the structure around the protease cleavage site.
• This PDE5* protein was used to produce crystals with inhibitors which diffract to higher resolution and have no disordered regions.
• the protein has also been used reproducibly to produce crystals with further inhibitors which routinely diffract to 1.8 A resolution or higher, making it an improved protein for use in structure based drug design.
• Helices 1 (HI 539-545) and 2 (H2 551-554) lie on the exterior of the protein and comprise the N-terminal region of the construct. These two helices do not overlay with the equivalent ones (HO, HI and H2) in the PDE4 structure. This region is not well conserved across the PDE protein family.
• Helices 3 (H3 568-582), 4 (H4 584-588), 5 (H5 592-604), 6 (H6 615-631) and 7 (H7 640-652) form the first sub-domain of the protein and are contained within the core of the protein. There is no observable electron density for helices 8 and 9 based on the PDE4 nomenclature.
• Helix 10 (H10 684-694) is again on the exterior and forms the dimer interface within the structure.
• Helices 10 and 11 are the visible portion of the second sub-domain.
• Helices 12 (H12a 725-731, H12b 733-741), 13 (H13 749- 765), 14 (H14 772-797), 15 (H15 813-824), 16 (H16 826-836) and 17 (H17 841-861) form the third sub-domain of the protein.
• helix H12 is not a contiguous helix as in PDE4 but is composed of two short helices with a kink in the middle and helix HI 5 is a contiguous helix in PDE5 but not in PDE4.
• the structure of the catalytic domain of PDE5* protein complexed with UK-088,800 was determined by molecular replacement using one molecule of the protein structure from the PDE5* complex with Sildenafil (as described in Application Number PCT/IB02/04426: Example 11, "crystallisation of PDE5* with Sildenafil", page 46, lines 8 to 25; Example 15, "data collection, structure determination and refinement PDE5* with Sildenafil", page 50, line 9 to page 51, line 6 and Table 6, "atomic coordinates for baculovirus-expressed PDE5* complex with Sildenafil", pages 328 to 408; incorporated herein by reference) as a basis for the search model, the co-ordinates for which are included in Table 3 of the present application.
• the PDE5* catalytic domain crystallises in the space group C2 as a monomer with one molecule present in the asymmetric unit.
• the structure of the PDE5* comprises 17 ⁇ helices and the overall fold is very similar to the wild-type structure with a number of important differences.
• helices H8 and H9 composed of the swapped portion from PDE4, residues 657-682. These fold in an identical way to that observed in the PDE4 structure and complete the second sub-domain of the protein.
• the entire C-terminal region of this construct can also be built into the electron density leaving just three disordered residues at the N- terminus of this structure. This is likely to contribute to its enhanced properties for crystallisation.
• PDE5* Active site and protein-inhibitor interactions
• UK-088,800 occupies the same region of the active site as observed for the previously described UK-092,480, Sildenafil, as detailed below (Application Number PCT/IB02/04426: Figure 4, "A view of compound Sildenafil bound to loop swapped PDE5 (PDE5*), page 425; incorporated herein by reference) and demonstrates the same mainly hydrophobic interactions with the protein ( Figure 3 "Comparison of the fo-fc electron density map contoured at 2.5 ⁇ of apo PDE5* (left) and PDE5* soaked with UK-088,800 (right), at the active site region" of the present application). The same two direct hydrogen bonds observed in the complex of PDE5* with Sildenafil are also observed between Gin 817 of the protein and inhibitor (O ⁇ l-N4 2.9 A and N ⁇ 2- Ol 3.1 A).
• Carbon atom C7 of the inhibitor points into a small hydrophobic pocket formed by Leu 765, Ala 767 and He 768. These residues, together with Phe 820, form a planar face to the binding site against which the purine ring of the inhibitor stacks. The opposite side of the purine packs against Val 782.
• the CIO propyl substituent form good van der Waals contacts with Phe 786.
• Phe 786 packs against Leu 804 which in turn forms additional hydrophobic interactions with the phenyl moiety of the inhibitor.
• the O-alkyl moiety occupies a small pocket bounded by Ala 779, Phe 786, Ala 783, Val 782, Leu 804, He 813, Met 816 and Gin 817.
• Wild-type PDE5-Sildenafil complex Active site and protein-inhibitor interactions
• the active site lies mainly within the third sub-domain of the protein and is bounded by helices H15, H14, the C-terminus of H13, and the C-terminus of Hll, along with the loop region between Hll and H12a.
• the majority of the interactions between the inhibitor and the protein are hydrophobic in nature, with only two direct hydrogen bonds observed (Application Number PCT/TB 02/04426: Figure 3, page 424, incorporated herein by reference).
• the first is between N17 of the purine ring of the inhibitor and O ⁇ l of Gin 817 (2.8 A) and the second is from the adjacent oxygen atom O16 of the inhibitor to N ⁇ 2 of the same residue Gin 817 (3.1 A).
• Carbon atom C12 of the inhibitor points into a small hydrophobic pocket formed by Leu 765, Ala 767 and He 768. These residues, together with Phe 820, form a planar face to the binding site against which the purine ring of the inhibitor stacks. The opposite side of the purine packs against Val 782.
• the C5 propyl substituent forms good van der Waals contacts with Val 782 and Phe 786 and Tyr 612.
• Phe 786 and Leu 804 form additional hydrophobic interactions with the phenyl moiety of the inhibitor.
• the O-alkyl moiety occupies a small pocket bounded by Ala 779, Phe 786, Ala 783, Val 782, Leu 804, ⁇ e 813, Met 816 and Gin 817.
• the sulphonamide group points out towards the solvent whilst the piperazine ring is bounded by the extended residues 662-665, although whether the conformation of this part of the structure is unaffected by the chain break is questionable.
• the structure confirms the competitive nature of the mode of inhibition of Sildenafil by binding in the active site therefore blocking access for the cGMP substrate (which has also been modelled - data not included).
• Wild type PDE5-Sildenafil Complex Metal Ions in the Active Site
• SEQ ID NO: 1 shows the amino acid sequence of the loop region from PDE5.
• SEQ ID NO: 2 shows the amino acid sequence of the wild-type PDE5 catalytic domain.
• SEQ ID NO: 3 shows the amino acid sequence of the full-length wild-type PDE5 sequence.
• SEQ ID NO: 4 shows the amino acid sequence of the loop region of PDE4.
• SEQ ID NO: 6 shows the amino acid sequence of full-length PDE5 sequence comprising PDE5*.
• SEQ ID NOS: 7-14 are oligonucleotide primers.
• Figure 1 shows an alignment of PDE5 (upper sequence) and PDE4b (lower sequence) catalytic domains. Positions and numbering of helices from the structures are marked for each. Residues in bold show a sequence alignment for the engineered region. The sequence from PDE4 has been used to replace the corresponding region in PDE5. This results in a residue insertion in this region. Underlining highlight C-terminal region absent in PDE5*.
• Figure 2 shows a ribbon representation of the overall fold of proteins showing secondary structure elements.
• the inhibitor is shown in an all atom stick representation and the metal ions as spheres.
• (A) PDE4b
• (B) wild-type PDE5 + Sildenafil
• (C) "loop-swapped" PDE5 (PDE5*) + Sildenafil.
• Helices are numbered using PDE4 structure as reference. Helices HO - H7 form sub-domain 1, helices H8- Hll form sub-domain 2, and helices H12-H16 form sub-domain 3.
• Figure 3 Comparison of the fo-fc electron density map contoured at 2.5 ⁇ of apo PDE5* ("loop-swapped" PDE5) (left) and PDE5* soaked with UK-088,800 (right), at the active site region.
• Figure 4 A two dimensional representation of the structure of UK-088,800, 5-(2- ethoxyphenyl)-l-methyl-3-propyl-l,6-dihydro-7H-pyrazolo[4,3-(f
• PDE5 3' Long Oligo CGTTCTAGACTATCAGTTCCGCTTGGCCTGGCCGCTTTCCCC (SEQ ID NO: 8)
• the PCR reaction was carried out for 30 cycles in a total volume of 50 ⁇ l in a solution containing 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 50 pmol of each primer and 2.5 units of Expand DNA polymerase (Roche, Eastshire, UK). Each cycle was 94°C, 1 min, 50°C, 1 min and 72°C, 2 mins.
• the final amplified DNA fragments for both constructs were separated on a 1% agarose gel and purified using a QIAquick gel extraction kit (Qiagen, West Wales, UK). The fragment was then digested using EcoRI and Xbal, and ligated into pFastbacl EcoRIVZb l-digested vector (Life Technologies, Paisley, UK). The ligation was carried out at 12°C for 16 hours. The ligation mix was then electroporated into E. coli (TOP 10) (Invitrogen, Gronigen, The Netherlands).
• Clones containing the desired insert were selected by using 2YT plates containing 100 ⁇ g/ml ampicillin and checked using endonuclease digestion for presence of correct size insert. DNA sequence analysis was carried out by Lark (Saffron Waldon, UK).
• Recombinant bacmid DNA was produced by transforming E.coli DH10BACTM with pFastbacl ::PD ⁇ 5 catalytic domain (534-875) plasmid DNA. This was carried out according to the method shown in the Bac to BacTM baculovirus expression manual (Life Technologies, Paisley, UK). PCR analysis was used to verify successful transposition to the bacmid using pUC/M13 amplification primers (Invitrogen, Gronigen, The Netherlands).
• the supernatant was harvested by centrifugation and stored at 4°C as the working virus stock.
• the titre of this working stock was determined by conventional plaque assay analysis as in the Bac to Bac baculovirus expression manual (Invitrogen, Gronigen, The Netherlands).
• Protein expression was optimised in Erlenmeyer flask cultures using Sf-9 and T.ni High5 insect cell cultures looking at different multiplicity's of infection (MOI) and harvest times, the optimal conditions found were then scaled up into fermenters.
• MOI multiplicity's of infection
• the fermenters used were autoclavable Applikon 3 litre stirred vessels controlled using Applikon 1030 biocontrollers.
• Inoculum of T.ni High5 cells was initially prepared from shake flask cultures.
• the fermenter was inoculated with 5 x 10 5 cells/ml, with an initial working volume of 1.8 litres made up in Excel 405 serum free medium (JRH Biosciences, Kansas, USA).
• Temperature was controlled at 27°C, dissolved oxygen concentration controlled at 60% and pH was measured but not controlled. Oxygen concentration was controlled throughout. Agitation was set at 150 rpm with a double impeller system of marine impeller within the culture and Rushton impeller at the liquid/headspace interface. Aeration was continuous to the headspace at 0.5 1/min.
• the culture was infected using an MOI of 1 from the titred baculovirus working stock.
• Harvest time for the culture was 48 hours post infection. This was achieved by centrifugation at 2000 g for 15 mins; the insect cell pellet was then stored at -80°C prior to purification.
• the PCR reaction was carried out for 30 cycles in a total volume of 50 ⁇ l in a solution containing 1.5 mM MgCl , 200 ⁇ M dNTPs, 50 pmol of each primer and 2.5 units of Expand DNA polymerase (Roche, Eastshire, UK). Each cycle was 94°C, 1 min, 50°C, 1 min and 72°C, 2 mins.
• the final amplified DNA fragments for both constructs were separated on a 1% agarose gel and purified using a QIAquick gel extraction kit (Qiagen, West Wales, UK). The fragment was then digested using EcoRI and Xbal, and ligated into pFastbacl EcoRIVZb l-digested vector (Life Technologies, Paisley, UK). The ligation was carried out at 12°C for 16 hours. The ligation mix was then electroporated into E. coli (TOP 10) (Invitrogen, Gronigen, The Netherlands).
• Clones containing the desired insert were selected by using 2YT plates containing 100 ⁇ g/ml ampicillin and checked using endonuclease digestion for presence of correct size insert. DNA sequence analysis was carried out by Lark (Saffron Waldon, UK). Methods to generate the recombinant baculovirus were as those for wild-type PDE5 catalytic domain (see EXAMPLE 1).
• the PDE5* construct was produced by using overlap extension PCR where the following oligonucleotides were used:
• Initial DNA fragments were generated using oligonucleotides A+B and C+D with the same template DNA as for the wild-type PDE5 catalytic domain construct.
• the PCR reaction was carried out for 30 cycles in a total volume of 50 ⁇ l in a solution containing 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 50 pmol of each primer and 2 units of Expand DNA polymerase (Roche, East Wales, UK). Each cycle was 94°C, 1 min, 50°C, 2 min, and 72°C, 3 min.
• DNA products from PCR A+B and C+D were used as template DNA with the oligonucleotides A+D used to amplify the full-length construct.
• the PCR conditions were the same as the initial PCR reaction. This generates a construct with the PDE4 swapped region and a C-terminal truncation (C-term 858) as compared to PDE5 catalytic domain (C-term 875).
• the PDE5* construct in E. coli was produced by using PCR where the following oligonucleotides were used and the template DNA being pFastbacl ::PDE5* plasmid DNA (sequence verified), produced in EXAMPLE 3.
• the PCR reaction was carried out for 30 cycles in a total volume of 50 ⁇ l in a solution containing 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 50 pmol of each primer and 2.5 units of Expand DNA polymerase (Roche, Eastshire, UK). Each cycle was 94°C, 1 min, 50°C, 1 min and 72°C, 2 mins.
• the final amplified DNA fragment was separated on a 1% agarose gel and purified using a QIAquick gel extraction kit (Qiagen, West Wales, UK). The fragments were then digested using Ndel and Xhol, and ligated into pET21C (Novagen, Nottingham, UK) Ndel/Xhol -digested vector. The ligation was carried out at 12°C for 16 hours. The ligation mix was then electroporated into E. coli (TOP 10) (Invitrogen, Gronigen, The Netherlands).
• TOP 10 E. coli
• Clones containing the desired insert were selected by using 2YT plates containing 100 ⁇ g/ml ampicillin. Plasmid DNA was also checked using endonuclease digestion for presence of correct size insert. DNA sequence analysis was carried out by Lark (Saffron Waldon, UK).
• E. coli BL21 (DE3) (Novagen, Nottingham, UK) for expression.
• Expression was carried out in 7 litre Applikon fermenters using 5 litre 2YT broth containing lOO ⁇ g/ml carbenicillin as the medium. Agitation was set at 1000 rpm using a double rushton impeller assembly and aeration to the sparger at 2 litres/min.
• the fermenter was inoculated with an overnight shake flask culture grown at 37°C and 200 rpm, the inoculation density was 1% vol/vol.
• the fermentation was pH controlled at 7.2 using 20% vol/vol NH OH solution and temperature initially set to 37°C.
• the temperature set-point was reduced to 25°C and then the culture was induced with IPTG at a final concentration of ImM. The fermentation was then harvested 4 hours post-induction by batch centrifugation (8,000 rpm for 10 minutes). The final pellet was then frozen (-80°C) to await subsequent purification.
• Pellet from the E. coli fermentation was resuspended into 10 mis lysis buffer per gram wet cell weight and mechanically broken using a continuous cell disrupter (Constant Systems, Warwickshire, UK) at a pressure of 20 kpsi.
• the lysis buffer consisted of 50 mM Tris HCl (pH 7.5), 100 mM NaCl, 1 mM DTT containing EDTA-free protease inhibitor cocktail tablets (Roche, East Wales, UK) and 10 ⁇ M E-64.
• the lysate was chilled and centrifuged at 14000 g for 45 min to remove cell debris. All purifications were subsequently carried out using an Akta Explorer purification system (Amersham Pharmacia, Buckinghamshire, UK).
• the supernatant was applied to a 50 ml Q- sepharose fast-flow column (Amersham Pharmacia, Buckinghamshire, UK) at 5 ml/min with the flow-through collected.
• the flow-through sample was then applied at 50 ml/min to a 2 litre G-25 superfine desalting column (Amersham Pharmacia, Buckinghamshire, UK) pre-equilibrated in Blue sepharose buffer A (50 mM Bis-Tris (pH 6.4), 50 mM NaCl, 2 mM EDTA, 2 mM EGTA and 1 mM DTT).
• Blue sepharose buffer A 50 mM Bis-Tris (pH 6.4), 50 mM NaCl, 2 mM EDTA, 2 mM EGTA and 1 mM DTT.
• the protein fraction was eluted in Blue sepharose buffer A.
• the next column step was carried out in series, loading the sample initially onto a 20 ml SP-sepharose high performance column (Amersham Pharmacia, Buckinghamshire, UK) then flow-through from this directly onto a 10 ml Blue sepharose fast-flow column (Amersham Pharmacia, Buckinghamshire, UK) at a flow- rate of 2 ml/min.
• the SP-sepharose column was taken out of line and the Blue sepharose column washed with 5 column volumes of Blue sepharose buffer A.
• the column was washed with Blue sepharose buffer A containing 1 M NaCl until the absorbance 280 nm reached baseline and then washed with 5 column volumes of Blue sepharose buffer A.
• PDE5* protein was step-eluted using Blue sepharose buffer containing 20 mM cGMP (Na-salt) (Sigma, Dorset, UK). Fractions were assayed on Tris-glycine SDS gels (Invitrogen, Gronigen, The Netherlands) and pooled accordingly. These fractions were concentrated to 2.5 mg/ml using centrifugal concentrators (Vivascience, Gloucestershire, UK) and loaded at 1.5 ml/min onto a Superdex-200 prep grade 26/60 column pre-equilibrated with 50 mM Bis-Tris (pH 6.8), 500 mM NaCl, 1 mM DTT and 2 ⁇ M E-64. The eluted fractions were analysed on Tris-glycine SDS PAGE gels.
• the PDE5* fractions from the final gel filtration column were pooled (total volume of 25mls) and the protein concentration was assayed (0.2mg/ml).
• the protein solution was supplemented with 10 ⁇ M E-64 and lmg/ml leupeptin (Sigma, Dorset, UK).
• the solution was concentrated to lOmg/ml using a Centriprep lOlcDa Molecular weight cut-off centrifugal concentrator (Amicon Bioseparations, Maine, USA) at 3,000rpm, 4°C. Prior to crystallisation, the protein solution was centrifuged for 5 min at 14,000rpm in an Eppendorf centrifuge.
• Plate-shaped crystals of apo PDE5* obtained according to methods detailed in EXAMPLE 6 were transferred to a 20 ⁇ l drop of 0.1M sodium HEPES and 20% Polyethylene glycol 4000 in square microbridges. To this, l.O ⁇ l of a 20mg/ml concentration of inhibitor dissolved in DMSO was added. Crystals were then transferred to a solution of 0.1M sodium HEPES pH 7.4, 20% Polyethylene glycol 4000 with 15% glycerol and then frozen during X-ray data collection.
• Molecular replacement was performed using AMORE (CCP4, J.Navaza 1994). The resulting map was of good quality and the structure was refitted using QUANTA® (Accelrys products).
• a representative X-ray diffraction data set was collected with a Rigaku Saturn92 CCD detector on an in-house FR-D rotating anode (Rigaku), with Osmic mirrors (MSC). Data were processed using the CrystalClear/D*Trek processing package (Rigaku- MSC). Data collection statistics are summarised in Table 1 (Apo).
• the co-ordinate set for the refined model of apo PDE5* is recorded in Table 4.
• the model contains 323 amino acid residues, 537-858 (residue Glu 681A has been numbered to maintain PDE5 numbering scheme), a zinc ion and a magnesium ion, as well as 222 water molecules.
• the co-ordinate set for the refined model of PDE5*-UK-088,800 is recorded in Table 5.
• the model contains 323 amino acid residues, 537-858 (residue Glu 681 A has been numbered to maintain PDE5 numbering scheme), the ligand UK-088,800, a zinc ion and a magnesium ion, as well as 183 water molecules.
• R, ,ym ⁇
• ATOM 40 CD GLN A 541 1. .709 29, .475 14, .868 1. .00 28. .37
• ATOM 46 CA SER A 542 3. .103 25, .440 20, .341 1. ,00 27. ,52
• ATOM 90 CA PRO A 549 11, .647 15. .414 26, .709 1. .00 28, .05
• ATOM 98 OG SER A 550 14. .611 11. .959 28, .843 1. ,00 32, .28
• ATOM 103 CB ALA A 551 12, .451 7, ,610 25, .756 1. .00 33, .59
• ATOM 104 C ALA A 551 12, .064 8. .389 28, .097 1. ,00 27, .99
• ATOM 110 CD GLN A 552 17. .068 9. .439 30. .310 1. ,00 51. ,35
• ATOM 135 CE LYS A 555 8, .106 9, .352 35. .455 1. .00 45, .10
• ATOM 140 CA IL ⁇ A 556 7, .222 5, .699 27. .740 1, .00 26 .45
• ATOM 148 CA THR A 557 9. .571 2, .718 27, .782 1. .00 26, .91
• ATOM 150 OG1 THR A 557 11, .653 3, .811 27, .179 1. .00 33, .53
• ATOM 156 CB ASP A 558 7, .564 2, .101 32, .145 1, .00 42 .16
• ATOM 160 C ASP A 558 6 .386 0 .797 30, .408 1. .00 23 .87
• ATOM 180 CA PHE A 561 3 .081 3. .711 31, .751 1 .00 25 .67
• ATOM 181 CB PHE A 561 3 .551 3 .982 30 .321 1 .00 26 .66
• ATOM 258 CD GLU A 570 2. .473 16. .953 30, .402 1. .00 28, .93
• ATOM 272 CB ALA A 572 1, .235 16, .037 21, .061 1, .00 23 .95
• ATOM 276 CA LEU A 573 5, .281 15, .215 22, .805 1. .00 25 .21
• ATOM 284 CA CYS A 574 5. .432 11, .581 23, .897 1. .00 21 .46
• ATOM 286 SG CYS A 574 4, .663 11, .173 26, .560 1, .00 22 .80
• ATOM 290 CA THR A 575 4, .334 10, .631 20, .384 1. .00 21 .88
• ATOM 306 CB ARG A 577 9 .560 11 .050 22 .609 1 .00 26 .95
• ATOM 308 CD ARG A 577 12 .031 10 .561 22 .731 1 .00 27 .06
• ATOM 316 CA MET A 578 8, .247 7, .552 20, .032 1, .00 21, .44 ATOM 317 CB MET A 578 6.,782 7.,122 20..042 1..00 25,.13
• ATOM 321 C MET A 578 8. ,820 7, ,370 18. ,630 1. .00 22. .27
• ATOM 324 CA PHE A 579 8. ,960 8. .194 16. ,355 1. ,00 22. .58
• ATOM 350 CA LEU A 582 12, .516 4, .231 16, .934 1. .00 25 .74
• ATOM 358 CA ASN A 583 14 .767 6 .026 14, .433 1. .00 32 .87
• ATOM 379 O VAL A 585 13, .059 11, .379 9, .693 1. ,00 29, .85
• ATOM 381 CA GLN A 586 15, .697 11, .132 10, .228 1. .00 38, .86
• ATOM 384 CD GLN A 586 18. .288 12. .833 12. .551 1. ,00 56. ,85
• ATOM 388 O GLN A 586 16. ,005 10. 336 7. ,985 1. ,00 38. ,63
• ATOM 390 CA ASN A 587 15. ,665 7. 652 8, ,685 1. ,00 34. ,34
• ATOM 391 CB ASN A 587 15. ,120 6. ,420 9. ,399 1, ,00 37. ,16
• ATOM 406 C PHE A 588 12, .535 10, .151 6. .304 1, .00 30, .36
• ATOM 409 CA GLN A 589 13, .489 12, .366 6 .750 1. .00 36, .58
• ATOM 412 CD GLN A 589 15, .680 11. .505 3. .839 1. .00 50, .27
• ATOM 423 C MET A 590 10 .515 15, .022 8 .759 1, .00 35 .35
• ATOM 426 CA LYS A 591 10 .016 17, .389 8 .648 1 .00 34 .67
• ATOM 435 CA HIS A 592 9 .332 18, .205 12 .310 1 .00 26 .86
• ATOM 461 CA LEU A 595 5. .062 15. ,187 11. ,918 1. ,00 25. ,41
• ATOM 469 CA CYS A 596 3. .603 17, .588 14, .479 1, .00 24. .03
• ATOM 471 SG CYS A 596 5. .699 18, .771 15, .892 1, .00 26, .30
• ATOM 478 CD ARG A 597 -0. .541 20, .392 9, .032 1, .00 38. .82
• ATOM 482 NH2 ARG A 597 1. .721 23, .278 9, .149 1, .00 38, .90
• ATOM 486 CA TRP A 598 0, .039 14 .802 11 .396 1, .00 20, .68
• ATOM 508 CA LEU A 600 -2 .486 16 .657 15 .802 1 .00 21 .13
• ATOM 529 CA LYS A 603 -5, .697 13, .303 17, .655 1. .00 19, .93
• ATOM 538 CA LYS A 604 -8. .325 15 .629 16 .174 1, .00 21 .57
• ATOM 552 C ASN A 605 -10, .506 11, .662 15, .116 1, .00 21, .57
• ATOM 553 O ASN A 605 -11, .072 10, .702 14, .590 1. .00 23, .07
• ATOM 570 CD ARG A 607 -17. .034 10. .010 16. .715 1. ,00 23. ,37
• ATOM 578 CA LYS A 608 -13. .484 13. .033 22. .349 1. ,00 25. ,95
• ATOM 588 CB ASN A 609 -18. .212 13. .066 23, .071 1, ,00 36, .30
• ATOM 602 CA ALA A 611 -15, .791 7, .078 26, .397 1. .00 19, .90
• ATOM 604 C ALA A 611 -14, .448 6, .363 26, .383 1. ,00 19, .34
• ATOM 607 CA TYR A 612 -13, .060 4, .599 25, .512 1. .00 15, .69
• ATOM 619 CA HIS A 613 -12, .197 4, .553 21, .793 1. .00 15, .66
• ATOM 634 C ASN A 614 -9, .265 8 .207 21 .628 1, .00 18 .30
• ATOM 642 CE3 TRP A 615 -4, ,307 10, .493 19, .542 1. ,00 20, .69
• ATOM 651 CA ARG A 616 -6 .063 8 .424 24 .764 1 .00 18 .73
• ATOM 662 CA HIS A 617 -6. .754 4 .812 23 .796 1, .00 17 .41
• ATOM 672 CA ALA A 618 -4 .897 5 .268 20 .499 1, .00 17 .60
• ATOM 693 C ASN A 620 -1, .450 2 .921 24 .113 1, ,00 18, .91
• ATOM 705 C ALA A 622 2. .151 3. .138 21, .529 1. ,00 17. .61
• ATOM 706 O ALA A 622 3. ,242 2. ,656 21. ,248 1. ,00 16. ,57
• ATOM 708 CA GLN A 623 2. .878 3. .578 23. ,803 1. ,00 19. .17

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