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WO2009076621A1 - Structures de haute résolution de chitinases mammifère acides et leurs utilisations - Google Patents

Structures de haute résolution de chitinases mammifère acides et leurs utilisations Download PDF

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Publication number
WO2009076621A1
WO2009076621A1 PCT/US2008/086649 US2008086649W WO2009076621A1 WO 2009076621 A1 WO2009076621 A1 WO 2009076621A1 US 2008086649 W US2008086649 W US 2008086649W WO 2009076621 A1 WO2009076621 A1 WO 2009076621A1
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WIPO (PCT)
Prior art keywords
atom
amcase
tyr
asp
glu
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PCT/US2008/086649
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English (en)
Inventor
Andrea Olland
James Strand
Margaret Fleming
Diane Joseph-Mccarthy
Rustem Krykbaev
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Wyeth
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Publication of WO2009076621A1 publication Critical patent/WO2009076621A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • Chitins a polymer of ⁇ -1 , 4-N-acetyl-glucosamine (GIcNAc), are abundantly produced in nature in the exoskeleton of crustaceans and insects, the sheath of nematodes, the cell wall of fungi, and other marine organisms, parasites, and pathogens; but not by mammals.
  • Chitin is degraded by chitinases (EC 3.2.1.14) that belong to members of the glycohydrolase family 18. This family is characterized by an eight fold I/ ⁇ barrel structure and includes bacterial, as well as plant chitinases.
  • CLPs chitinase-like proteins belonging to the glycohydrolase family 18 in mice and human, including, acidic mammalian chitinase (AMCase), chitinase 3-like-l (CHI3L1), chitotriosidase, YKL-39, YmI, oviduct-specific glycoprotein, and stabilin-1 -interacting chitinase-like protein (Hakala, et al, J. Biol.
  • AMCase acidic mammalian chitinase
  • CHI3L1 chitinase 3-like-l
  • chitotriosidase chitotriosidase
  • YKL-39 chitotriosidase
  • YmI chitotriosidase
  • oviduct-specific glycoprotein oviduct-specific glycoprotein
  • stabilin-1 -interacting chitinase-like protein stabilin-1 -interacting
  • Chitotriosidase and AMCase possess chitinase enzymatic activity, whereas other currently identified mammalian chitinases, including CLPs, do not possess this activity (Chang, et al, J. Biol. Chem., 276:17497-17506, 2001).
  • the consensus catalytic site in enzymatically active chitinases is DGXDXDXE (SEQ ID NO: 6) on strand M. W2025-7029WO / AM102903-PCT
  • AMCase The prototypic chitinase, catalyses the hydrolysis of artificial chitin-like substrates. It is unique among mammalian enzymes in that it has an acidic pH optimum. For example, AMCase catalyzes the conversion of chitin to N-acetyl-D-glucosamine. AMCase has been shown to be expressed predominately in the gastrointestinal tract, particularly the stomach, and to a lesser extent in the lung and is thought to play a role in digestion or possibly anti-parasitic defense mechanism.
  • AMCase is a secreted enzyme about 52.2 kD encoded by chromosome Ip 13.1- p21.3 (Boot, et al, J. Biol. Chem., 270:26252-26256, 1995; Saito A., et al, Gene, 239:325-331, 1999). Optimum AMCase activity is seen at pH 4-5, and it is typically found in stomach, salivary gland, and lung. AMCase is induced during T H 2 inflammation through an IL- 13 -dependent mechanism (Elias J. A., et al, J. Allergy Clin. Immunol, 116:497-500, 2005; Elias J.
  • AMCase mRNA is significantly increased in an animal model of asthma compared to control, non-asthmatic animals (Zhu Z., et al., Science, 304:1678-1682, 2004).
  • the mRNA encoding AMCase protein is elevated by intrathecal ovalbumin challenge or direct pulmonary instillation of IL- 13, and blocking binding of IL- 13 to the IL-13 receptor inhibits AMCase expression (Zhu Z., et al., Science, 304:1678-1682, 2004).
  • AMCase neutralization has been shown to reduce asthmatic pathology in animal models; for example, lowered BAL inflammation has been observed in a mouse asthma model and reduced airway hyperactivity and dynamic compliance has been detected in OVA models in response to AMCase inhibition (WO 2004/092404).
  • AMCase acidic mammalian chitinase
  • apo uncomplexed
  • ligand-bound forms of the catalytic domain of human AMCase have been determined.
  • the structures disclosed herein enable the identification and/or design of agents that modulate, e.g., antagonize, AMCase activity.
  • crystal compositions, methods of selecting and/or designing agents, e.g., antagonists, of AMCase, and computer and software programs are disclosed.
  • the agents identified by the methods disclosed herein can be used to treat or prevent chitinase-associated disorders or conditions, such as inflammatory and respiratory disorders (e.g., asthma, chronic obstructive pulmonary disease (COPD), and emphysema).
  • COPD chronic obstructive pulmonary disease
  • the invention features a crystallized AMCase, e.g., a human AMCase polypeptide or fragment thereof.
  • the invention features a crystallized AMCase-ligand complex that includes an AMCase (e.g., a human AMCase polypeptide or fragment thereof) and a ligand, e.g., an antagonist, of the AMCase.
  • AMCase e.g., a human AMCase polypeptide or fragment thereof
  • a ligand e.g., an antagonist
  • the invention features a method that includes using a three- dimensional model of an AMCase to design an agent that binds the AMCase.
  • the invention features a method that includes using a three- dimensional model of an AMCase to identify (e.g., select and/or screen for) an agent that binds the AMCase.
  • the invention features a method that includes using a three- dimensional model of a complex including an AMCase to design an agent that binds the AMCase.
  • the invention features a method that includes using a three- dimensional model of an AMCase-ligand complex to identify (e.g., select and/or screen for) an agent that binds the AMCase.
  • the AMCase in the compositions and methods disclosed herein includes (or consists essentially of) a chitinase catalytic domain, a flexible linker and/or a chitin-binding domain, or a combination thereof.
  • the AMCase includes (or consists essentially of) the amino acid sequence from a mammalian (e.g., human) or non- mammalian AMCase; for example, the AMCase polypeptide can include (or consist essentially of) the amino acid sequence of residues about 1 to 389 of SEQ ID NO:2, or about residues 1 to 471 (full length), about 22 to 408 (chitinase catalytic domain), about W2025-7029WO / AM102903-PCT
  • the AMCase polypeptide is a fragment that includes (or consists essentially of) one or more of a chitinase catalytic domain, a flexible linker and/or a chitin-binding domain, or a combination thereof.
  • the AMCase fragment includes at least 90, 100, 150, 200, 250, 300, 350, 400 or more contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4.
  • the AMCase fragment includes one or more of ligand binding site, a chitin-binding site, and/or a catalytic site that includes the amino acid sequence DXXDXDXE (SEQ ID NO: 5).
  • the AMCase polypeptide or fragment thereof has up to 50, 40, 30, 20, 15, 10, 5, 3, 2, or 1 change(s), e.g., deletion(s), insertion(s), or substitution(s) (e.g., conservative substitution(s)) from the amino acid sequence of SEQ ID NO: 2 or 4.
  • the change is located in a region that does not substantially alter the structure and/or activity of the AMCase polypeptide or fragment thereof.
  • one or more changes can be found at a site of the human AMCase that is exposed to a solvent, e.g., a site that has about 60 to 80%, 40 to 60%, 20 to 40%, 30 to 40%, 35 to 40%, 38 to 40%, 39 to 40%, or less than 20% access to the solvent.
  • the AMCase is crystallized, e.g., is capable of diffracting
  • X-rays to a resolution of at least about 3.7 A or less (e.g., about 3.6 A or less, about 3.5 A or less, about 3.2 A or less, about 3.0 A or less, about 2.7 A or less, about 2.4 A or less, about 2.3 A or less, about 2.2 A or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.8 A or less, about 1.7 A or less, about 1.6 A or less, about 1.5 A or less, or about 1.4 A or less).
  • a crystal of the human AMCase polypeptide can diffract X-rays to a resolution of from about 1.7 A to about 3.7 A (e.g., the crystal of the W2025-7029WO / AM102903-PCT
  • human AMCase polypeptide can diffract X-rays to about 2.7 A, about 3.5 A or about 3.6 A).
  • the crystal of the AMCase belongs to space group chosen from P2i or V2 ⁇ l ⁇ l ⁇ .
  • the crystallized AMCase has a three-dimensional structure that includes the structural coordinates according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A.
  • the crystallized AMCase has a three-dimensional structure that includes the structural coordinates of an atom chosen from one or more of amino acids: TYR-27, TRP-31, PHE-58, ILE-69, GLU-70, GLY-97, GLY-98, TRP-99, ASN-100, PHE-101, ASP-136, ASP-138, GLU-140, ALA-183, MET-210, TYR-212, ASP-213, TYR-267, ALA-295, LYS-296, GLU-297, ILE-300, MET-358, TRP-360, and/or LEU- 364 according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A.
  • the crystallized AMCase has a three-dimensional structure that includes the structural coordinates of an atom chosen from one or more of amino acids: TYR-27, TRP-31, PHE
  • AMCase has a three-dimensional structure that includes the structural coordinates of an atom chosen from one or more of amino acids: THR-25, CYS-26, TYR-27, PHE-28, THR-29, ASN-30, TRP-31, ALA-32, GLN-33, TYR-34, ARG-35, ILE-55, TYR-56, ALA-57, PHE-58, ALA-59, GLY-60, THR-68, ILE-69, GLU-70, ASP-73, LEU-93, LEU-94, ALA-95, ILE-96, GLY-97, GLY-98, TRP-99, ASN-100, PHE-101, GLY-102, PHE-106, LEU-135, ASP-136, PHE-137, ASP-138, TRP-139, GLU-140, TYR-141, W2025-7029WO / AM102903-PCT
  • the crystallized AMCase has a three-dimensional structure that includes the structural coordinates of o amino acid residues HIS-208, HIS-269 and ARG-145, according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A, and one or more coordinate values from the aforesaid listed residues.
  • residues HIS-208, HIS-269 and ARG-145 are believed to alter (e.g., lower) the pH optimum of the enzyme, e.g., by influencing the pK a of ASP1385 and/or GLU140.
  • the crystallized AMCase has a three- dimensional structure that includes the structural coordinates of amino acid residues HIS- 208, HIS-269, ARG-145, ASP138 and GLU140, according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A, and one or more coordinate values from the aforesaid listed residues.
  • the methods disclosed herein include identifying (e.g., selecting) or designing the agent (e.g., one or more candidate agents) by one or more of: generating a three-dimensional model of the AMCase three-dimensional structure (e.g., utilizing the X-ray three-dimensional coordinates according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A, to 5 generate a three-dimensional model of the AMCase three-dimensional structure); identifying the amino acid residues forming the ligand binding pocket of the AMCase ligand binding domain from the three-dimensional model in order to generate a three- dimensional representation of the ligand binding pocket of AMCase; evaluating an interaction between the three dimensional model or structure of AMCase and one or more0 candidate agents, e.g., by performing computer fitting analysis, the structure of the
  • AMCase the AMCase-ligand complex or the one or more candidate agents; or altering, W2025-7029WO / AM102903-PCT
  • the three-dimensional structure of the AMCase, the AMCase-ligand complex, or the one or more candidate agents disclosed herein to thereby select or design the agent.
  • the three dimensional structure of AMCase or the AMCase-ligand complex e.g., the ligand binding pocket of AMCase
  • the three dimensional structure of AMCase or the AMCase-ligand complex includes an atom chosen from one or more atoms of amino acids: TYR-27, TRP-31,
  • the three dimensional structure of AMCase or the AMCase-ligand complex (e.g., the ligand binding pocket of AMCase) includes an atom chosen from one or more atoms of amino acids: THR-25, CYS -26, TYR-27, PHE-28, THR-29, ASN-30, TRP-31, ALA-32, GLN-33, TYR-34, ARG-35, ILE-55, TYR-56, ALA-57, PHE-58, ALA-59, GLY-60, THR-68, ILE-69, GLU-70, ASP- 73, LEU-93, LEU-94, ALA-95, ILE-96, GLY-97, GLY-98, TRP-99, ASN-100, PHE- 101, GLY-102, PHE-106, LEU-135, ASP-136, PHE-137, ASP-138, TRP-139, GLU-140,
  • HIS-208, HIS-269 and ARG-145 according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A, and one or more coordinate values from the aforesaid listed residues.
  • residues HIS-208, HIS-269 and ARG-145 are believed to alter (e.g., lower) the pH optimum of the enzyme, e.g., by influencing the pKa of ASP138 and/or GLU140.
  • the three-dimensional structure that includes the structural W2025-7029WO / AM102903-PCT
  • the methods disclosed herein include the step of contacting the candidate agent(s) with the AMCase, and evaluating, e.g., detecting, a change in one or more activities of the AMCase in the presence of the candidate agent(s), relative to a predetermined level, e.g., a control sample without the candidate agent(s).
  • the contacting step can be effected in vitro (in cultured cells or a reconstituted system) or in vivo (e.g., by administering the candidate agent to a non-human subject).
  • the contacting step(s) and/or the administration of the test compound can be repeated.
  • the activities evaluated include one or more of: a change in binding (e.g., direct binding or ability to compete for binding with a known AMCase ligand), or an enzymatic activity of the AMCase.
  • Exemplary known AMCase ligands that can be evaluated include methyallosamidin, allosamidin, glucoallosamindin-A or -B, methyl-N- demethylallosamidin, demethylallosamidin, didemethylallosamidin, styloguanidine, dipeptide cyclo-(L-Arg-D-Pro), (L-Arg-L-Pro), (D-Arg-D-Pro), riboflavin, antibody molecules (e.g., antibody molecule that bind to a catalytic or a chitinase-binding domain), or analogues thereof.
  • the enzymatic activity of AMCase evaluate includes: (1) the ability to bind chitin and/or chi tin- like substrates; (2) the ability to catalyze the hydrolysis of chitin and/or chi tin-like substrates; (3) the ability to induce lung inflammation, e.g., airway hyperresponsiveness, in a subject (e.g., a non-human subject).
  • the method includes administering the candidate agent to a subject, e.g., a non-human animal (e.g., a sheep, a non-human primate (e.g., a cynomolgus monkey), or a rodent), and evaluating a change in one or more of parameters of a chitinase-associated disorder.
  • a subject e.g., a non-human animal (e.g., a sheep, a non-human primate (e.g., a cynomolgus monkey), or a rodent)
  • one or more of the following can be evaluated in the presence or absence of the candidate agent: (i) detecting a change in the number of inflammatory cells (e.g., eosinophils, macrophages, neutrophils) into the airways; (ii) measuring eotaxin levels; (iii) detecting in basophil histamine release; and/or (iv) detecting IgE titers.
  • a change e.g., a reduction, in the level W2025-7029WO / AM102903-PCT
  • a predetermined level e.g., comparing before and after treatment
  • the candidate agent is effectively reducing airway eosinophilia in the subjects, and thus is a candidate for treating inflammatory pulmonary conditions (e.g., asthma, COPD and emphysema).
  • the methods disclosed herein can additionally include one or more of the following steps: calculating a distance between an atom of the AMCase (e.g., the AMCase polypeptide or fragment thereof) and an atom of the agent; determining the interaction of the agent with the AMCase; comparing the interaction of the agent with the AMCase to an interaction of a second agent with the AMCase; selecting the agent via computer modeling; designing the candidate agent (e.g., de novo design or altering the three dimensional structure of a known compound, e.g., a known AMCase ligand as disclosed herein); obtaining the candidate agent, e.g., synthesizing the agent; detecting the ability of the agent to inhibit one or more AMCase activities (e.g., inhibit one or more of the AMCase enzymatic activities described herein).
  • the candidate agent e.g., de novo design or altering the three dimensional structure of a known compound,
  • the method further includes one or more of: obtaining a supplemental crystalline complex that includes the AMCase and one or more candidate agents; determining the three-dimensional structure of the supplemental crystalline complex; evaluating, e.g., selecting, a second candidate agent by, e.g., performing rational drug design with the three-dimensional structure of the supplemental crystalline complex; contacting the second agent with the AMCase; and/or detecting the ability of the second agent to bind to or alter an activity of the AMCase.
  • the method can further include one or more of: synthesizing the second agent; detecting an ability of the second agent to modulate, e.g., inhibit AMCase activity in vitro or in vivo (e.g., inhibit an AMCase enzymatic activity as described herein).
  • the agent designed or identified using the methods disclosed herein inhibits AMCase activity.
  • the agent can bind AMCase at a ligand binding site or a chitin-binding site, and/or interfere with an interaction between AMCase and the ligand or chitin.
  • the agent preferentially displaces the beta-anomer form of the ligand, e.g., the chitin ligand.
  • the ligand binding site bound by the agent is located on the chitinase catalytic domain, e.g., in proximity to the chitinase active site having a DGXDXDXE (SEQ ID NO: 6) motif, or W2025-7029WO / AM102903-PCT
  • the ligand binding site bound by the agent is located in proximity to one or more of: HIS-208, HIS-269, ARG-145, ASP138 and/or GLU140, according to Table 1 or Table 2, +/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A, 1 A, or 0.5 A, and one or more coordinate values from the aforesaid listed residues.
  • residues HIS-208, HIS-269 and ARG- 145 are believed to alter (e.g., lower) the pH optimum of the enzyme, e.g., by influencing the pKa of ASP138 and/or GLU140.
  • the methods disclosed herein include providing a composition including an AMCase, or a AMCase -ligand complex, and/or crystallizing the composition to form a crystalline complex that includes the AMCase, or the AMCase- ligand complex (e.g., a crystallized form of AMCase or an AMCase complex as disclosed herein).
  • the crystalline complex can diffract X-rays to a resolution of at least about 3.7 A (e.g., about 3.6 A or less, about 3.5 A or less, about 3.2 A or less, about 3.0 A or less, about 2.7 A or less, about 2.4 A or less, about 2.3 A or less, about 2.2 A or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.8 A or less, about 1.7 A or less, about 1.6 A or less, about 1.5 A or less, or about 1.4 A or less).
  • 3.7 A e.g., about 3.6 A or less, about 3.5 A or less, about 3.2 A or less, about 3.0 A or less, about 2.7 A or less, about 2.4 A or less, about 2.3 A or less, about 2.2 A or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.8 A or less, about 1.7 A or less, about 1.6 A or less, about 1.5 A or less
  • the invention features an agent designed or identified using the methods disclosed herein. W2025-7029WO / AM102903-PCT
  • the invention features a method for crystallizing an AMCase (e.g., a human AMCase polypeptide or fragment thereof).
  • the method includes (i) providing the AMCase;
  • a chitinase ligand e.g., a chitinase inhibitor, such as allosamidin-like ligand or an analogue thereof
  • a chitinase ligand e.g., a chitinase inhibitor, such as allosamidin-like ligand or an analogue thereof
  • step (iii) mixing a solution of the AMCase, and (optionally) the chitinase ligand, in the presence of polyethylene glycol (e.g. PEG 2000, PEG 3350, PEG4000, PEG 6000), a salt (e.g., a sodium salt and/or ammonium salt), and a buffer (e.g. acetate, citrate, Tris buffer); and (iv) crystallizing the solution of step (iii), e.g., by sitting drop or hanging drop vapor diffusion to form a crystalline precipitate.
  • polyethylene glycol e.g. PEG 2000, PEG 3350, PEG4000, PEG 6000
  • a salt e.g., a sodium salt and/or ammonium salt
  • a buffer e.g. acetate, citrate, Tris buffer
  • the solution of step (iii) is mixed with a crystallization buffer chosen from one or more of: 25% PEG4000, 0.1 M Sodium Acetate, 0.2 M Ammonium Sulfate; 20% PEG 3350, 0.2 M Ammonium Formate; 24.5% PEG 3350, 0.1 M bis-tris buffer at pH 6.5; 20% PEG 3350, 0.2 Ammonium Phosphate; 20% PEG 3350, 0.2 Ammonium Fluoride; or 20% PEG 3350, 0.2 di- Ammonium citrate.
  • a crystallization buffer chosen from one or more of: 25% PEG4000, 0.1 M Sodium Acetate, 0.2 M Ammonium Sulfate; 20% PEG 3350, 0.2 M Ammonium Formate; 24.5% PEG 3350, 0.1 M bis-tris buffer at pH 6.5; 20% PEG 3350, 0.2 Ammonium Phosphate; 20% PEG 3350, 0.2 Ammonium Fluoride; or 20% PEG 3350, 0.2 di-
  • the crystallization is performed at a temperature of from about 4°C to about 60 0 C (e.g., from about 10 0 C to about 45°C, such as at about 12°C, about 15°C, about 18°C, about 20 0 C, about 25°C, about 30 0 C, about 32°C, about 35°C, about 37°C).
  • the invention features a software system that includes instructions for causing a computer system to accept information relating to the structure of an AMCase (e.g., a human AMCase polypeptide or fragment thereof), accept information relating to a candidate agent, and determine binding characteristics of the candidate agent to the AMCase.
  • the determination of binding characteristics can be based on the information relating to the structure of the AMCase, and the information relating to the candidate agent.
  • the invention features a computer program residing on a computer readable medium having a plurality of instructions stored thereon, which, when executed by one or more processors, cause the one or more processors to accept information relating to the structure of a complex comprising an AMCase, accept W2025-7029WO / AM102903-PCT
  • the invention features a method that includes accepting information relating to the structure of an AMCase and modeling the binding characteristics of the AMCase with a candidate agent.
  • the method can be implemented by a software system.
  • the invention features a computer program residing on a computer readable medium having a plurality of instructions stored thereon, which, when executed by one or more processors, cause the one or more processors to accept information relating to a structure of an AMCase and model the binding characteristics of the AMCase with a candidate agent.
  • the invention features a software system that includes instructions for causing a computer system to accept information relating to a structure of an AMCase and model the binding characteristics of the AMCase with a candidate agent.
  • the invention features a method of modulating AMCase activity in a subject that includes: selecting or designing, e.g., using the methods described herein (e.g., by rational drug design), an agent that is capable of modulating AMCase activity and administering an effective amount of the agent to the subject, such that the AMCase activity is modulated.
  • the invention features a method of treating or preventing in a subject having, or at risk of having, a chitinase-associated disorder or condition that includes: selecting or designing, e.g., using the methods described herein (e.g., by rational drug design), an agent that is capable of affecting (e.g., inhibiting) AMCase activity and administering a therapeutically effective amount of the agent to a subject in need thereof.
  • the agent used in the methods disclosed herein e.g., the therapeutic and prophylactic methods disclosed herein, inhibit one or more AMCase enzymatic activity in vivo (e.g., reduce, ameliorate, or cure one or more symptoms) of a chitinase-associated disorder or condition in a subject.
  • a chitinase-associated W2025-7029WO / AM102903-PCT e.g., W2025-7029WO / AM102903-PCT
  • disorders include inflammatory disorders (e.g., lung inflammation), respiratory disorders (e.g., asthma, including allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), emphysema), as well as conditions involving airway inflammation, eosinophilia, interstitial lung disease, chronic obstructive lung disease, bronchitis, pneumonia, fibrotic disorders (e.g., cystic fibrosis, liver fibrosis, and pulmonary fibrosis); scleroderma; atopic disorders (e.g., atopic dermatitis, urticaria, eczema, allergic rhinitis, and allergic enterogastritis), and inflammatory bowel disease.
  • lung inflammation e.g., lung inflammation
  • respiratory disorders e.g., asthma, including allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), emphysema
  • COPD chronic obstructive pulmonary
  • FIGs IA- IB depict the nucleotide and amino acid sequences of a truncated human AMCase polypeptide used for crystallization (SEQ ID NO:1 and SEQ ID NO: 2, respectively).
  • This human AMCase sequence has a mutation at position 354 to replace a serine for a phenylalanine and a histidine tag at the C-terminal end.
  • FIGs. 2A-2B depict the full length nucleotide and amino acid sequences, respectively, of an exemplary human acidic chitinase acidic isoform C precursor (SEQ ID NO:3 and SEQ ID NO:4, respectively).
  • the AMCase amino acid sequence used for crystallization encompassed the catalytic domain of human AMCase located at about amino acids 22 to 408 of the amino acid sequence of FIG. 2A (SEQ ID NO:4). Amino acids 22 to 408 of the full length amino acid sequence shown in Figure 2B has some minor differences from the AMCase truncated sequence shown in Figure IB at 5 positions.
  • FIG. 3A illustrates a ribbon representation of human AMCase polypeptide bound with methyallosamidin. The location of the N-terminus and the C-terminus is indicated.
  • FIG. 3B illustrates an electrostatic surface potential map of AMCase showing the active site cleft, in the same orientation as FIG. 3A. Positive and negative charge is depicted at +15.0 kT/e, respectively. The long negatively charged groove that accommodates chitin binding is visible.
  • FIG. 4 illustrates a 2Fo-Fc electron density map, contoured at 1.2 ⁇ , showing methyallosamidin bound to human AMCase polypeptide, at a resolution of 1.7 A. W2025-7029WO / AM102903-PCT
  • FIG. 5 is a table summarizing the characteristics of the AMCase crystals disclosed herein, including crystallization conditions, space group and unit cell dimensions.
  • FIG. 6 is a summary of interactions between methylallosamidin and AMCase active site residues.
  • FIG. 7A illustrates a ribbon diagram of the AMCase active site, depicting side chains of certain residues. ASP-136, ASP-138, GLU-140, ASP-213, and TRP-360 are highly conserved active site residues, ARG-145, HIS-208, and HIS-269 are residues specific to AMCase. Dotted lines indicate hydrogen bonds, with the distance in angstroms indicated.
  • FIG. 7B illustrates ARG-145 in the context of the active site. ARG-145 affects substrate binding via a network of hydrogen bonds mediated by TRP-99, ASN-100, and GLU-140.
  • FIG. 8 illustrates a space-filled representation of AMCase molecules showing the crystal packing of Apo AMCase. The C-terminal end of one molecule binds in the active site cleft of the neighboring molecule.
  • FIG. 9 illustrates the modeled binding of the alpha-anomer vs the beta-anomer of a chitin fragment (chitopentose).
  • This figure shows the structural basis of the preference for the beta-anomer of the substrate by AMCase.
  • the terminal hydroxyl of the alpha- anomer, (labeled as alpha-chitopentose, terminal hydroxyl) makes an unfavorable electrostatic interaction with the -NH of the indole ring of Trp-218, and thus binding of the beta-anomer (labeled as beta-chitopentose, terminal hydroxyl) is energetically more favorable.
  • the present invention relates to human AMCase polypeptides, human
  • the invention provides high resolution, three dimensional structures of uncomplexed and ligand-bound forms of the catalytic domain of human AMCase.
  • the structures disclosed herein can be useful for designing or identifying inhibitors that can interact with human AMCase polypeptides.
  • Structure-based modeling can allow the identification of an agent capable of interacting with AMCase, without the need to experimentally test a large variety of compounds in vivo or in vitro with the goal of identifying a candidate agent that can interact with AMCase. Such screening can be expensive and time-consuming.
  • Modifications to a known agent that interacts with AMCase can be examined with structure based design to identify an agent with more desirable properties, such as tighter binding or greater selectivity for AMCase over other chitinases, without the need to prepare and test each modified agent.
  • FIG. 1A-1B Shown in FIG. 1A-1B is the nucleotide and amino acid sequence (SEQ ID NO:1 and SEQ ID NO:2, respectively) of a fragment of human AMCase polypeptide (human AMCase) that includes the catalytic domain of family 18 glycosylases The hexahistidine tag facilitated purification. Not present in the human AMCase polypeptide is a 21 amino acid signal sequence that is found at the N-terminus of the full-length human AMCase.
  • FIG. 2 is a ribbon diagram illustrating the structure of methylallosamidin-bound human AMCase polypeptide (amino acid residues 22-400 of the catalytic domain of human AMCase).
  • FIG. 1A-1B Shown the nucleotide and amino acid sequence (SEQ ID NO:1 and SEQ ID NO:2, respectively) of a fragment of human AMCase polypeptide (human AMCase) that includes the cata
  • FIG. 3 is a 2Fo-Fc electron density map, depicting the high quality of the electron density for the methylallosamidin in the bound human AMCase.
  • the coordinates of a crystal structure of the human AMCase polypeptide are provided in Table 1.
  • the coordinates of a crystal structure of the methylallosamidin-bound human AMCase polypeptide are provided in Table 2.
  • AMCases belong to a family of chitinases of the glycohydrolase family 18. Recent studies have identified chitinases and chitinase-like proteins (CLPs) belonging to the glycohydrolase family 18 in mice, human and non-human primates.
  • the members of the mammalian chitinases and CLPs include acidic mammalian chitinase (AMCase), chitinase 3-like-l (CHI3L1), chitotriosidase, YKL-39, YmI, oviduct-specific glycoprotein, and stabilin-1 -interacting chitinase-like protein (Hakala, et ah, J. Biol.
  • the consensus catalytic site in enzymatically active chitinases is DGXDXDXE (SEQ ID NO: 6) on strand M.
  • AMCase polypeptides or fragments can include one or more of the following domains: chitinase catalytic domain, a linker region, and/or a chi tin-binding domain, or regions substantially identical thereto.
  • chitinase catalytic domain includes an amino acid sequence about 200 to 400, more typically about 300 to 400 amino acids in length (e.g., from about amino acid residues 1 to 401 of SEQ ID NO: 2, or about residues 22 to 408 of SEQ ID NO:4 and which typically includes a consensus catalytic site having the amino acid sequence DGXDXDXE (SEQ ID NO: 6); or an amino acid sequence substantially identical thereto (e.g., an amino acid sequence about 85%, 87%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4), or a fragment thereof (e.g., a biologically active portion thereof as described herein); or an amino acid sequence encoded by a nucleic acid molecule comprising, or consisting essentially of, the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a nucleotide sequence substantially identical thereto (e.g.,
  • An AMCase polypeptide can further include a "chitin-binding domain.”
  • a "chitin-binding domain” includes an amino acid sequence about 20 to 70, more typically about 40 to 50 amino acids in length and which has at least about 90% 95%, 99%, or 100% identity with amino acid residues 436 to 471 of SEQ ID NO:4.
  • An AMCase polypeptide can also include a linker region connecting the catalytic and chitinase-binding domains.
  • the linker region can have an amino acid sequence about 10 to 30, 15 to 28 amino acid residues in length, and having an W2025-7029WO / AM102903-PCT
  • Particular AMCase polypeptides or fragments of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • substantially identical is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:4 are termed substantially identical.
  • nucleotide sequence in the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • nucleotide sequences having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or 3 are termed substantially identical.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the W2025-7029WO / AM102903-PCT
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) /. MoI. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) /. MoI. Biol. 215:403-10.
  • BLAST protein searches can be W2025-7029WO / AM102903-PCT
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402.
  • XBLAST and NBLAST the default parameters of the respective programs. See http://www.ncbi.nlm.nih.gov.
  • Conservative substitutions are amino acid substitutions which are functionally or structurally equivalent to the substituted amino acid residue in terms of polarity, steric arrangement, or belonging to the same class (e.g., hydrophobic, acidic or basic) as the substituted residue. Classes of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Conservative substitutions include substitutions having an inconsequential effect on the three-dimensional structure of AMCase with respect to identification and design of agents that interact with AMCase.
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.
  • the term “about” refers to a sequence having up to 5, 4, 3, 2, or 1 more or less amino acid residues or nucleotides at the N- or C-terminus of the sequence specified.
  • agents identified using the methods described herein may modulate, e.g., inhibit, one or more chitinase -associated activities, they may be useful for developing novel therapeutic agents for chitinase-associated disorders, as described below.
  • an AMCase or a chitinase activity, biological or enzymatic activity of AMCase refers to an activity exerted by an AMCase polypeptide.
  • AMCase or chitinase activity can be determined in vivo or in vitro.
  • an AMCase activity is a direct activity, such as an association with an AMCase target molecule or binding partner, e.g., a target molecule or binding partner with which the AMCase binds or interacts in nature.
  • AMCase is an enzyme for a chitin-like substrate.
  • the AMCase proteins can have one or more of the following activities or properties: (1) has the ability to bind chitin and/or chitin-like substrates; (2) catalyzes the hydrolysis of chitin and/or chitin-like substrates; (3) has optimal catalytic properties in acidic pH; (4) is inhibited by a chitinase-like inhibitor, such as methylallosamidin, allosamidin, glucoallosamindin-A or -B, methyl-N- demethylallosamidin, demethylallosamidin, didemethylallosamidin, styloguanidine, dipeptide cyclo-(L-Arg-D-Pro) or (L-Arg-L-Pro), or (D-Arg-D-Pro), riboflavin; and/or (5) induces lung inflammation, e.g., airway hyperresponsiveness in a subject, e.g., asthmatic patients, particularly patients having allergic asthma.
  • lung inflammation e.
  • the agents identified using the methods disclosed herein can act as novel therapeutic agents for detecting and/or treating chitinase-associated diseases, including, but not limited to, inflammatory disorders (e.g., lung inflammation), respiratory disorders (e.g., asthma, including allergic and non-allergic asthma, chronic obstructive pulmonary disease (COPD), emphysema), as well as conditions involving airway inflammation, eosinophilia, interstitial lung disease, chronic obstructive lung disease, bronchitis, pneumonia, fibrotic disorders (e.g., cystic fibrosis, liver fibrosis, and pulmonary fibrosis); scleroderma; atopic disorders (e.g., atopic dermatitis, urticaria, eczema, allergic rhinitis, and allergic enterogastritis), and inflammatory bowel disease.
  • inflammatory disorders e.g., lung inflammation
  • respiratory disorders e.g., asthma, including allergic and
  • An agent e.g., an antagonist, that interacts with (e.g., binds) the AMCase polypeptide can be identified or designed by a method that includes using a representation of the polypeptide or a fragment of the polypeptide.
  • Exemplary types of representations include the representations discussed herein.
  • the representation can be of an analog polypeptide or polypeptide fragment.
  • a candidate agent that interacts with the representation can be designed or identified by performing computer fitting analysis of the candidate agent with the representation.
  • the agent e.g., a candidate agent, includes a protein, polypeptide, peptide, nucleic acid
  • An agent can act as a ligand, W2025-7029WO / AM102903-PCT
  • An agent that interacts with a polypeptide can interact transiently or stably with the polypeptide.
  • the interaction can be mediated by any of the forces noted herein, including, for example, hydrogen bonding, electrostatic forces, hydrophobic interactions, and van der Waals interactions.
  • An active site is a region of a molecule or molecular complex that can interact or associate with an agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound or drug).
  • An active site may include, for example, the site of agent binding, as well as accessory binding sites adjacent or proximal to the actual site of binding that may affect activity upon interaction or association with a particular agent.
  • the active site can include a site of ligand, e.g., inhibitor, binding.
  • the inhibitor can inhibit either by direct interference with the actual site of substrate binding (i.e., by competing for substrate binding) or by indirectly affecting the steric conformation or charge potential, thereby preventing or reducing binding of substrate at the actual site of substrate binding.
  • Ligands that interact with human AMCase polypeptides can inhibit or increase human AMCase activity.
  • AMCase activity can be inhibited or reduced by an antagonist, such as allosamidin, methylallosamidin, glucoallosamindin-A or -B, methyl-N-demethylallosamidin, demethylallosamidin, didemethylallosamidin, styloguanidine, dipeptide cyclo-(L-Arg-D-Pro) or (L-Arg-L-Pro), or (D-Arg-D-Pro), riboflavinhuman, or an analog thereof.
  • an antagonist such as allosamidin, methylallosamidin, glucoallosamindin-A or -B, methyl-N-demethylallosamidin, demethylallosamidin, didemethylallosamidin, styloguanidine, dipeptide cyclo-(L-Arg-D-Pro) or (L-Arg-L-Pro), or (D
  • Allosamidin-like ligands can have general formula (I)
  • one of Ri and R 2 is hydrogen, and the other is hydroxyl or C 1 -C 4 alkoxy; one of R 3 and R 4 is hydrogen, and the other is hydroxyl or C 1 -C 4 alkoxy; each of R 5 and Rio is, independently, hydrogen or C 1 -C 3 alkyl; each of R 6 and Rn is, independently, Ci-C 6 alkyl; each Of R 7 , R 12 , and R 1 3 is, independently, hydrogen; Ci-C 6 alkyl; C 1 -C 4 haloalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkenyl; C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, heterocyclyl including from 3-6 atoms, phenyl, or heteroaryl including from 5-6 atoms, each of which is optionally substituted with one or more substituents; one of Rg and R 9 is hydrogen, and the other is hydroxyl or
  • X is O, NH, or S
  • R 14 is Ci-C 6 alkyl
  • Ri 5 is hydrogen or Ci-C 6 alkyl.
  • Embodiments can include one or more of the following features. Variables R 1 , R 2 , R 3 , R4, Rs, and RQ
  • each of Ri and R 3 can be hydrogen, and each of R 2 and R 4 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ). In certain embodiments, each of Ri and R 3 can be hydrogen, and each of R 2 and R 4 can be hydroxyl. In some embodiments, each of Ri and R 4 can be hydrogen, and each of R 2 and R 3 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ). In certain embodiments, each of Ri and R 4 can be hydrogen, and each of R 2 and R 3 can be hydroxyl.
  • each of R 2 and R 3 can be hydrogen, and each of Ri and R 4 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ).
  • each of R 2 and R 3 can be hydrogen
  • each of Ri and R 4 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ).
  • each of R 2 and R 3 can be hydrogen, and each of Ri and R 4 can be hydroxyl.
  • each of R 2 and R 4 can be hydrogen, and each of Ri and R 3 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ). In certain embodiments, each of R 2 and R 4 can be hydrogen, and each of Ri and R 3 can be hydroxyl.
  • Rs is hydrogen
  • R 9 is hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ).
  • Rg is hydrogen
  • R 9 is hydroxyl.
  • R 9 is hydrogen, and Rg is hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ). In certain embodiments, R 9 is hydrogen, and Rg is hydroxyl.
  • Embodiments can include any combination of (i) definitions for variables R 1 -R 4 as delineated above and (ii) definitions for variables Rg and R 9 as delineated above.
  • each of Ri, R 3 , and Rg can be hydrogen, and each of R 2 , R 4 , and R 9 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ).
  • each of Ri, R 3 , and Rg can be hydrogen, and each of R 2 , R 4 , and R 9 can be hydroxyl.
  • each of R 1 , R 3 , and R 9 can be hydrogen, and each of R 2 , R 4 , and Rg can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ).
  • each of R 1 , R 3 , and R 9 can be hydrogen, and each of R 2 , R 4 , and Rg can be hydroxyl.
  • R 5 can be hydrogen or CH 3 .
  • Rio can be hydrogen or CH 3 .
  • each of R 5 and Rio can be hydrogen.
  • R 6 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ).
  • R 5 can be straight or branched C 3 -C 6 alkyl.
  • Rn can be CH 3 or CH 2 CH 3 (e.g., CH 3 ).
  • R 5 can be straight or branched C 3 -C 6 alkyl.
  • each of R 6 and Rn can be CH 3 .
  • one (e.g., R 7 ), two (e.g., R 7 and R 12 or R 7 and R 13 ), or three of R 7 , R 12 , and R i3 can be Ci-C 6 alkyl; Ci-C 4 haloalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkenyl; C 3 - C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, heterocyclyl including from 3-6 atoms, phenyl, or heteroaryl including from 5-6 atoms, each of which can be optionally substitutedwith one or more substituents; and the others can be hydrogen.
  • R 7 can be Ci-C 6 alkyl; C 1 -C 4 haloalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkenyl; C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, heterocyclyl including from 3-6 atoms, phenyl, or heteroaryl including from 5-6 atoms, each of which can be optionally substituted with one or more substituents.
  • R 7 can be Ci-C 6 (e.g., C 1 -C 4 ) alkyl, e.g.., R 7 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ); or straight or branched C 3 -C 6 alkyl.
  • R 7 can be C 1 -C 4 haloalkyl (e.g., CH 2 F or CF 3 ).
  • Ri 2 can be hydrogen.
  • Ri 3 can be hydrogen.
  • X can be O.
  • R 14 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ). In other embodiments, R 14 can be straight or branched C 3 -C 6 alkyl.
  • R 15 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ). In other embodiments, R 15 can be straight or branched C 3 -C 6 alkyl.
  • R 14 can be CH 3
  • R 15 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ); or straight or branched C 3 -C 6 alkyl.
  • each of R 14 and R 15 can be CH 3 .
  • a subset of formula (I) compounds includes in which: • each of R 1 , R 3 , and R 8 can be hydrogen, and each of R 2 , R 4 , and R 9 can be, independently of one another, hydroxyl or C 1 -C 4 alkoxy (e.g., OCH 3 ); and
  • R 7 can be C x -C 6 alkyl; C x -C 4 haloalkyl; C 2 -C 6 alkenyl; C 2 -C 6 alkenyl; C 3 - C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, heterocyclyl including from 3-6 atoms, phenyl, or heteroaryl including from 5-6 atoms, each of which can be optionally substituted with one or more substituents; and
  • X can be O; and W2025-7029WO / AM102903-PCT
  • R 15 can be Ci-C 6 alkyl.
  • Each of R 1 , R 3 , and Rg can be hydrogen, and each of R 2 , R 4 , and R 9 can be hydroxyl.
  • Each of R 5 and Rio can be hydrogen.
  • Each of R 6 and Rn can be CH 3 .
  • R 7 can be C x -C 6 (e.g., C x -C 4 ) alkyl, e.g.., R 7 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ); or straight or branched C 3 -C 6 alkyl.
  • Each of R 12 and Ri 3 can be hydrogen.
  • Ri 4 can be CH 3
  • R 15 can be CH 3 or CH 2 CH 3 (e.g., CH 3 ); or straight or branched C 3 -C 6 alkyl.
  • each of R 1 , R 3 , and Ks can be hydrogen, and each of R 2 , R 4 , and R 9 can be hydroxyl; and each of R 5 and Rio can be hydrogen; and each of R 6 and Rn can be CH 3 ; and R 7 can be CH 3 ; and X can be O; and each of Ri 2 and Ri 3 can be hydrogen; and each of R M and Ri 5 can be CH 3 .
  • the allosamidin-like ligand has the following structure:
  • Ri in the case of allosamidin, Ri can be CH 3 , R 2 and R 4 can be hydrogen and R 3 can be hydroxyl; wherein, in the case of demethylallosamidin, R 1 , R 2 and R 4 can be hydrogen and
  • R 3 can be hydroxyl
  • Ri and R 2 can be CH 3 , R 3 can be hydroxyl, and R 4 can be hydrogen; wherein, in the case of glucoallosamidin B, Ri and R 3 can be hydrogen, R 2 can be CH 3 and R 4 can be hydroxyl.
  • Allosamidin-like ligands are described in Rao, F.V., et ah, J. Biol. Chem. 278,
  • a human AMCase-polypeptide can be prepared and crystallized as described below.
  • the human AMCase polypeptide can be prepared as desired.
  • the human AMCase polypeptide is expressed from a DNA plasmid. The expression can be driven by a promoter, such as an inducible promoter or a constitutive promoter, such as a cytomegalovirus promoter.
  • the human AMCase polypeptide can be expressed as a fusion protein with a suitable tag, such as a polyhistidine (e.g., hexahistidine), glutathione-S-transferase (GST), myc, HA, Strep, or FLAG tag.
  • a suitable tag such as a polyhistidine (e.g., hexahistidine), glutathione-S-transferase (GST), myc, HA, Strep, or FLAG tag.
  • GST glutathione-S-transferase
  • myc e.g., myc
  • HA hexahistidine
  • Strep hexahistidine
  • FLAG tag can facilitate isolation of the human AMCase polypeptide from cells, such as from bacterial cells or from a mammalian cell line.
  • the human AMCase polypeptide can be expressed in and isolated from Chinese Hamster Ovary (CHO)
  • the human AMCase polypeptide can be fused to a hexahistidine tag and isolated by contacting a cell extract with a Nickel resin (e.g., a Nickel-nitrilotriacetic acid (Ni-NTA) resin) to bind the hexahistidine tag, and then releasing the polypeptide by washing the resin with a buffer containing imidazole.
  • a fusion human AMCase polypeptide can be cleaved at a protease site engineered into the fusion protein, such as at or near the site of fusion between the polypeptide and the tag.
  • the human AMCase polypeptide can be placed in solution for collecting spectral data, NMR data, or for growing a crystal.
  • the human AMCase polypeptide can be crystallized in the presence of a salt (e.g., a sodium salt and/or ammonium salt).
  • a salt e.g., a sodium salt and/or ammonium salt.
  • the human AMCase polypeptide can be crystallized in the presence of polyethylene glycol (e.g. PEG 2000, PEG 3350, PEG 6000, PEG 8000, PEG 10,000,
  • PEG 20,000 polyethylene glycol monomethylether
  • PEG MME 750 polyethylene glycol monomethylether
  • the human AMCase polypeptide can be crystallized in the presence of polyethylene glycol, salts, and buffers (e.g. acetate, citrate, Tris buffers, such as a buffer chosen from one or more of: 25% PEG4000, 0.1M Sodium Acetate, 0.2 M Ammonium Sulfate; 20% PEG 3350, 0.2 M Ammonium Formate; 24.5% PEG 3350, 0.1 5 M bis-tris buffer at pH 6.5; 20% PEG 3350, 0.2 Ammonium Phosphate; 20% PEG 3350, 0.2 Ammonium Fluoride; or 20% PEG 3350, 0.2 di-Ammonium citrate).
  • buffers e.g. acetate, citrate, Tris buffers, such as a buffer chosen from one or more of: 25% PEG4000, 0.1M Sodium Acetate, 0.2 M Ammonium Sulfate; 20% PEG 3350, 0.2 M Ammonium Formate; 24.5% PEG 3350, 0.1 5
  • Crystals can be grown by various methods, such as, for example, sitting or hanging drop vapor diffusion.
  • crystallization can be performed at a temperature of from about 4°C to about 60 0 C (e.g., from about 10 0 C to about 45°C, such as at about 12°C, about 15°C, about o 18°C, about 20 0 C, about 25°C, about 30 0 C, about 32°C, about 35°C, about 37°C).
  • a crystal of the human AMCase polypeptide can diffract X-rays to a resolution of about 3.7 A or less (e.g., about 3.6 A or less, about 3.5 A or less, about 3.2 A or less, about 3.0 A or less, about 2.7 A or less, about 2.4 A or less, about 2.3 A or less, about 2.2 A or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.85 A or less, about 1.7 A or less, about 1.6 A or less, about 1.5 A or less, or about 1.4 A or less).
  • a crystal of the human AMCase polypeptide can diffract X- rays to a resolution of from about 1.7 A to about 3.7 A (e.g., the crystal of the human AMCase polypeptide can diffract X-rays to about 2.7 A, about 3.5 A or about 3.6 A).
  • the crystal of the human AMCase polypeptide belongs to0 space group chosen from P2i or V1 ⁇ 1 ⁇ 1 ⁇ .
  • the space group refers to the overall symmetry of the crystal, and includes point symmetry and space symmetry.
  • a crystal of the human AMCase polypeptide belongs to space group P2i and contains four molecules of the human AMCase polypeptide in the asymmetric unit.
  • a crystal of the 5 human AMCase polypeptide belongs to space group P2i2i2 and contains two molecules of the human AMCase polypeptide in the asymmetric unit.
  • the asymmetric unit is the smallest unit from which the crystal structure can be generated by making use of the symmetry operations of the space group.
  • a crystal is generally made up of the motif defined by the space-group symmetry operations on the asymmetric units, and a o translation of that motif through the crystal lattice.
  • Structural data describing a crystal can be obtained, for example, by X-ray diffraction and phases.
  • X-ray diffraction data can be collected by a variety of sources, X- ray wavelengths and detectors.
  • rotating anodes and synchrotron sources e.g., Advanced Light Source (ALS), Berkeley, California; or Advanced Photon5 Source (APS), Argonne, Illinois
  • ALS Advanced Light Source
  • APS Advanced Photon5 Source
  • Argonne, Illinois can be used as the source(s) of X-rays.
  • X-rays for generating diffraction data can have a wavelength of from about 0.5 A to about 1.6 A (e.g., about 0.7 A, about 0.9 A, about 1.0 A, about 1.1 A, about 1.3 A, about 1.4 A, about 1.5 A, or about 1.6 A).
  • area detectors and/or charge-couple devices can be used as the detector(s).
  • 0 X-ray diffraction data of a crystal of the human AMCase polypeptide can be used together with phases to obtain the structural coordinates of the atoms in the crystal.
  • the structural coordinates are Cartesian coordinates that describe the location of atoms in three-dimensional space in relation to other atoms in the polypeptide or complex.
  • the structural coordinates listed in Table 1 are the structural coordinates of a5 crystalline human AMCase polypeptide.
  • the structural coordinates listed in Table 2 are the structural coordinates of a crystalline methylallosamidin-bound human AMCase polypeptide.
  • the structural coordinates of Table 1 describe the location of atoms of the human AMCase polypeptide in relation to each other.
  • the structural coordinates can be modified by mathematical manipulation, such as by inversion or0 integer additions or subtractions.
  • structural coordinates are relative coordinates.
  • structural coordinates describing the location of atoms in the human W2025-7029WO / AM102903-PCT are relative coordinates.
  • AMCase polypeptide are not specifically limited by the actual x, y, and z coordinates of Table 1.
  • structural coordinates describing the location of atoms in the methylallosamidin-bound human AMCase polypeptide are not specifically limited by the actual x, y, and z coordinates of Table 2.
  • the structural coordinates of the human AMCase polypeptide can be used to derive a representation (e.g., a two dimensional representation or three dimensional representation) of the polypeptide or a fragment of the polypeptide.
  • a representation e.g., a two dimensional representation or three dimensional representation
  • Such representations can be useful for a number of applications, including, for example, the visualization, identification and characterization of an active site of the polypeptide.
  • a three-dimensional representation can include the structural coordinates of the human AMCase polypeptide according to Table 1, + a root mean square (rms) deviation from the alpha carbon atoms of amino acids of not more than about 1.5 A (e.g., not more than about 1.0 A, not more than about 0.5 A).
  • a three- dimensional representation can include the structural coordinates of the methylallosamidin-bound human AMCase polypeptide according to Table 2, + a root mean square (rms) deviation from the alpha carbon atoms of amino acids of not more than about 1.5 A (e.g., not more than about 1.0 A, not more than about 0.5 A)
  • Root mean square deviation is the square root of the arithmetic mean of the squares of the deviations from the mean, and is a way of expressing deviation or variation from structural coordinates.
  • Conservative substitutions of amino acid residues can result in a molecular model having structural coordinates within the stated root mean square deviation.
  • two molecular models of polypeptides that differ from one another by conservative substitutions can have coordinates of backbone atoms within a stated rms deviation, such as less than 1.5 A, less than 1.0 A, or less than 0.5 A.
  • Backbone atoms of a polypeptide include the alpha carbon (C ⁇ or CA) atoms, carbonyl carbon (C) atoms, carbonyl oxygen (O) atoms, and amide nitrogen (N) atoms.
  • C ⁇ or CA alpha carbon
  • C carbonyl carbon
  • O carbonyl oxygen
  • N amide nitrogen
  • the rmsd between uncomplexed AMCase and methylallosamidin-bound AMCase (C ⁇ ) is 1.063.
  • the rmsd between uncomplexed AMCase and uncomplexed human chitriosidase (C ⁇ ) is 0.0664.
  • the numbering of the amino acid residues of AMCase may be different than set forth here, and may contain certain conservative amino acid substitutions, additions or W2025-7029WO / AM102903-PCT
  • deletions that yield the same three-dimensional structures as those defined by Table 1 or 2, + an rmsd for backbone atoms of less than 1.5 A.
  • Corresponding amino acids and conservative substitutions in other isoforms or analogs are easily identified by visual inspection of the relevant amino acid sequences or by using commercially available homology software programs (e.g., MODELLAR, MSI, San Diego, CA).
  • An isoform is any of several multiple forms of a protein that differ in their primary structure.
  • An analog is a polypeptide having conservative amino acid substitutions.
  • the representation is a two-dimensional figure, such as a stereoscopic two- dimensional figure.
  • the representation is an interactive two- dimensional display, such as an interactive stereoscopic two-dimensional display.
  • An interactive two-dimensional display can be, for example, a computer display that can be rotated to show different faces of a polypeptide or a fragment of a polypeptide.
  • the representation is a three-dimensional representation.
  • a three-dimensional model can be a physical model of a molecular structure (e.g., a ball- and-stick model).
  • a three dimensional representation can be a graphical representation of a molecular structure (e.g., a drawing or a figure presented on a computer display).
  • a two-dimensional graphical representation e.g., a drawing
  • a representation can be modeled at more than one level.
  • the three-dimensional representation includes a polypeptide, such as a human AMCase polypeptide
  • the polypeptide can be represented at one or more different levels of structure, such as primary (amino acid sequence), secondary (e.g., I-helices and ⁇ -sheets), tertiary (overall fold), and quaternary (oligomerization state) structure.
  • primary amino acid sequence
  • secondary e.g., I-helices and ⁇ -sheets
  • tertiary overall fold
  • quaternary oligomerization state
  • representation includes a polypeptide and an inhibitor, such as a methylallosamidin- bound human AMCase polypeptide
  • the polypeptide can be represented at one or more different levels of structure, such as primary (amino acid sequence), secondary (e.g., I- helices and ⁇ -sheets), tertiary (overall fold), and quaternary (oligomerization state) structure
  • the inhibitor can be represented as ball-and-stick, space-filling, or pharmacophores.
  • a representation can include different levels of detail.
  • the representation can include the relative locations of secondary structural features of a protein without specifying the positions of atoms.
  • a more detailed representation could, for example, include the positions of atoms.
  • a representation can include information in addition to the structural coordinates of the atoms in the human AMCase polypeptide or in an inhibitor- bound human AMCase polypeptide, e.g. methylallosamidin-bound human AMCase polypeptide.
  • a representation can provide information regarding the shape of a solvent accessible surface, the van der Waals radii of the atoms of the model, and the van der Waals radius of a solvent (e.g., water).
  • Other features that can be derived from a representation include, for example, electrostatic potential, the location of voids or pockets within a macromolecular structure, and the location of hydrogen bonds and salt bridges.
  • X-ray crystallography can be used to obtain structural coordinates of the human AMCase polypeptide and inhibitor-bound human AMCase polypeptide, e.g. methylallosamidin-bound human AMCase polypeptide.
  • structural coordinates can be obtained using other techniques including NMR techniques.
  • Some structural information can also be obtained from spectral techniques (e.g., optical rotary dispersion (ORD), circular dichroism (CD)), homology modeling, and computational methods (e.g., computational methods that can include data from molecular mechanics, computational methods that include data from dynamics assays).
  • the X-ray diffraction data together with its phases can be used to construct an electron density map of the human AMCase polypeptide or a fragment of the polypeptide, and the electron density map can be used to derive a representation (e.g., a two dimensional representation, a three dimensional representation) of the human AMCase polypeptide.
  • a representation e.g., a two dimensional representation, a three dimensional representation
  • Phase information can be extracted, for example, either from the diffraction data or from supplementing diffraction experiments to complete the construction of the electron density map.
  • Methods for calculating phase from X-ray diffraction data include, for example, multiwavelength anomalous dispersion (MAD), multiple isomorphous replacement (MIR), multiple isomorphous replacement with anomalous scattering (MIRAS), single isomorphous replacement with anomalous scattering (SIRAS), or any combination thereof.
  • phase information by making isomorphous structural modifications to the native protein, such as by including a heavy atom or changing the scattering strength of a heavy atom already present, and then measuring the diffraction amplitudes for the native protein and each of the modified cases. If the position of the additional heavy atom or the change in its scattering strength is known, then the phase of each diffracted X-ray can be determined by solving a set of simultaneous phase equations.
  • the location of heavy atom sites can be identified using a computer program, such as SHELXD (Bruker-AXS, Madison, WI) or SHELXS (Sheldrick, Institut Anorg.
  • the phases of the diffracted X-rays of crystalline human AMCase polypeptide can be obtained by molecular replacement techniques, such as AMORE, with a starting model based on a similar protein of the glycohydrolase family 18, e.g. human chitotriosidase, and refined against the observed structure factors.
  • the resulting model can then be used to solve a different crystal structure of human AMCase poylpeptide and/or methylallosamidin-bound human AMCase polypeptide and/or inhibitor-bound human AMCase polypeptide by molecular replacement.
  • a model derived from a crystalline human AMCase polypeptide having space group P2i can be used as a search model to solve a crystalline methylallosamidin-bound human AMCase polypeptide having space group P2 1 2 1 2 1 .
  • a model derived from W2025-7029WO / AM102903-PCT can be used as a search model to solve a crystalline methylallosamidin-bound human AMCase polypeptide having space group P2 1 2 1 2 1 .
  • either crystalline human AMCase polypeptide having space group P2i or a crystalline methylallosamidin-bound human AMCase polypeptide having space group P2 1 2 1 2 1 can be used as a search model to solve by molecular replacement techniques the crystal structure of an inhibitor-bound human AMCase polypeptide in any space group available to molecules that have at least one chiral atom, e.g.
  • the electron density map can be used to derive a representation of a polypeptide or a fragment of a polypeptide by aligning a three- dimensional model of a polypeptide with the electron density map.
  • the electron density map corresponding to the native crystalline human AMCase polypeptide can be aligned with the electron density map corresponding to the platinum derivative of the crystalline human AMCase polypeptide complex derived by an isomorphous replacement method.
  • the alignment process results in a comparative model.
  • the comparative model is then refined over one or more cycles (e.g., two cycles, three cycles, four cycles, five cycles, six cycles, seven cycles, eight cycles, nine cycles, ten cycles) to generate a better fit with the electron density map.
  • Software programs such as CNS (Brunger et al, Acta Crystallogr. D54:905-921, 1998) and REFMAC (Collaborative Computational Project, Number 4.
  • the CCP4 suite Programs for Protein Crystallography, Acta Crystallogr. D50:760-776, 1994
  • the quality of fit in the comparative model can be measured by, for example, an Rf ac tor or Rf ree value.
  • a smaller value of Rfactor or Rf ree generally indicates a better fit.
  • Misalignments in the comparative model can be adjusted to provide a modified comparative model and a lower Rfactor or R free value.
  • the adjustments can be based on information relating to variations of the human AMCase polypeptide (e.g., sequence variation information, alternative structure information, heavy atom derivative information) as appropriate.
  • sequence variation information e.g., sequence variation information, alternative structure information, heavy atom derivative information
  • an adjustment can include fitting an approximate model of W2025-7029WO / AM102903-PCT
  • an adjustment can include replacing an amino acid in the previously known human AMCase polypeptide with an amino acid having a similar structure (a conservative amino acid change).
  • the resulting model is that which is determined to describe the polypeptide or complex from which the X-ray data was derived.
  • a machine such as a computer, can be programmed in memory with the structural coordinates of the human AMCase polypeptide together with a program capable of generating a graphical representation of the structural coordinates on a display connected to the machine.
  • a software system can also be designed and/or utilized to accept and store the structural coordinates.
  • the software system can be capable of generating a graphical representation of the structural coordinates.
  • the software system can also be capable of accessing external databases to identify one or more candidate agents likely to interact with the human AMCase polypeptide.
  • a machine having a memory containing structure data or a software system containing such data can aid in the rational design or selection of a human AMCase polypeptide agonist of a human AMCase polypeptide antagonist.
  • such a machine or software system can aid in the evaluation of the ability of an agent to associate with the human AMCase polypeptide, or can aid in the modeling of compounds or proteins related by structural or sequence homology to the human AMCase polypeptide.
  • an agonist refers to a compound that enhances at least one activity of the human AMCase polypeptide
  • an antagonist refers to a compound that inhibits or counteracts at least one activity of the human AMCase polypeptide.
  • a compound may function as an antagonist of the human AMCase polypeptide by, for example, increasing the rate of myelin production by a neural cell, or by inhibiting interaction of the human AMCase polypeptide with the Nogo receptor complex.
  • W2025-7029WO / AM102903-PCT W2025-7029WO / AM102903-PCT
  • the machine can produce a representation (e.g., a two dimensional representation, a three dimensional representation) of the human AMCase polypeptide or a fragment of the polypeptide.
  • a software system for example, can cause the machine to produce such information.
  • the machine can include a machine-readable data storage medium including a data storage material encoded with machine-readable data.
  • the machine- readable data can include structural coordinates of atoms of the human AMCase polypeptide.
  • Machine-readable storage media include, for example, conventional computer hard drives, floppy disks, DAT tape, CD-ROM, DVD, and other magnetic, magneto-optical, optical, and other media which may be adapted for use with a machine (e.g., a computer).
  • the machine can also have a working memory for storing instructions for processing the machine-readable data, as well as a central processing unit (CPU) coupled to the working memory and to the machine-readable data storage medium for the purpose of processing the machine-readable data into the desired three-dimensional representation.
  • CPU central processing unit
  • a display can be connected to the CPU so that the three-dimensional representation can be visualized by the user.
  • the machine when used with a machine programmed with instructions for using the data (e.g., a computer loaded with one or more programs of the sort described herein) the machine is capable of displaying a graphical representation (e.g., a two dimensional graphical representation, a three-dimensional graphical representation) of any of the polypeptides, polypeptide fragments, complexes, or complex fragments described herein.
  • a graphical representation e.g., a two dimensional graphical representation, a three-dimensional graphical representation
  • a display e.g., a computer display
  • the X-ray coordinates of the human AMCase ligand binding domain as shown in Tables 1 and 2 can be transformed into a visual depiction, e.g., a three-dimensional representation or model.
  • Such representation or model can be visualized, e.g., electronically in a computer. The user can inspect the representation and, using information gained from the representation, generate a model of the human AMCase polypeptide or polypeptide fragment bound to a inhibitor.
  • the model can be generated, for example, by altering a previously existing representation of the human AMCase polypeptide.
  • the user can superimpose a three-dimensional model of an agent on the representation of the human AMCase W2025-7029WO / AM102903-PCT
  • the agent can be an agonist (e.g., a candidate agonist) of the human AMCase polypeptide.
  • the agent can be a known compound or a fragment of a known compound.
  • the agent can be a previously unknown compound, or a fragment of a previously unknown compound.
  • the agent, alone or complexed with the AMCase polypeptide can be displayed, e.g., in a computer. It can be desirable for the agent to have a shape that complements the shape of the active site. There can be a preferred distance, or range of distances, between atoms of the agent and atoms of the human AMCase polypeptide.
  • Distances longer than a preferred distance may be associated with a weak interaction between the agent and active site (e.g., the active site of the human AMCase polypeptide). Distances shorter than a preferred distance may be associated with repulsive forces that can weaken the interaction between the agent and the polypeptide.
  • a steric clash can occur when distances between atoms are too short. A steric clash occurs when the locations of two atoms are unreasonably close together, for example, when two atoms are separated by a distance less than the sum of their van der Waals radii.
  • the user can adjust the position of the agent relative to the human AMCase polypeptide (e.g., a rigid body translation or rotation of the agent) until the steric clash is relieved.
  • the user can adjust the conformation of the agent or of the human AMCase polypeptide in the vicinity of the agent in order to relieve a steric clash.
  • Steric clashes can also be removed by altering the structure of the agent, for example, by changing a "bulky group," such as an aromatic ring, to a smaller group, such as to a methyl or hydroxyl group, or by changing a rigid group to a flexible group that can accommodate a conformation that does not produce a steric clash.
  • Electrostatic forces can also influence an interaction between an agent and an inhibitor-binding domain.
  • electrostatic properties can be associated with repulsive forces that can weaken the interaction between the agent and the human AMCase polypeptide.
  • Electrostatic repulsion can be relieved by altering the charge of the agent, e.g., by replacing a positively charged group with a neutral group.
  • Forces that influence binding strength between a candidate agent and the human AMCase polypeptide, respectively, can be evaluated in the polypeptide/agent model. These can include, for example, hydrogen bonding, electrostatic forces, hydrophobic interactions, van der Waals interactions, dipole-dipole interactions, ⁇ -stacking forces, and W2025-7029WO / AM102903-PCT
  • the user can evaluate these forces visually, for example by noting a hydrogen bond donor/acceptor pair arranged with a distance and angle suitable for a hydrogen bond. Based on the evaluation, the user can alter the model to find a more favorable interaction between the human AMCase polypeptide and the agent.
  • Altering the model can include changing the three-dimensional structure of the polypeptide without altering its chemical structure, for example by altering the conformation of amino acid side chains or backbone dihedral angles.
  • Altering the model can include altering the position or conformation of the agent, as described above.
  • Altering the model can also include altering the chemical structure of the agent, for example by substituting, adding, or removing groups. For example, if a hydrogen bond donor on the human AMCase polypeptide is located near a hydrogen bond donor on the agent, the user can replace the hydrogen bond donor on the agent with a hydrogen bond acceptor.
  • the relative locations of an agent and the human AMCase polypeptide, or their conformations, can be adjusted to find an optimized binding geometry for a particular agent to the human AMCase polypeptide.
  • An optimized binding geometry is characterized by, for example, favorable hydrogen bond distances and angles, maximal electrostatic attractions, minimal electrostatic repulsions, the sequestration of hydrophobic moieties away from an aqueous environment, and the absence of steric clashes.
  • the optimized geometry can have the lowest calculated energy of a family of possible geometries for the human AMCase polypeptide/agent complex.
  • An optimized geometry can be determined, for example, through molecular mechanics or molecular dynamics calculations.
  • a series of representations of the human AMCase polypeptide having different bound agents can be generated.
  • a score can be calculated for each representation.
  • the score can describe, for example, an expected strength of interaction between the human AMCase polypeptide and the agent.
  • the score can reflect one of the factors described above that influence binding strength.
  • the score can be an aggregate score that reflects more than one of the factors.
  • the different agents can be ranked according to their scores. Steps in the design of the agent can be carried out in an automated fashion by a machine.
  • a representation of the human AMCase polypeptide can be W2025-7029WO / AM102903-PCT
  • the machine can find an optimized binding geometry for each of the candidate agents to an active site, and calculate a score to determine which of the agents in the series is likely to interact most strongly with the human AMCase polypeptide.
  • a software system can be designed and/or implemented to facilitate these steps.
  • Software systems used to generate representations or perform the fitting analyses include, for example: MCSS, Ludi, QUANTA® (macromolecular X- ray crystallography software), Insight II® (biological compound modeling and simulation software), Cerius ® (modeling and simulation software), CHARMm® (software for simulation of biological macromolecules), and Modeler from Accelrys, Inc. (San Diego, CA); SYBYL® (molecular modeling software), Unity, FIeXX, and LEAPFROG from TRIPOS, Inc. (St.
  • the structural coordinates can also be used to visualize the three-dimensional structure of the human AMCase polypeptide using MOLSCRIPT, RASTER3D, or PYMOL (Kraulis, /. Appl. Crystallogr. 24: 946-950, 1991; Bacon and Anderson, /. MoI. Graph. 6: 219-220, 1998; DeLano, The PYMOL Molecular Graphics System (2002) DeLano Scientific, San Carlos, CA).
  • the inhibitor can, for example, be selected by screening an appropriate database, can be designed de novo by analyzing the steric configurations and charge potentials of a methylallosamidin-bound human AMCase polypeptide and/or human AMCase polypeptide, in conjunction with the appropriate software systems, and/or can be designed using characteristics of known inhibitors, such methylallosamidin, as revealed by the methylallosamidin-bound human AMCase polypeptide crystal structure.
  • the method can be used to design or select inhibitors of the human AMCase polypeptide.
  • software system can be designed and/or implemented to facilitate database searching, and/or agent selection and design.
  • an agent Once an agent has been designed or identified, it can be obtained or synthesized and further evaluated for its effect on the activity of the human AMCase polypeptide.
  • the agent can be evaluated by contacting it with the human AMCase polypeptide and measuring the effect of the agent on polypeptide activity.
  • a method for evaluating the agent can include an activity assay performed in vitro or in vivo.
  • an activity of an AMCase can be assayed as follows.
  • the sample e. g., tissue, cell culture, or amount of AMCase
  • a test agent for a time period sufficient to inhibit the activity or expression of the chitinase. This time period may vary depending on the nature of the test agent, the AMCase, the activity or expression detection method selected, and the sample tissue selected. The skilled artisan without undue experimentation may readily determine such times.
  • An exemplary test agent is one that binds to or otherwise decreases the catalytic activity of an AMCase, although test agents that inhibit AMCase activity or expression by, for example, binding to the substrate for AMCase, or binding to a component of the signal pathway, such as IL- 13R, IL-4R or IL- 13 are also envisioned.
  • An exemplary substrate for chitinase is chitin, although derivatives thereof that may participate in a reaction catalyzed by chitinase are also encompassed by the invention.
  • assays may be utilized to determine whether the candidate agent inhibits the activity of the AMCase.
  • the amount of reactants remaining and/or products formed in reactions catalyzed by AMCase may be quantified.
  • a non- limiting example of such a reaction is the conversion of chitin or a chitin-like compound to N-acetyl-D-glucosamine.
  • Other reactions include, without limitation, the release of 4- methylumbelliferyl from 4-methylumbelliferyl- tri-N-acetyl chitotrioxide, 4- methylumbelliferyl -D-N, N'-diacetylchitobiose or 4-methylumbelliferyl -D-N, N', N"- triacetylchitotriose; or the release of p- nitrophenylfrom p-nitrophenyl P-D-O-D-N, N'- diacetylchitobiose or p-nitrophenyl -D-N,N', N"-triacetylchitotriose.
  • the amount of chitin remaining after contacting AMCase with the test agent as a function of time may be determined.
  • the amount of N-acetyl-D-glucosamine or 4- methylumbelliferyl or p-nitrophenyl produced after contacting chitinase with the test W2025-7029WO / AM102903-PCT may be determined.
  • a agent in the presence of, for example, chitin as a function of time may be determined.
  • Various assays may be used to determine the quantity of these products and/or reactants.
  • colorimetric assays may be utilized to determine the quantity of N-acetyl- D-glucosamine as described in, for example, Reissig, J. L.,J. Biol.Chers. 217:959-966, 1955.
  • the amount of glucosamine may be determined by chromatographic methods known to the skilled artisan, including high performance liquid chromatography, as described in, for example, Ekblad, A. (1996) Plant and Soil 178:29-35.
  • Fluorometric assays may be utilized to determine the quantity of 4- methylumbelliferylorp-nitrophenyl as described in, for example, U. S. Patent No. 5,561, 051 (Silverman); Houston, D. R., et ai, PNAS 99:9127-9232, 2002; Hollak, C. E. M., et al, J. Clin. Invest. 93: 1288-1292, 1994; and Hu et al, J. Biol. Chem. 271: 19415-194520.
  • Methods of quantitating chitin are known to the art, including use of various immunoassays, such as enzyme-linked immunosorbents assays.
  • candidate agents may be tested in the screening methods of the present invention.
  • small molecule compounds known in the art, synthetic small molecule chemicals, nucleic acids such as antisense oligonucleotides, RNA inhibitors such as siRNA, ribozymes, and aptamers, peptides and proteins such as hormones, antibodies, and portions thereof, may act as candidate agents.
  • the candidate agents of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-pep tide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al. (1994) /. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection.
  • Example 1 Crystallization and Structural Resolution of Human AMCase
  • AMCase Human acidic mammalian chitinase
  • the X-ray structures of a human AMCase polypeptide and an inhibitor-bound form of human AMCase human AMCase polypeptide were determined to a resolution of 2.0 A and 1.7 A, respectively (see Example 2).
  • Example 3 summarizes crystallization conditions and crystal space unit and parameters for four additional complexes of human AMCase with four different compounds. Experimental conditions used in expressing and crystallizing the human AMCase are provided in more detail herein. W2025-7029WO / AM102903-PCT
  • a nucleotide sequence encoding truncated human AMCase mutated at position 354 to replace a serine by a phenylalanine (S354F) was cloned into pTMED vector.
  • a histidine tag is attached at the C-terminal end to facilitate protein purification.
  • the vector was expressed in CHO cells. Culture conditions for the CHO cells are provided in more detail below.
  • the nucleotide and amino acid sequence of the truncated human AMCase protein used for the crystallization (SEQ ID NO:1 and SEQ ID NO: 2, respectively) has the following nucleotide and amino acid sequences:
  • the S354 mutated human AMCase was generated because the cloned human cDNA had a serine residue at position 354, which resulted in an N-linked glycosylation site.
  • position 354 is a polymorphism, and that a phenylalanine at that position is at least as prevalent as the S. Therefore, when the truncated form was made for crystallization, the serine was changed to a phenylalanine at position 354 in order to avoid an N-linked glycosylated protein.
  • a Chinese Hamster Ovary cell line (CHO PA-DUKX) was used to express the truncated human AMCase polypeptide, with an N-terminal HBML signal sequence and a C-terminal 6-His-tag (SEQ ID NO: 7).
  • the cell line was maintained in alpha media with 10% heat inactivated and dialyzed FBS, 2mM Glutamine, 100 U/ml Penicillin, 100ug/ml Streptomycin, 2.5ug/ml Fungizone and 20OnM Methotrexate. Cells were then scaled up and seeded in roller bottles (1700 sq. cm) in growth media. When the cells reached confluence, the media was replaced with R5CD1 serum- free media. Conditioned media was collected every 48-72 hrs.
  • the truncated human AMCase was purified from serum- free CHO conditioned media using Ni-NTA Superflow resin (Qiagen Inc., Valencia, CA). One liter of media was applied, batch-wise, to 6 mL Ni-NTA resin at 4 0 C within 1 hr. Unbound protein was washed away using wash buffer (50 mM Tris, pH 8.0, 0.3 M NaCl, and 10 mM Imidazole). Human AMCase was then eluted using wash buffer plus 200 mM Imidazole. Human AMCase was further purified by Superdex-200 (Amersham Biosciences Inc.,
  • Methylallosamidin production and isolation A strain of streptomyces was grown in 10-L fermentation vessels on a medium containing nutrasoy (12.5 g/L), dextrose (12.5 g/L), N-Z amine (12.5 g/L), NH 4 Cl (1.5 g/L), and CaCl 2 (1.0 g/L) with the pH adjusted to 6.8, to produce mainly methylallosamidin and other allosamidins (Sakuda et al. (1987) /. Antibio. (Tokyo) 40(3):296-300) at titers of - 5mg/L after 5 days. The titers of the allosamidins and the progress of methylallosamidin isolation and purification were followed by LCMS using positive ion ms-peaks in the 609 to 637 range.
  • the fermentation batch was stored at 4 0 C for 3 to 5 days following fermentation.
  • the mash was then centrifuged to remove the pellet cell paste.
  • the pH of the supernatant was subsequently adjusted to pH 9.2 using NH 4 OH and passed over an open HP-20 column (4 x 36 cm) and the column was washed with 2 L of 0.01 m NH 4 OH.
  • a methylallosamidin- rich fraction was then eluted from the column using 1 L of 0.01 m CH 3 COOH solution containing 20% methanol. This fraction was concentrated under vacuum to remove methanol.
  • AMCase purified as above and concentrated to 21 mg/ml in 25 mM Tris, pH 7.5, 50 mM NaCl, and well solution (15% PEG 3350, 120 mM Ammonium Sulfate, 60 mM Na Acetate pH 4.6) were mixed in the hanging drop. Rectangular crystals at 18°C grew in about one week, and generally measured -80 microns, or micrometers across. Crystals of human AMCase polypeptide, grown in the absence of exogenously added inhibitor, were drawn through a solution of 25% glycerol and 75% well solution, and cooled rapidly in liquid nitrogen.
  • the amino acid range visible and modeled for the apo AMCase corresponded to amino acids 22-400 of the full length human AMCase sequence for all 4 molecules of the apo structure, with the exception of amino acids 231 to 236 of chain B.
  • the crystal structure of human AMCase polypeptide was determined by molecular replacement, using the protein model of the human chitotriosidase (Fusetti et al. (2002) Journal of Biological Chemistry 277(28):25537-25544) as the search model, PDB ID code IGUV (all atoms were used not just C alpha backbone).
  • Example 2 Crystallization and Resolution of Methylallosamidin-bound Human AMCase Co-crystals of human AMCase polypeptide and methylallosamidin were grown by hanging drop vapor diffusion at 18°C in drops containing 0.2 ⁇ l protein stock solution (21 mg/ml protein, 25 mM Tris pH 7.5, 50 mM NaCl, 1 mM methylallosamidin) and mixed with 0.2 ⁇ l well solution (20% PEG 3350, 200 mM Ammonium Formate) and equilibrated against 0.2 mL well solution. Slate-like crystals grew in about 1 week, and generally measured -80 microns, or micrometers, across.
  • Co-crystals of human AMCase polypeptide and methylallosamidin were drawn through a solution of 25% glycerol and 75% well solution, and cooled rapidly in liquid nitrogen. Diffraction data were recorded at Advanced Light Source Beamline 5.0.1 on a q-210 ccd camera (Berkeley, CA). Intensities were integrated and scaled using the programs Denzo and Scalepack (Otwinowski and Minor, 1997).
  • amino acid range visible and modeled for the methylallosamidin-bound AMCase corresponded to amino acids 22-398 for both molecules A and B, as well as for the bound methylallosamidin.
  • the crystal structure of the co-crystals of human AMCase polypeptide and methylallosamidin was solved by molecular replacement using the crystal structure of the human AMCase polypeptide alone.
  • the molecular replacement solution was refined using CNX (Brunger et al, 1998) and Refmac5 (CCP4), using Coot (Emsley, 2004) for model refinement.
  • the final R work and Rf ree values were 17.48 % and 19.39 % for methylallosamidin-bound human AMCase polypeptide (see Table 4).
  • Angles (°) 1.058 a Rmerge Hh - ⁇ Ih>l / Ih, where ⁇ Ih> is the average intensity over symmetry equivalents. Numbers in parentheses reflect statistics for the last shell.
  • Rwork 11 Fobs I " IFcalcll / I Fobs I c R f ree is equivalent to R WOrk , but calculated for a randomly chosen 5% of reflections omitted from the refinement process.
  • Beta-chitopentose with central NAG unit in a boat-like conformation mimicking the transition state was then minimized using eMBrAcE with the OPLS force field (Jorgensen et al. (1996) Journal of the American Chemical Society 118(45): 11225-11236) and implicit water solvent (Ghosh et al. (1998) /. Phys. Chem. B. 102: 10983-10990).
  • the substrate and all residues with an atom within 3 A of the ligand were subject to up to 1000 steps of full minimization, the next shell of residues within 5 A of the ligand was subject to harmonic constraints during the minimization, and a second shell of residues within 7 A were fixed.
  • a large excess of DEPC, 200 microM was added to 1 microM AMCase and allowed to incubate for 10 to 15 minutes in a citric acid/phosphate buffer pH 6.9.
  • the spectrum of unlabeled AMCase was compared to spectrum of labeled AMCase.
  • a spectral change at 278 nm represents modifications of tyrosines while a spectral change at 242 nm represents modified histidines.
  • Modified His has extinction coefficient of 3.2 xlO 3 m "1 cm " .
  • the change in absorbance at 242 nm is 0.059 AU, corresponding to 18 microM His labeled.
  • AMCase The activity of AMCase was determined after incubation with varying amounts of DEPC (0 to 1000 microM). Inactivation was done at pH 6.9 with 0.75 microM AMCase in which the reaction was quenched by dilution (1:200) in buffer pH 6.0. Activity was measured with 30 microM substrate and change in activity (Ao/A) was plotted against ratio of DEPC to AMCase ([DEPC]/[AMCase]). Reaction time was 5 and 15 min. This W2025-7029WO / AM102903-PCT
  • the pK a of the imidazole of His is 6.9, therefore the pH dependence of inactivation should mirror the pK a of His.
  • the inactivation assay was done with 0.75 microM AMCase and 200 microM DEPC at varying pH from 5.7 to 7.8 and quenched at different times from 0 to 15 min in assay buffer pH 6.0.
  • the pK a determined was 6.9 + 0.1, which corresponds to a His.
  • the kinetic constants of the di-sugar substrate were determined for labeled and 5 non-labeled AMCase. This will show what effect the labeling of AMCase has on the enzyme.
  • 10 microM DEPC was used with 0.75 microM AMCase. The reaction was quenched by dilution and the kinetics was determined. Resulting data was plotted in a double-recipical plot and shows that there is an effect seen on V max but not on K 1n .
  • PH rate profile Labeled versus unlabeled AMCase.
  • the pH dependence of the kinetic parameters was determined for the labeled and5 non-labeled AMCase.
  • the samples were treated the same with the exception of the addition of 250 microM DEPC in the inactivation assay.
  • the inactivation assay was performed at pH 6.9 and then quenched by dilution into buffer at varying pH (pH 2.8 to 7.3).
  • the steady state kinetic parameters were then determined at each pH and the log V max and log V/K were plotted against pH. There appears to be a "hollow" in the logV/K0 vs pH plot when the enzyme is labeled.
  • the hollow appears on the ascending limb and typically suggest substrate stickiness.
  • Co-crystals of Compound 1 and AMCase were generated in 24.5% PEG 3350, 0.1 M solution of bis-tris pH 6.5. These co-crystals belong to space group V1 ⁇ l ⁇ ⁇ ⁇ and have o unit cell parameters 45.2 x 81.5 x 91.9. They contain one molecule of Compound 1- bound human AMCase polypeptide in the asymmetric unit.
  • Co-crystals of Compound 3 and AMCase were generated in 20% PEG 3350, 0.2 M solution of Ammonium Fluoride. These co-crystals belong to space group V1 ⁇ l ⁇ ⁇ and have unit cell parameters 45.3 x 81.9 x 91.7. They contain one molecule of Compound 3- bound human AMCase polypeptide in the asymmetric unit. 0 Co-crystals of Compound 4 and AMCase were generated in 20% PEG 3350, 0.2
  • Figure 5 provides crystallization conditions, and space groups and unit5 cell parameters for human AMCase in uncomplexed form (apo) and five ligand-bound forms of AMCase.
  • AMCase was determined to a resolution of 2.0 A in the apo form and W2025-7029WO / AM102903-PCT
  • the construct that was successfully crystallized was a C- terminal truncation including the catalytic domain.
  • the Ser residue originally at position 354 was mutated to Phe, a polymorphism that is at least as common as the Ser, to avoid N-linked glycosylation.
  • the apo protein crystallized in space group P2i, and contains four molecules of AMCase in the asymmetric unit, implying a solvent content of 39.5%.
  • the resulting map showed good electron density for the residues 22 to 400 (monomer A, B, C, and D, with the exception of AA 231-236 in molecule B).
  • Residues C-terminal to 400, including the His tag, are not seen in the electron density maps and are presumably disordered.
  • AMCase in complex with methylallosamidin crystallized in space group ⁇ 2 ⁇ l ⁇ l ⁇ and contains two molecules of AMCase in the asymmetric unit, implying a solvent content of 39.7%.
  • the resulting map shows good electron density for residues 22 through 398 in both molecules A and B, as well as for the bound methylallosamidin ( Figure 4).
  • Residues C-terminal to 398, including the His tag are not seen in the electron density maps and are presumably disordered
  • the structures of AMCase alone and in complex with the inhibitor methylallosamidin reveal an eight stranded beta-barrel structure (TIM-barrel) with an alpha/beta lobe inserted between beta-sheet 7 and alpha-helix I, that is composed of a 5- stranded anti-parallel beta sheet and an alpha-helix (Figure 3A).
  • the overall tertiary structure is very similar to the structure of the related human chitotriosidase (Fusetti et al. 2002 /. Biol. Chem. 277(28): 25537-44; Rao et al. 2003 /. Biol. Chem.
  • Allosamidin and its variant methylallosamidin are broad-spectrum natural product inhibitors. They are pseudo-trisaccharides with two NAG sugars coupled to allosamizoline.
  • Glu-140 Furthermore there is a rotation of the Glu-140 side chain from the apo-structure where it is pointing towards the main chain N of Trp-99 (3.3 A) and the side chain hydroxyl of Tyr-141.
  • Glu-140 Upon binding of methylallosamidin, Glu-140 rotates about 60 degrees to form hydrogen-bonds with Asp-138 and with the allosamizoline N. It is the protonation of Glu-140 that is the final step of chitin hydrolysis.
  • the interaction energy of beta-chitopentose with AMCase is 2.95 kcal/mol more favorable than that of the alpha-chitopentose.
  • DEPC diethyl pyrocarbonate
  • AMCase and AMCase treated with DEPC show an increase in absorbance at 245 nm, a lack of a decrease in absorbance at 278 nm demonstrates that very few if any tyrosine are labeled. From these data it has been determined that ⁇ 3 histidines are labeled.
  • AMCase activity has been demonstrated at a pH of 3. Assays done at lower pH, such as 3 may give more incite into the function of the histidine at lower pH.
  • the pH dependence on DEPC inactivation shows a pK a of 6.9, which corresponds with an imidazole group of histidine.
  • AMCase and the other human chitinase, chitotriosidase are secreted enzymes, only AMCase is implicated in asthma.
  • AMCase and human chitotriosidase are three residue differences near the active site. Two of these three residues, His-208 and His-269 of SEQ ID NO:4, are conserved in AMCase genes across species (Bussink et al., 2007 Genetics 177(2):959-70) but are different in human chitotriosidase, suggesting that they together have an important structural role in AMCases.
  • AMCase His-208 is a hydrogen-bonding partner of the conserved Asp- 136 ( Figures 6 and 7A), part of the DXXDXDXE (SEQ ID NO:5) motif common to all chitinases.
  • human chitotriosidase has an Asn at this position. Mutation of this His-208 to Asn in AMCase has been shown to play an important role in the acidic pH optimum of AMCase (Bussink et al. (2008) FEBS Lett. 582(6): 931-5)).
  • the pK a of Asp-136 is extremely low, ensuring that Asp-138 carries the H as it swings from Asp-136 in the apo protein to Asp-138 in the liganded structure.
  • the protonated GIu- 140 then participates in the cleavage at the beta(l,4) linkage between NAG sugars.
  • AMCase His-208 is 2.8/2.9 A from Asp-136 and is well within H- bonding range. While the position of chitotriosidase' s Asn-208 is very similar, the respective H donor and acceptor atoms slightly further apart 3.2/3.4 A apart.
  • Asp-136 The other residues contacting Asp-136 are essentially identical between AMCase and chitotriosidase; H-bond donor groups from Tyr-27 and Thr-181 (Ser-181 in chito). His-269 has not previously been remarked upon, yet represents another key difference between AMCase and chitotriosidase. His-269 is observed to form a hydrogen-bond with Asp-213, a conserved and important residue in the active site ( Figures 6 and 7A). Asp-213 lies on the bottom of the active site and coordinates the boat conformation of the substrate sugar in the -1 subsite by accepting a hydrogen bond from a hydroxyl of the substrate sugar in the -2 subsite, in preparation for hydrolysis.
  • AMCase equivalent to AMCase's 269.
  • AMCase we see replacement of this Arg with His-269, with the side chain N of His being positioned to H-bond with Asp-213 as the Arg side chain guanidyl group does.
  • the pK a of Asp-213 is likely not as low. This consequently would render Asp-213 less able to increase the pK a of the Asp-138/Glu-140 system.
  • the net result of a lower pK a in the Asp-138/Glu-140 system would be manifest as a lower pH optimum for activity.
  • Arg- 145 side chain forms H-bonds with the main chain carbonyls of Trp-99 and Glu-140.
  • Glu-140, Trp-99 and the neighboring Asn-100 of SEQ ID NO:4 in turn H- bond with methylallosamidin (and by extension, the substrate) in five separate places, transmitting the effects of Arg- 145 ( Figure 7B).
  • Having a strongly basic residue influencing these active site residues would have the effect of lowering the pK a of the Asp-138/Glu-140 system, again lowering the optimal pH of the enzyme.
  • methylallosamidin in the methylallosamidin-bound human AMCase polypeptide crystal structure TYR-27, TRP-31, PHE-58, ILE-69, GLU-70, GLY-97, GLY-98, TRP- 99, ASN-100, PHE-101, ASP-136, ASP-138, GLU-140, ALA-183, MET-210, TYR-212, ASP-213, TYR-267, ALA-295, LYS-296, GLU-297, ILE-300, MET-358, TRP-360, LEU-364 of Table 2.
  • ATOM 636 CA ASN A 100 70.043 -0.675 9.227 1.00 11.34 C

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Abstract

L'invention concerne des structures de haute résolution de chitinase mammifère acide (AMCase). Les structures révélées ici permettent la sélection et la conception médicamenteuse rationnelle à base de structure, d'agents qui modulent, par exemple, antagonisent, l'activité de l'AMCase. Ainsi, des compositions cristallines, des procédés de sélection et/ou de conception d'agents, par exemple des antagonistes, d'AMCase, et des programmes informatiques et logiciels sont révélés.
PCT/US2008/086649 2007-12-13 2008-12-12 Structures de haute résolution de chitinases mammifère acides et leurs utilisations WO2009076621A1 (fr)

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CN110358781A (zh) * 2019-07-31 2019-10-22 湖北大学 一种酸性哺乳动物几丁质酶编码基因和应用
CN115216829A (zh) * 2022-07-07 2022-10-21 淄博众晓新材料科技有限公司 一种莫来石晶须的制备方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358781A (zh) * 2019-07-31 2019-10-22 湖北大学 一种酸性哺乳动物几丁质酶编码基因和应用
CN115216829A (zh) * 2022-07-07 2022-10-21 淄博众晓新材料科技有限公司 一种莫来石晶须的制备方法
CN115216829B (zh) * 2022-07-07 2023-11-21 淄博众晓新材料科技有限公司 一种莫来石晶须的制备方法

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