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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P3/04—Anorexiants; Antiobesity agents
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
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  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

• This invention is directed to a crystalline form of human insulin-like growth factor- 1 (IGF-1) and more particularly to a crystal of human IGF-1, a method of crystallization thereof, and its structure, obtained by x-ray diffraction.
• the invention relates to methods of identifying new IGF-1 agonist molecules based on biophysical and biochemical data suggesting that a single detergent molecule that contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions binds to IGF-1 specifically, and blocks binding of IGFBP-1 and IGFBP-3.
• IGF-1 IGF-1, IGF-2, and IGF variants.
• Human IGF-1 is a serum protein of 70 amino acids and 7649 daltons with a pi of 8.4
• IGFs share a high sequence identity with insulin, being about 49% identical thereto. Unlike insulin, however, which is synthesized as a precursor protein containing a 33-amino-acid segment known as the C-peptide (which is excised to yield a covalently linked dimer of the remaining A and B chains), IGFs are single polypeptides (see Figure 1).
• IGF-1 is a powerful mitogen, regulating diverse cellular functions such as cell cycle progression, apoptosis, and cellular differentiation (LeRoith, Endocrinology, 141: 1287-1288 (2000)).
• IGFs have been implicated in a variety of cellular functions and disease processes, including cell cycle progression, proliferation, differentiation, and insulin-like effects in insulin-resistant diabetes. Thus, IGF has been suggested as a therapeutic tool in a variety of diseases and injuries (for review, see Lowe, Scientific American (March/April 1996), p. 62). Due to this range of activities, IGF-1 has been tested in mammals for such widely disparate uses as wound healing, treatment of kidney disorders, treatment of diabetes, reversal of whole-body catabolic states such as AIDS-related wasting, treatment of heart conditions such as congestive heart failure, and treatment of neurological disorders (Guler et al, Proc. Natl. Acad. Sci.
• IGF-1 in vivo is mostly found in complex with a family of at least six serum proteins known as IGFBPs (Jones and Clemmons, supra; Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)), that modulate access of the IGFs to the IGF-IR. They also regulate the concentrations of IGF-1 and IGF-2 in the circulation and at the level of the tissue IGF-IR (Clemmons et al, Anal. NY Acad. Sci. USA, 692:10-21 (1993)).
• the IGFBPs bind IGF-1 and IGF-2 with varying affinities and specificities (Jones and Clemmons, supra; Bach and Rechler, supra).
• IGFBP-1 the growth- stimulating effect of estradiol on the MCF-7 human breast cancer cells is associated with decreased IGFBP- 3 mRNA and protein accumulation, while the anti-estrogen ICI 182780 causes growth inhibition and increased IGFBP-3 mRNA and protein levels (Huynh et al, J Biol. Chem., 271:1016-1021 (1996); Oh et al, Prog. Growth Factor Res., 6:503-512 (1995)).
• IGFBP-3 is a target gene of the tumor suppressor, p53 (Buckbinder et al, Nature, 377:646-649 (1995)). This suggests that the suppressor activity of p53 is, in part, mediated by IGFBP-3 production and the consequential blockade of IGF action (Buckbinder et al, supra). These results indicate that the IGFBPs can block cell proliferation by modulating paracrine/autocrine processes regulated by IGF- l/IGF-2.
• IGFs have mitogenic and anti-apoptotic influences on normal and transformed prostate epithelial cells (Hsing et al, Cancer Research, 56: 5146 (1996); Culig et al, Cancer Research, 54: 5474 (1994); Cohen et al., Hormone and Metabolic Research, 26: 81 (1994); Iwamura et al, Prostate, 22: 243 (1993); Cohen et al, J. Clin. Endocrin. & Metabol., 73: 401 (1991); Rajah et al, J. Biol. Chem., 272: 12181 (1997)).
• the IGFs are present in high concentrations in the circulation, but only a small fraction of the IGFs is not protein bound. For example, it is generally known that in humans or rodents, less than 1% of the IGFs in blood is in a "free" or unbound form (Juul et al, Clin. Endocrinol., 44: 515-523 (1996); Hizuka et al, Growth Regulation, 1: 51-55 (1991); Hasegawa et al, J. Clin. Endocrinol. Metab., 80: 3284-3286 (1995)).
• Cascieri et al, Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-1, three of which have reduced affinity to IGF-IR. These mutants are: (Phe 23 ,Phe 24 ,Tyr 25 )IGF-l (which is equipotent to human IGF-1 in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu 24 )IGF-l and (Ser 24 )IGF-l (which have a lower affinity than IGF-1 to the human placental IGF-IR, the placental insulin receptor, and the IGF-IR of rat and mouse cells), and desoctapeptide (Leu 24 )IGF-l (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D-region of hIGF-1, which has lower affinity than (Leu 24 )IGF-l for the IGF-IR and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum
• the invention includes a method of designing a compound, such as a peptidomimetic, that mimics the 3-dimensional surface structure of IGF-1 comprising the steps of: (a) dete ⁇ nining the 3-dimensional structure of the IGF-1; and (b) designing a compound that mimics the 3-dimensional surface structure of the IGF-1.
• Figure 9 A shows a non-linear least-squares analysis of sedimentation equilibrium data for IGF-1 in solution. Data collected at rotor speeds of 30,000 rpm (open triangles) and 35,000 rpm (open squares) were fit as an ideal monomer-dimer self-association model. The solid lines are the fits of the data.
• Figure 9B shows the residuals plotted for both rotor speeds after accounting for the data by the fitting procedure. They are randomly distributed around zero, indicating that the monomer-dimer model is correct for this interaction.
• IGFBP-1 and IGFBP-3 bind to different residues of IGF-1.
• agonist disorders for purposes herein include any condition that would benefit from treatment with an IGF-1, including but not limited to, for example, lung diseases, hyperglycemic disorders as set forth below, renal disorders, such as acute and chronic renal insufficiency, end-stage chronic renal failure, glomerulonephritis, interstitial nephritis, pyelonephritis, glomerulosclerosis, e.g., Kimmelstiel- Wilson in diabetic patients and kidney failure after kidney transplantation, obesity, GH-insufficiency, Turner's syndrome, Laron's syndrome, short stature, undesirable symptoms associated with aging such as obesity and increased fat mass-to-lean ratios, immunological disorders such as immunodeficiencies including decreased CD4 counts and decreased immune tolerance or chemotherapy-induced tissue damage, bone marrow transplantation, diseases or insufficiencies of cardiac structure or function such as heart dysfunctions and congestive heart failure, neuronal, neurological, or neuromuscular disorders, e.g., peripheral
• space group refers to the arrangement of symmetry elements of a crystal
• molecular replacement refers to a method that involves generating a preliminary structural model of a crystal whose structural coordinates are unknown, by orienting and positioning a molecule whose structural coordinates are known, e.g., the IGF-1 coordinates in Appendix 1, within the unit cell of the unknown crystal, so as to best account for the observed diffraction pattern of the unknown crystal Phases can then be calculated from this model, and combined with the observed amplitudes to give an approximated Fourier synthesis of the structure whose coordinates are unknown. This in turn can be subject to any of the several forms of refinement to provide a final accurate structure of the unknown crystal.
• the ribbon structure thereof is shown in Figure 2 having three helices, with the N-terminal B-region corresponding to residues 3-28, the C-region from residues 29-34, a stretch of poorly ordered residues from residues 35-40, and the A-region from residues 42-62.
• the D-region (residues 63-70) is essentially disordered.
• Figures 4 and 7 show that the detergent used in the crystallization binds into a small hydrophobic cleft at the base of the B-helix of the structure.
• the IGF-1 can form a dimer in the crystal, as shown in Fig. 5, wherein the two tails are positioned at the dimer interface.
• the buried surface area is 689 A 2 /monomer, which is 1378 A 2 total
• the residues important for IGF-IR binding cluster at the dimer interface as shown in Figure 6.
• the reservoir solution further comprises a detergent.
• the detergent is present in an amount of about 10 to 50 mM.
• the detergent is N, N-bis(3-D-gluconamidopropyl)-deoxycholamine.
• the pH of the reservoir solution may also be varied, preferably between about 4 to 10, most preferably about 6.5.
• Various methods of crystallization can be used in the claimed invention, including vapor diffusion, batch, liquid-bridge, or dialysis crystallization. Vapor diffusion crystallization is preferred. See, e.g.
• a small volume i.e., a few milliliters
• a solution containing a precipitant i.e., a small amount, i.e. about 1 ml, of precipitant. Vapor diffusion from the drop to the well will result in crystal formation in the drop.
• the batch methods generally involve the slow addition of a precipitant to an aqueous solution of protein until the solution just becomes turbid; at this point the container can be sealed and left undisturbed for a period of time until crystallization occurs.
• a precipitant to an aqueous solution of protein until the solution just becomes turbid; at this point the container can be sealed and left undisturbed for a period of time until crystallization occurs.
• the claimed invention encompass any and all methods of crystallization.
• One skilled in the art can choose any of such methods and vary the parameters such that the chosen method results in the desired crystals.
• the most preferred method of crystallization involves the method wherein the IGF-1, after isolation from the cell and formulation in, for example, an acetate, citrate, or succinate buffer, as described, for example, in U.S. Pat. No. 5,681,814 and WO 99/51272, is optionally desalted if necessary to apH of about 4-5, preferably about 4.5, to fo ⁇ n an aqueous solution.
• a droplet of the aqueous solution is mixed with about 24% polyethylene glycol buffered to about pH 6.5 with either about 0.1M sodium citrate or about 0.1M sodium cacodylate and with about 1 ⁇ l of about 1.4 mM N, N-bis(3-D-gluconamidopropyl)- deoxycholamine as detergent.
• This solution is then equilibrated by vapor diffusion crystallization with about 1 mL of about 24%) polyethylene glycol buffered to about pH 6.5 with either about 0.1M sodium citrate or about 0.1M sodium cacodylate until crystallization droplets are formed, usually about 4-5 days.
• the crystal structure was determined by combined anomalous scattering from intrinsic sulfur and fortuitous bromide ion as discussed in detail in the Example ' below. c. Methods of using an IGF-1 crystal and its coordinates
• the crystalline IGF-1 herein can be used for various purposes.
• the crystallization process itself further purifies the IGF-1 to homogeneity.
• one such purpose is to provide a highly purified IGF-1 that can be used as a standard or control in a diagnostic setting, for example, as a molecular weight marker, or as an ELISA, radioassay, or radioreceptor assay control.
• crystalline IGF-1 is stable at room temperature, can be lyophilized readily, and is less apt to degrade than less pure compositions.
• crystals of IGF-1 of a size and quality to allow performance of x-ray diffraction studies enable those of skill in the art to conduct studies relating to the binding properties of IGF-1, as well as the binding properties of IGFBPs, IGF-1 receptors, and ALS that associate with the IGF-1.
• structural information derived from a peptide crystal structure can be used for the identification of chemical entities, for example, small organic and bioorganic molecules such as peptidomimetics and synthetic organic molecules that bind IGF-1 and preferably block or prevent an IGF-1- mediated or -associated process or event, or that act as IGF-1 agonists.
• small organic and bioorganic molecules such as peptidomimetics and synthetic organic molecules that bind IGF-1 and preferably block or prevent an IGF-1- mediated or -associated process or event, or that act as IGF-1 agonists.
• One approach enabled by this invention is the use of the structural coordinates of IGF-1 to design chemical entities that bind to or associate with IGF-1 and alter the physical properties of the chemical entities in different ways.
• properties such as, for example, solubility, affinity, specificity, potency, on/off rates, or other binding characteristics may all be altered and/or maximized.
• An unknown crystal structure which may be any unknown structure, such as, for example, another crystal form of IGF-1, an IGF-1 mutant or peptide, or a co-complex with IGF-1, or any other unknown crystal of a chemical entity that associates with IGF- 1 that is of interest, may be determined using the structural coordinates as set forth in Appendix 1.
• Co-complexes with IGF-1 may include, but are not limited to, IGF-1 -IGFBP-3, IGF-1- IGFBP-3-ALS, IGF-1 -receptor, IGF-1- ⁇ eptide, or IGF-1-small molecule. This method will provide an accurate structural form for the unknown crystal far more quickly and efficiently than attempting to determine such information without the invention herein. The information obtained can thus be used to obtain maximally effective inhibitors or agonists of
• IGF-1 insulin growth factor-1. Although not all portions of the chemical entity will necessarily participate in the association with IGF-1, those non-participating portions may still influence the overall conformation of the molecule. This in turn may have a significant impact on the desirability of the chemical entity. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the binding site.
• the potential inhibitory or binding effect of a chemical entity on IGF-1 may be analyzed prior to its actual synthesis and testing by the use of computer-modeling techniques. If the theoretical structure of the given chemical entity suggests insufficient interaction and association between it and IGF-1, the need for synthesis and testing of the chemical entity is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to IGF-1. Thus, expensive and time-consuming synthesis of inoperative compounds may be avoided.
• Specialized computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, for example, GRID, available from Oxford University, Oxford, UK; MCSS or CATALYST, available from Molecular Simulations, Burlington, MA; AUTODOCK, available from Scripps Research Institute, La Jolla, CA; DOCK, available from University of California, San Francisco, CA, and XSITE, available from University College of London, UK.
• GRID available from Oxford University, Oxford, UK
• MCSS or CATALYST available from Molecular Simulations, Burlington, MA
• AUTODOCK available from Scripps Research Institute, La Jolla, CA
• DOCK available from University of California, San Francisco, CA
• XSITE available from University College of London, UK.
• Assembly may be by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen, in relation to the structural coordinates disclosed herein.
• any molecular modeling techniques may be employed in accordance with the invention; these techniques are known, or readily available to those skilled in the art. It will be understood that the methods and compositions disclosed herein can be used to identify, design, or characterize not only entities that will associate or bind to IGF-1, but alternatively to identify, design, or characterize entities that, like
• IGF-1 will bind to the receptor, thereby disrupting the IGF-1 -receptor interaction.
• the claimed invention is intended to encompass these methods and compositions broadly.
• the efficiency with which that compound may bind to IGF-1 may be tested and modified for the maximum desired characteristic(s) using computational or experimental evaluation.
• Various parameters can be maximized depending on the desired result. These include, but are not limited to, specificity, affinity, on/off rates, hydrophobicity, solubility, and other characteristics readily identifiable by the skilled artisan.
• the invention is useful for the production of small-molecule drug candidates.
• the claimed crystal structures may be also used to obtain information about the crystal structures of complexes of the IGF-1 and small-molecule inhibitors. For example, if the small-molecule inhibitor is co- crystallized with IGF-1, then the crystal structure of the complex can be solved by molecular replacement using the known coordinates of IGF-1- for the calculation of phases. Such information is useful, for example, for determining the nature of the interaction between the IGF-1 and the small-molecule inhibitor, and thus may suggest modifications that would improve binding characteristics such as affinity, specificity, and kinetics. d. Other methods
• the invention herein is also useful in providing a method of identifying indirect agonists of IGF-1 based on the inhibitory properties of N, N-bis(3-D-gluconamido ⁇ ropyl)-deoxycholamine with respect to IGFBPs.
• This method comprises the steps of: comparing the ability of N, N-bis(3-D-gluconamidopro ⁇ yl)- deoxycholamine to inhibit binding of IGFBP-1 or -3 to IGF-1 with the ability of a candidate IGF-1 indirect agonist to inhibit such binding; and determining whether the candidate IGF-1 indirect agonist can inhibit such binding at least as well as N, N-bis(3-D-gluconamidopropyl)-deoxycholamine can so inhibit the binding.
• the comparison is accomplished by competition assay between N, N-bis(3-D- gluconamidopropyl)-deoxycholamine and the candidate IGF-1 indirect agonist, using IC J0 to measure ability to inhibit IGFBP binding.
• inhibition of binding is measured by pre-incubating N, N-bis(3-D-gluconamidopropyl)-deoxycholamine or the candidate agonist molecule with IGF-1 expressed on bacteriophage particles and measuring residual binding of IGF-1 to IGFBP-1 or IGFBP- 3 in a plate-based assay, such as an ELISA.
• the invention further provides a method of identifying indirect agonists of IGF-1 comprising co- crystallizing the candidate agonist with IGF-1 to form a co-crystalline structure and determining if the candidate agonist molecule binds to one or both of two patches on IGF-1.
• the first patch contains the amino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13, Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57
• the second patch contains the amino acid residues Val 11, Ghi 15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55.
• bmding means that there is at least one contact between each listed amino acid residue of a given patch and the candidate agonist molecule that is less than or equal to 6 angstroms in the co-crystalline structure.
• a candidate agonist molecule will have the property of inhibiting binding of IGFBP-1 or IGFBP-3 to IGF-1.
• the preferred such candidate agonist molecule will inhibit binding of IGFBP-1 or -3 to IGF-1 at least as well as N, N-bis(3-D- gluconamidopropyl)-deoxycholamine. More preferred is the method wherein inhibition of binding is measured using a competition assay between N, N-bis(3-D-gluconamidopro ⁇ yl)-deoxycholamine and the candidate agonist molecule.
• N, N-bis(3-D-gluconamidopropyl)-deoxycholamine detergent herein can be used as a template to perform design of small-molecule drugs that elicit the same effect as the detergent (compete with IGF-1 for IGFBP binding and subsequent disruption of the interaction of IGFBP with IGF-1 to free IGF-1 in situ so that it is active and will interact with the receptor.
• N, N-bis(3-D-gluconamidopropyl)-deoxycholamine lacks an oxygen atom at position CIO. This region of the detergent is in close contact with the side-chain atoms of residues Leu 5, Leu 54, and Leu 57 of IGF-1. Molecules with this same type of conformation would work as indirect IGF-1 agonists.
• the indirect agonist so identified can be used in a method for treating an agonist disorder wherein an effective amount of the indirect agonist of IGF-1 is administered to a mammal with such a disorder.
• the formulation of the indirect agonist herein can be used to treat any condition that would benefit from treatment with IGF-1, including, for example, diabetes, chronic and acute renal disorders, such as chronic renal insufficiency, necrosis, etc., obesity, hyperinsulinemia, GH-insufficiency, Turner's syndrome, short stature, undesirable symptoms associated with aging such as increasing lean-mass-to-fat ratios, immuno-deficiencies including increasing CD4 counts and increasing immune tolerance, catabolic states associated with wasting, etc., Laron dwarfism, insulin resistance, and so forth.
• the indirect agonist composition herein may be directly administered to the mammal by any suitable technique, including orally, parenterally, intranasally, or intrapulmonarily, and can be administered locally or systemically.
• suitable technique including orally, parenterally, intranasally, or intrapulmonarily, and can be administered locally or systemically.
• the specific route of administration will depend, e.g., on the medical history of the patient, including any perceived or anticipated side or reduced effects using IGF-1, and the disorder to be treated.
• parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration. Most preferably, the administration is by continuous infusion (using, e.g., minipumps such as osmotic pumps), or by injection (using, e.g., intravenous or subcutaneous means).
• the administration may also be as a single bolus or by slow-release depot formulation.
• the direct agonist is administered orally or by infusion or injection, at a frequency of, preferably, one-half, once, twice, or three times daily, most preferably daily.
• the agonist composition to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient
• the site of delivery of the agonist composition is thus determined by such considerations and must be an amount that treats the disorder in question.
• the total pharmaceutically effective amount of agonist administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day up to about 100 mg/kg/day, preferably 10 ⁇ g/kg/day up to about 10 mg/kg/day.
• the agonist is generally administered in doses of about 1 ⁇ g/kg/hour up to about 100 ⁇ g/kg/hour, either by about 1-4 injections per day or by continuous subcutaneous infusions, for example, using a minipump or a portable infusion pump. An intravenous bag solution may also be employed.
• the key factor in selecting an appropriate dose is the result obtained as measured by criteria as are deemed appropriate by the practitioner. If the agonist is administered together with insulin, the latter is used in lower amounts than if used alone, down to amounts which by themselves have little effect on blood glucose, i.e., in amounts of between about 0.1 IU/kg/24 hour to about 0.5 IU/kg/24 hour.
• the agonist is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• a pharmaceutically acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
• the formulation is prepared by contacting the agonist uniformly and intimately with a liquid carrier or a finely divided solid carrier or both.
• the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
• carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
• Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
• the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
• Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low-molecular-weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; nonionic surfactants such as polysorbates, poloxamers, or PEG; and/or neutral salts, e.g.,
• the agonist is typically formulated individually in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at apH of about 4.5 to 8.
• the final formulation if a liquid, is preferably stored at a temperature of about 2-8 °C for up to about four weeks.
• the formulation can be lyophilized and provided as a powder for reconstitution with water for injection that is stored as described for the liquid formulation.
• the agonist to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
• Therapeutic agonist compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• the agonist ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
• Recombinant human IGF-1 (rhIGF-1) was obtained as described in the Examples of U.S. Pat. No. 5,723,310 using a polymer/salt combination for phase-forming species and formulated as described in the Examples of U.S. Pat. No. 5,681,814 (acetate, NaCl, polysorbate 20, and benzyl alcohol), and placed in a vial containing 7 ml of 10 mg/ml rhIGF-1. It was desalted into 0.15 M NaCl, 20 mM NaOAc, pH 4.5, and diluted to a final concentration of 10 mg/ml.
• a 4- ⁇ l droplet of the IGF-1 solution was mixed with 5 ⁇ l of reservoir solution (24% polyethylene glycol 3350 buffered to pH 6.5 with 0.1M sodium cacodylate) and 1 ⁇ l of 14 mM of N, N-bis(3-D-gluconamidopropyl)-deoxycholamine, which is obtained in a CRYSTAL
• SCREENTM reagent kit used for crystallization condition screenings and available from Hampton Research, Madison Nigel, CA. This solution was allowed to equilibrate via vapor diffusion (Jancarik et al, supra) with 1 mL of reservoir solution. Thus, a drop of the mixture was suspended under a plastic cover slip over the reservoir solution. Small crystals with a thin, plate-like morphology appeared within 4-5 days. At this point, 2 ⁇ l of 100% methyl pentanediol (MPD) (to a final concentration of 20%) was added to the crystallization droplet, and the crystals dissolved overnight. Within 1 week, crystals reappeared and grew to final dimensions of 0.2 mm X 0.1mm X 0.05 mm with noticeably sharper edges. These crystals were used for all subsequent analysis.
• MPD methyl pentanediol
• crystallization conditions can be varied. By varying the crystallization conditions, other crystal forms of IGF-1 may be obtained. Such variations may be used alone or in combination, and include, for example, varying final protein concentrations between about 5 and 35 mg/ml; varying the IGF-1 -to-precipitant ratio, varying precipitant concentrations between about 20 and 30% for polyethylene glycol, varying pH ranges between about 5.5 and 7.5, varying the concentration or type of detergent, varying the temperature between about -5 and 30°c, and crystallizing IGF-1 by batch, liquid bridge, or dialysis methods using the above conditions or variations thereof. See McPherson et al. (1982), supra. Characterization of IGF-1 crystals
• a single crystal was transferred from the mother liquor to a cryo-protectant solution consisting of 25% (w/v) polyethylene glycol 3350, 30% MPD, 0.2 M sodium cacodylate pH 6.5, 2.8 mM of N, N-bis(3- D-gluconamido ⁇ ropyl)-deoxycholamine, and 1 M NaBr.
• the diffraction was to 1.8 A.
• the crystals were flash-cooled by plunging the solution into liquid nitrogen. The technique of freezing the crystals essentially immortalizes them and produces a much higher quality data set. All subsequent manipulations and x-ray data collection were performed at 100° Kelvin.
• a 4-wavelength MAD data set was collected at beamline 9-2 at the Stanford Synchrotron Radiation Laboratory, with the order of the data sets as follows: Br peak ( ⁇ l), low-energy remote ( ⁇ 2), Br inflection ( ⁇ 3), and high-energy remote ( ⁇ 4).
• the Br peak and inflection points were estimated from fluorescence scans of the crystal, and the low-energy remote was chosen to be 1.54 angstroms, to maximize the small sulfur anomalous signal at this wavelength while minimizing absorption effects. No inverse beam geometry was used.
• Data reduction was performed using Denzo and Scalepack (Otwinowski and Minor, Methods in Enzymology, 276: 307-326 (1997)). To determine the most accurate scale and 5-factors possible, data for all four wavelengths were initially scaled together, assuming no anomalous signal. The scale and -3-factors determined from this scaling run were then applied to each of the four data sets.
• the asymmetric unit of the crystals contained a monomer of IGF-1 bound to a single detergent molecule, yielding a Matthew's coefficient of 2.4 A 3 /Da, or 48.1% solvent.
• the solvent content of the crystals was about 55%.
• the coordinates of the single-bound bromide were determined by manual inspection of the anomalous and dispersive difference Patterson maps.
• the hand ambiguity was resolved by phase refinement using the program SHARP (De La Fortelle and Bricogne, Methods in Enzymology, 276: 472- 494 (1997)) from Global Phasing Limited, 43 Newton Road, Cambridge CB2 2AL, ENGLAND, followed by examination of anomalous-difference Fourier maps calculated using the ⁇ 2 Bijvoet differences.
• SHARP De La Fortelle and Bricogne, Methods in Enzymology, 276: 472- 494 (1997)
• Density modification (solvent flattening and histogram mapping) was performed using DM (Collaborative Computational Project Number 4, Acta Crystallogr., D50: 760-763 (1994); Cowtan, Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31: 34-38 (1994)), and the resulting electron-density maps were of high quality. Approximately 50% of the structure, corresponding to the three helical regions of IGF-1, was built directly into the experimental electron-density maps using the programs O (Jones et al, Acta Crystallogr., A47: 110-119 (1991)) and QUANTA (version 97.0, MSI, San Diego, CA).
• the final model contains residues 3-34 and 41-64 of IGF-1, one N, N-bis(3-D- gluconamidopro ⁇ yl)-deoxycholamine molecule, one Br, and 50 water molecules.
• the model was refined against the ⁇ 3 data set, since the data statistics demonstrated this data set to be of higher quality than the others. All data from 20- to 1.8-angstrom resolution were included in the refinement, with no application of a sigma cutoff. Secondary structure assignments were made with the program PROMOTIF (Hutchinson and Thornton, Protein Science, 5: 212-220 (1996)).
• the R factor to 1.8 A is 23.7%, and the free R factor is 26.9%, with good stereochemistry.
• the N-terminal B-region corresponds to residues 3-28, the C-region from 29-34, a stretch of poorly ordered residues from 35-40, and the A-region from 42-62.
• the D-region (63-70) is essentially disordered.
• the structure of IGF-1 is similar to insulin (see Figure 3), with a Root-Mean-Squared-Deviation (RMSD) of 3A over backbone atoms that are conserved between the two molecules. Most of these deviations occur in the flexible regions, and when only the helical regions are considered, the RMSD between alpha-carbon atoms is about 0.47A.
• the major difference is the extension of the C-region, for which there is no counterpart in mature insulin, away from the body of the molecule. This loop contains many of the residues that are known to be important for receptor binding. ,
• the detergent molecule binds into a small hydrophobic cleft at the base of the B-helix.
• the preliminary results suggest, without being limited to any one theory, that the detergent does not inhibit binding of these proteins to IGF-1.
• the opposite face of the detergent is making a symmetry contact to the opposite face of IGF-1.
• IGF-1 molecules which results in a symmetric homodimer.
• the buried surface area is 1378 A 2 , which is in the range of physiologically relevant protein-protein interfaces.
• Figure 6 shows that the residues known to be important for receptor binding cluster at this dimer interface. Shown are Tyr24, Thr29, Tyr31, and Tyr60. Mutation of these residues results in anywhere from 6-20X loss in affinity for receptor for individual mutations, or 240->1200X loss in affinity for double mutations. Also shown are Phe23 and Phe25, which are interchangeable with Phe24 and Tyr26 of insulin, with no loss of affinity.
• IGF-1 is composed primarily of three helical segments corresponding to the B-helix (IGF-1 residues 7-18) and two A-helices (IGF-1 residues 43-47 and 54-58) of insulin.
• the hydrophobic core is essentially identical to that described for the NMR structures of IGF-1, including the three disulfide linkages between Cys 6 and Cys 48, Cys 18 and Cys 61, and Cys 47 and Cys 52, as noted in the references above.
• Residues 3 through 6 do not form any regular secondary structure and hence the structure described herein can be classified as being most similar to the T-form of insulin (Derewenda et al, Nature, 338: 594-596 (1989)); indeed, when IGF-1 and the T-form of insulin are superimposed on the C ⁇ positions of their respective helical segments (IGF-1 residues 8-19, 42-49, and 54-61; insulin residues B9-B20, A1-A8, and A13-A20) the RMSD is only 0.93 angstroms. As in insulin, residues 18-21 at the end of the B-helix form a type IF ⁇ -turn, which redirects the backbone from the B-helix into an extended region.
• Residues 24-27 form a type VIII ⁇ -turn in to accommodate the C-region, which extends away from the core of IGF-1, and interacts with a symmetry-related molecule.
• Residues 30-33 form a well-defined type II beta-turn, prominently displaying Tyr 31 at the i+1 position.
• Residues 35-40 have not been modeled, as the electron density in this region is weak and disconnected. Only the first two residues of the D-region (residues 63 and 64) are ordered in the structure.
• the C-region of IGF-1 mediates a two-fold symmetric crystal-packing interaction across the ⁇ -axis of the unit cell.
• This interaction buries 689 A 2 of solvent-accessible surface area from each molecule of IGF-1, or 1378 A 2 total, and is the largest interface in the crystal.
• a total of 28 intermolecular contacts of distance 3.6 A or less are formed via this interface, with the next most extensive crystal packing interaction forming only nine contacts.
• the core of the interface is dominated by Tyr24 and Pro28 from each monomer, which bury 39 A 2 and 57 A 2 of solvent-accessible surface area, respectively.
• Tyr 31 which lies at the tip of the loop at the furthest point from the core of IGF-1, packs against the phenolic rings of Phe 23 and Phe 25 of the symmetry-related molecule.
• two main-chain hydrogen bonds (Tyr 31 N-Phe 23 O; Ser 34 N-Asp 20 O) are present in the dimer interface.
• Residues from the D-region (62-64) are also partially sequestered by dimer formation. Because of these interactions, most of the C-region in the crystal is well-ordered, providing the first high- resolution view of the conformation of this biologically important loop.
• N, N-bis(3-D- gluconamido ⁇ ropyl)-deoxycholamine interacts with residues, forming a small hydrophobic cleft on one surface of IGF-1 (Leu 5, Phe 16, Val 17, Leu 54, and Leu 57) (Fig. 7A).
• the preference for N, N-bis(3-D- gluconamidopropyl)-deoxycholamine is explained, without being limited to any one theory, by the absence of an oxygen atom at position CIO in the detergent molecule. This region of the detergent is in close contact with the side chain atoms of residues Leu 5, Leu 54, and Leu 57 in IGF-1.
• the opposite face of the detergent mediates a symmetry contact with residues Val 11 , Leu 14, and Gin 15 of a symmetry-related
• One patch has the amino acid residues Glu 3, Tlir 4, Leu 5, Asp 12, Ala 13, Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57, and the second patch has the amino acid residues Val 11, Gin 15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55. Binding is defined by having at least one contact between each listed amino acid residue and the candidate agonist molecule that is less than or equal to 6 angstroms.
• the C-region in the IGF-1 crystal structure extends out from the core of the molecule, with residues 30-33 forming a canonical type II beta-turn, and the remainder of the C-region forming a crystallographic dimer with a symmetry-related molecule.
• Tyr 31 has been implicated as being a critical determinant for IGF-IR binding, and its location at the tip of this extension places it in an ideal location to interact with a receptor molecule.
• this region of IGF-1 is not well-defined by NMR data, the conformation of the C-region in the crystal is likely to reflect the solution conformation. There is evidence of a reverse turn at the tip of the loop and a hinge bending at the loop termini of IGF-2 (Torres et al, supra).
• crystal packing forces undoubtedly help stabilize the orientation of this loop, its conformation is consistent with the solution structure of the closely related IGF-2.
• the size of the interface formed by the crystallographic dimer is well within the range of buried surface area in known biological complexes (Janin and Chothia, J. Biol Chem., 264: 16027-16030 (1990)). In addition, this interaction partially excludes from solvent several of the residues known to be important for binding to the IGF-IR, including Phe 23 (69% buried), Tyr 24 (64%), Phe 25 (29%), and Tyr 31 (38%). Other groups have also reported homodimeric interactions of IGF-1 and IGF-2.
• the known binding stoichiometry of one IGF-1 molecule per receptor dimer makes it difficult to rationalize the biological significance of IGF-1 dimerization.
• the IGF-1 dimer in this crystal form results from the high concentration of IGF-1 in the crystallization experiment, and does not represent a physiologically relevant form of the molecule.
• the crystal structure of IGF-1 has been determined using anomalous scattering from the intrinsic sulfur atoms and a Br- ion bound at a fortuitous halide-binding site.
• the structure is very similar to insulin, with the only major difference being the C-region, which protrudes from the body of the protein and mediates a homodimeric interaction.
• the amount of buried surface area is consistent with the feet that at neutral pH, IGF-1 undergoes self-association in a concentration-dependent manner.
• several residues that are important for receptor binding are found at this dimer interface, suggesting, without being limited to any one theory, that effects on receptor binding by mutation of these residues may be a result of disruption of the dimer, rather than direct contact with the receptor surface.
• NMR-derived diffusion measurements were used to estimate the K . for the interaction between IGF-1 and N, N-bis(3-D-gluconamidopropyl)-deoxycholamine.
• Samples were prepared in 50 mM phosphate buffer in D 2 0, pH 6.5 (uncorrected meter reading), and contained: 1.0 mM N, N-bis(3-D- gluconamidopropyl)-deoxycholamine + 0.5 mM IGF-1; 0.5 mM N, N-bis(3-D-gluconamidopropyl)- deoxycholamine + 0.25 mM IGF-1; 0.25 mM N, N-bis(3-D-gluconamido ⁇ ropyl)-deoxycholamine + 0.125 mM IGF-1; or N, N-bis(3-D-gluconamidopro ⁇ yl)-deoxycholamine only (1.0, 0.5, or 0.25 mM).
• Spectra were collected with 128 to 1024 transients as the z-gradient strength was increased from 0.009 to 0.45 T» ⁇ n l in 18 equal increments; measurements were made at least twice on each sample. Spectra were processed and peak heights extracted with the program FELLX (v98.0, MSI, San Diego). Diffusion constants, proportion of bound detergent, and resulting K. were extracted as described by Fejzo et al, Chemistry & Biology, 6: 755-769 (1999).
• E. coli cells (XLl-Blue, Stratagene) freshly transformed with the phage vector pIGF-g3 displaying human IGF-1 as described in Dubaquie and Lowman, supra, were grown overnight in 5 ml of 2YT medium (Sambrook et al, Molecular Cloning: A Laboratory Handbook (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)).
• the phage particles displaying IGF-1 were titered against IGFBP-1 and
• IGFBP-3 to obtain a 500- 1000-fold dilution for preincubation with serial dilutions of the detergents and binding protein standards for 35 minutes.
• Microwell clear polystyrene immunoplates with a MAXISORPTM surface (Nunc, Denmark) were coated with IGFBP-1 or IGFBP-3 protein overnight at 4°C (50 ⁇ l at 3 ⁇ g/mL in 50 mM carbonate buffer, pH 9.6), blocked with 0.5% TWEEN® 20 polyoxyethylene sorbitan monolaurate (Atlas Chemical Co.), and PBS and washed eight times with PBS, 0.05% TWEEN® 20 polyoxyethylene sorbitan monolaurate. The samples were added to the plates for 30 minutes. Plates were washed eight times with PBS, 0.05% TWEEN® 20 polyoxyethylene sorbitan monolaurate, incubated with
• the self-association of IGF-1 was determined by sedimentation equilibrium analysis.
• the experiments were conducted at 20°C in an OPTIMATM XL-A XL-I analytical ultracentrifuge (Beckman Coulter, Inc.).
• the samples were prepared in 0.1 M citrate buffer, pH 6.5, 75 mM NaCl with a loading concentration from 1 mM to 0.01 mM.
• the concentration gradients were measured at rotor speeds of 25000 and 30000 rpm at 280 nm or 285 nm using a scanning absorption optical system.
• the attainment of an equilibrium state was verified by comparing successive scans after approximately 16 hours.
• the partial specific volume of IGF-1 was calculated from its amino acid composition.
• K. values of 220, 440, and 430 ⁇ M were obtained, respectively.
• This technique has routinely been applied to small molecules (several hundred Daltons molecular weight or less) binding to large proteins.
• the ligand is relatively large (862 Da) and the protein is relatively small (7648 Da); hence, the differential decrease in diffusion constant on binding is small This increases the uncertainty with which the dissociation constant can be measured.
• the data described above suggest that the K. for the interaction between N, N- bis(3-D-gluconamidopro ⁇ yl)-deoxycholamine and IGF-1 is 300+150 ⁇ M.
• a similar analysis of the (3-(3- cholamidopropyl) dimethylammonio)-l -propane sulphonate diffusion data suggests that that K. in this case is greater than 3 mM.
• N, N-bis(3-D-gluconamidopropyl)-deoxycholamine blocks IGFBP-1 and IGFBP-3 binding.
• the detergent was preincubated with IGF-1 expressed on bacteriophage particles, and the level of residual binding to IGFBP-1 and IGFBP-3 was measured in a plate-based assay (ELISA).
• ELISA plate-based assay
• N N-bis(3-D-gluconamidopropyl)-deoxycholamine completely inhibited IGF-1 on phage from binding to IGFBP-1 and IGFBP-3 with IC 50 values of 740+260 ⁇ M and 231+29 ⁇ M, respectively. These numbers must be interpreted conservatively, however, since the critical micelle concentration of N, N-bis(3-D- gluconamidopropyl)-deoxycholamine (1.4 mM) presents an upper limit on the curve in Figure 8.
• the sedimentation equilibrium data show that IGF-1 undergoes self-association in solution.
• the average molecular weight increased with increasing protein concentration from 0.01 mM to 1 mM.
• the average molecular weight at the highest concentration studied (1 mM) is about 37% higher than the monomer molecular weight (10.4 KDa at 1 mM versus 7.6 KDa monomer molecular weight).
• concentrations below 0.05 mM no self-association was observed, and IGF-1 exists only as a monomer in solution at neutral pH. If it is assumed that the higher-molecular-weight species are IGF-1 dimers, the sedimentation data can be fit as a monomer-dimer model with a K d of 3.6+ 1.0 mM ( Figure 9) .
• Patch I consists of Glu 7, Leu 10, Val 11, Leu 14, Phe 25, lie 43, and Val 44, while patch 2 consists of Glu 3, Thr 4, Leu 5, Phe 16, Val 17, and Leu 54. In the crystal structure of IGF-1, these two patches are involved in detergent-mediated crystal packing contacts.
• Patch 1 of the crystal structure of IGF-1 consists of amino acid residues Glu 3, Thr 4, Leu 5, Asp 12, Ala 13, Phe 16, Val 17, Cys 47, Ser 51, Cys 52, Asp 53, Leu 54, and Leu 57
• Patch 2 of the crystal structure of IGF-1 consists of amino acid residues Val 11, Gin 15, Phe 23, Phe 25, Asn 26, Val 44, Phe 49, and Arg 55, wherein binding occurs if there is at least one contact between each listed amino acid residue and the candidate agonist molecule that is less than or equal to 6 angstroms.
• N, N-bis(3-D-gluconamidopro ⁇ yl)-deoxycholamine does not inhibit IGF-1R- mediated signaling in a cell-based receptor activation assay.
• N, N-bis(3- D-gluconamidopropyl)-deoxycholamine as an inhibitor of IGFBP interactions allows the ability to develop small-molecule drugs or peptidomimetics that disrupt the IGF-1/IGFBP complex in vivo, thereby releasing receptor-active IGF-1 from the systemic, inactive pool.
• drugs include orally bioavailable therapy for metabolic disease such as diabetes.
• Zeslawski et al. EMBO J., 20: 3638-3644 (2001) published the crystal structure of IGF- 1 in complex with the N-terminal domain of IGFBP-5.
• IGF-F 1-1 The complex between peptide IGF-F 1-1 and IGF- 1 was determined from NMR spectroscopy data collected at 600 and 800 MHz.
• IGF-1 uniformly labeled with 13 C and I5 N was prepared using the scheme outlined by Reilly and Fairbrother, J. Biomol NMR, 4: 459-462 (1994) and purified according to the protocol in Vajdos et al, supra.
• a slight molar excess of unlabeled IGF-Fl-1 was mixed with a 1.5 mM solution of 13 C/ 15 N IGF-1 and 'H, 13 C, and 15 N N NMR resonances assigned from double- and triple-resonance NMR experiments as described by Cavanagh et al.
• Intermolecular restraints between IGF-1 and the peptide were obtained from an ⁇ l -filtered, ⁇ 2- edited 13 C HSQC-NOESY spectrum (Lee et al, FEBS Lett., 350: 87-90 (1994)).
• Intrapeptide distance restraints were obtained from a 2-D 13 C-filtered NOESY spectrum.
• ⁇ dihedral angle restraints were obtained from an HNHA spectrum (Cavanagh et al, supra), and ⁇ l restraints were derived from HNHB and short-mixing-time TOCSY spectra (Clore et al, J. Biomolec. NMR, 1: 13-22 (1991)).
• IGF-Fl-1 adopts a conformation very similar to that determined for the peptide by itself in solution.
• the conformation of IGF- 1 contains three helices (residues 7-18, 43-49, and 54-60) and is similar to that seen at lower resolution in previous NMR studies of uncomplexed IGF-1 (see e.g. Cooke et al, supra; Sato et al, supra; and Laajoki et al, supra).
• Fig. 10 shows the comparison for the detergent and phage peptide complexes.
• Fig. 10A shows a ribbon diagram of a complex of IGF-1 and N, N-bis(3-D-gluconamidopropyl)- deoxycholamine), and
• Fig. 10B shows a complex of IGF-1 bound to the phage-derived peptide IGF-Fl-1.
• the B-region (helix I) adopts a very similar conformation in both complexes.
• the C-loop is only partially ordered in the detergent complex, and ill defined in the peptide complex.
• AATTOOMM 8800 CCGG LLEEUU AA 1144 26.457 13.638 31.695 1.00 15.59
• ATOM 214 N TYR A 31 32.265 -8.32529.275 1.0023.05
• ATOM 215 CA TYR A 31 32.723 -9.57628.690 1.0025.72
• ATOM 309 CA ARG A 50 25.381 16.219 17.640 1.00 26.05
• ATOM 310 CB ARG A 50 25.106 14.832 17.157 1.0023.82

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