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WO2008054947A2 - Structure de la bêta-glucosidase acide et procédés d'identification d'agents thérapeutiques - Google Patents

Structure de la bêta-glucosidase acide et procédés d'identification d'agents thérapeutiques Download PDF

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WO2008054947A2
WO2008054947A2 PCT/US2007/080187 US2007080187W WO2008054947A2 WO 2008054947 A2 WO2008054947 A2 WO 2008054947A2 US 2007080187 W US2007080187 W US 2007080187W WO 2008054947 A2 WO2008054947 A2 WO 2008054947A2
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structural coordinates
phe
giu
asn
leu
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WO2008054947A8 (fr
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Raquel L. Lieberman
Dagmar Ringe
Gregory A. Petsko
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Brigham and Womens Hospital Inc
Brandeis University
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Brigham and Womens Hospital Inc
Brandeis University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • Gaucher Disease a lysosomal storage disorder, is caused by mutations in the human lysosomal enzyme acid ⁇ -glucosidase (glucocerebrosidase, GCase, locus GBA).
  • GCase is a peripheral membrane protein that catalyses the hydrolysis of glucosylceramide to D-glucose and ceramide in lysosomes (FIG 1A). Maximal activity of the enzyme requires the coordinated action of another protein, saposin C, and negatively-charged lipids. Mutations in GCase lead to diminished activity within the lysosome due to impaired trafficking of the enzyme, altered stability of the enzyme, and/or defective intrinsic activity (1,2).
  • glucosylceramide in macrophages leads to proinflammatory responses and altered glycosphingolipid distribution (3).
  • CNS Central Nervous System
  • GCase protein proteotoxicity
  • mutations in the gene coding for GCase may represent one of the most important risk factors for sporadic, late onset PD, the second most common neurologic disorder.
  • a number of mutations in the GBA gene locus mostly missense, are associated with GD.
  • the symptoms of Gaucher patients cluster into three broad categories depending on the sites of mutation. The most prevalent lesions include a substitution of serine for asparagine at position 370 in the protein sequence (N370S), and leucine to proline at position 444 (L444P).
  • N370S mutation Patients with the N370S mutation display multiple organ involvement, but generally lack the neuropathies found in the other two categories of GD, such as those manifested in the L444P mutation. Interestingly, it is the N370S mutation that has been most strongly associated with PD in Gaucher carriers. Biochemical data suggest that these mutations and most of the others in GD do not act by removing a key catalytic residue in the enzyme's active site, but likely by destabilizing the native conformation, thereby rendering the protein more susceptible to mistrafficking and degradation (21).
  • GD non-neuronopathic
  • ERT enzyme replacement therapy
  • GCase recombinant human GCase
  • Disadvantages of ERT include regular intravenous infusions, little direct effect on the CNS affected variants of GD (Types 2 and 3), and high cost (2).
  • substrate reduction therapy (SRT) through inhibition of glucosylceramide synthesis with N-butyl- deoxynojirimycin (NB-DNJ, miglustat, Zavesca®), has a lower therapeutic index (9).
  • NB-DNJ has significant adverse effects, including diarrhea, abdominal pain and tremor (10). Both therapeutic approaches address substrate accumulation but not the potential contributions of mutant GCase proteo toxicity and mistrafficking to the pathogenesis of GCase.
  • GD therapy is the use of small molecules that stabilize mutant GCase and consequently restore trafficking and activity (2).
  • This general approach called pharmacological chaperoning, is also being evaluated as a therapeutic strategy for other defective lysosomal enzymes including ⁇ - galactosidase A (mutated in Anderson-Fabry Disease) and ⁇ -hexosaminidase (mutated in Tay-Sachs Disease) (11,12), as well as for non-catalytic proteins in other diseases, i.e., mutant G-protein coupled receptors and ion channels (12).
  • GCase falls into the broad category of enzymes that hydrolyze glycosidic bonds many of which, including GCase, are built on the classic TIM barrel fold, with the active site at the terminal mouth of the ⁇ -barrel (11,7) (see also CaZY database, on the internet at cafmb.cnrs-mrs.fr/CaZY/). Subtle differences among glycosidase structures govern substrate specificity and mechanistic details. While the crystal structure of some glycosidases have been known since the mid 1960 's (18), that of the human acid- ⁇ -glucosidase was determined only in 2003, well after the enzyme had been approved for use in treatment of GD (19).
  • IFG isofagomine
  • a well- studied transition-state analog inhibitor of glycosidases effectively chaperones mutant GCase to the lysosome in vivo and increases enzymatic activity.
  • a high- resolution crystal structure of human GCase in the presence and absence of this compound is also presented.
  • the structure and supporting in silico studies show that, in contrast to other small molecules that bind to the same site on the enzyme such as glycerol or CBE, isofagomine induces what appears to be the active conformation of the enzyme onto which the substrate can be readily docked.
  • the results also suggest a direct biochemical role for N370 in stabilizing this active conformation.
  • these studies offer a structural explanation for the chaperone behavior of IFG, a compound with therapeutic potential in an important human disease, and tools and information for the design of agents for the treatment of GD.
  • Described herein is a method for identifying a candidate chaperone for Case, the method comprising:
  • the test compound is selected by assembly of molecular fragments; the test compound is selected by de novo ligand design; the test compound is selected from a database of compounds; the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of the atoms of: GIu 50, Met 49, Tyr 418, Asp 24, Phe 26 and Asp 27 of Case, according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 ; and the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of the atoms of Case according to FIG 9.
  • a candidate chaperone i.e., a molecule that stabilized GCase in vivo and/or improves proper intracellular trafficking oof GCase
  • the method comprising:
  • the test compound is selected by assembly of molecular fragments; the test compound is selected by de novo ligand design; the test compound is selected from a database of compounds; the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of the atoms of: Asp 127, Trp 179, GIu 235, GIu 340 and Asn 396 of Case, according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 ; the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of atoms: CB, CG, CD, NE, CZ, NH1 and NH2 of Arg 120; CB and SG of Cys 126; CB, CG, CD1 and OD2 of Asp 127; CB, CG, CD1, CE1, CZ, CE2 and CD2 of Phe 128; CB, CG, CD1, NE1, CE2, CD2, CE3, CZ3, CH2, and
  • a candidate therapeutic agent for Gaucher disease comprising:
  • the test compound is selected by assembly of molecular fragments; the test compound is selected by de novo ligand design; the test compound is selected from a database of compounds; the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of atoms the atoms of: GIu 50, Met 49, Tyr 418, Asp 24, Phe 26, Asp 27 of Case, according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 ; and the plurality of structural coordinates comprise the structural coordinates of the atoms of Case according to FIG 9.
  • Also described is a method for identifying a candidate therapeutic agent for treatment of Gaucher disease comprising:
  • the method of claim 24 wherein the test compound is selected by assembly of molecular fragments; the test compound is selected by de novo ligand design; the test compound is selected from a database of compounds; the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of atoms: CB, CG, CD, NE, CZ, NH1 and NH2 of Arg 120; CB and SG of Cys 126; CB, CG, CD1 and OD2 of Asp 127; CB, CG, CD1, CE1, CZ, CE2 and CD2 of Phe 128; CB, CG, CD1, NE1, CE2, CD2, CE3, CZ3, CH2, and CZ2 of Trp 179; CB, CG, OD1 and ND2 of Asn 234; CB, CG, CD, OE1 and OE2 of GIu 235; CB, CG, CD1, CE1, CZ, OH, CE2 and CD2 of Tyr 24
  • a crystal comprising a polypeptide having the sequence of SEQ ID NO:1, wherein the atomic coordinates of CB, CG, OD1 and OD2 of Asp 127; CB, CG, CD1, NE1, CE2, CD2, CE3, CZ3, CH2, and CZ2 of Trp 179; CB, CG, CD, OE1 and OE2 of GIu 235; CB, CG, CD, OE1 and OE2 of GIu 340; CB, CG, OD1 and ND2 of Asn 396, all of Case, differ from those according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 .
  • a crystal comprising a polypeptide having the sequence of SEQ ID NO:1, wherein the atomic coordinates of: CB, CG, CD, OE1 and OE2 of GIu 50; CB, CG, SD and CE Met 49; CB, CG, CD1, CE1, CZ, OH, CE2 and CD2 of Tyr 418; CB, CG, OD1 and OD2 of Asp 24; CB, CG, CD1, CE1, CZ, CE2 and CD2 of Phe 26; CB, CG, OD1 and OD2 of Asp 27, all of Case, differ from those according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 .
  • the crystal further comprises isofagomine;
  • the crystal further comprises glycerol;
  • the atomic coordinates defined for the crystal differ from those according FIG 9 or FIG 11 by a root mean square deviation that is less than about 1.5 angstroms 2 ;
  • the crystal is grown by vapor diffusion from a solution containing a polypeptide comprising SEQ ID NO:1 wherein the pH of the solution is between 7.0 and 7.7.
  • a data processing device comprising:
  • a central processing unit coupled to the working memory and to the machine-readable data storage medium for processing the machine-readable data and a means for generating three-dimensional structural information from the structural coordinates;
  • the plurality of structural coordinates comprise structural coordinates of at least 10 amino acids of Case, according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 ;
  • the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of atoms: CB, CG, OD1 and OD2 of Asp 127; CB, CG, CD1, NE1, CE2, CD2, CE3, CZ3, CH2, and CZ2 of Trp 179; CB, CG, CD, OE1 and OE2 of GIu 235; CB, CG, CD, OE1 and OE2 of GIu 340; CB, CG, OD1 and ND2 of Asn 396, all of Case, according FIG 9 or FIG 11 by a root mean square deviation that is less than about 2.5 angstroms 2 ;
  • the plurality of structural coordinates comprise structural coordinates that differ from the structural coordinates of the atoms of: CB,
  • the organic solvent is selected from phenol, methanol, DMS, DMSO, and dioxane; and the crystal is grown by vapor diffusion from a solution containing a polypeptide comprising SEQ ID NO:1 wherein the pH of the solution is between 7.0 and 7.7.
  • FIGS 1A-1D depict the effects of IFG on GCase activity and trafficking in primary fibroblasts.
  • FIGG 1A Schematic of the reaction performed by GCase and the chemical structure of IFG.
  • FIGG 1B Inhibition curve for IFG with rhGCase (Cerezyme®)
  • FIGG 1C Dose-response for GCase-N370S fibroblasts treated with IFG for 5 days.
  • FIG 1D Fluorescence intensities from untreated and IFG-treated wildtype (WT) and GCase-N370S primary fibroblasts labeled with anti-GCase (top panels) and dual labeling of fibroblasts with antibodies against GCase (green) and the lysosomal marker Lampl (red). Overlap (yellow) indicates co-localization of GCase and Lampl (bottom panels). Values from lysates of IFG treated cells were compared to those of untreated control cells using a two-tailed Student's t-test assuming equal variances. Differences with P values ⁇ 0.05 were considered significant, as indicated by the asterisks.
  • FIG 2A depicts a stereoview of inh (slate) superimposed with native (yellow). IFG is displayed as ball-and-stick and GCase as cartoon.
  • FIG 2B depicts a cartoon representation of Family GH-1 ⁇ -glucosidase (PDB code 1OIF) with ball-and-stick representation of IFG.
  • PDB code 1OIF Family GH-1 ⁇ -glucosidase
  • FIG 3A is a ball-and-stick representation of IFG-bound inh active site. Asn 396 and Trp 381 are omitted here.
  • FIG 3B is a depiction of the glycerol-bound native active site. Difference (Fo - Fc ) electron density (green) is contoured at 3A and was calculated using only respective GCase coordinates. Asn 396 and Trp 381 are omitted here.
  • FIG 3C depicts hydrogen bonding interactions involved in stabilizing IFG in the active site of GCase (left) and Family GH-1 ⁇ -glucosidase (right). Distances are in A.
  • FIGs 4A-4C are surface representations of GCase near the active site.
  • FIG 4A IFG-bound inh.
  • FIG.4B Glycerol-bound native GCase.
  • FIG 4C Glucoceramide-docked inh. Glucoceramide has been modified by truncating its alkyl chain. IFG, glycerol, and modified glucoceramide are presented in ball-and- stick.
  • FIGs 5A and 5B depict stereoviews of region near N370, highlighting changes in loop 1.
  • FIG 5A IFG bound.
  • FIG 5B glycerol (GOL) bound. Residues in dark orange derive from 1OGS, those in yellow are native.
  • FIGs 6A-6D depict the results of studies comparing the effects of IFG and CBE treatment on GCase localization in the lysosomes in primary fibroblasts.
  • FIG 6B N370S-GCase untreated.
  • FIG 6C N370S-GCase treated with 100 ⁇ M IFG.
  • FIG 6D N370S-GCase treated with 100 ⁇ M CBE.
  • FIG 7 depicts the amino acid sequence of GCase used in the studies described herein.
  • the third amino acid from the carboxy terminus is His.
  • this amino acid is Arg.
  • the sequence of the GCase used in the studies described herein has the amino acid shown in (GenBank® P04062; GI 55584151).
  • the GCase used in the studies described here is Cerezyme ® (Genzyme Corporation; Cambridge, MA), a recombinant analog of acid ⁇ -glucosidase.
  • FIG 8 depicts a ball-and-stick representation of the binding a molecule of glycerol to the remote binding site of GCase.
  • FIG 9 is a table is a table providing the coordinates of various atoms in a complex containing human GCase bound to IFG.
  • atoms in the protein and are identified using standard PDB format and nomenclature.
  • a line reading (ATOM 901 N ARG B 120 22.563 -8.039 33.493 1.00 24.54 N) refers to Arg 120 in GCase.
  • each line includes: atom number, atom identity (e.g., C, N, O etc.) amino acid name, amino acid number, x coordinate, y coordinate, z coordinate, occupancy, thermal B factor, atom identity).
  • the amino acid numbering of the proteins in this table corresponds to that in FIG 7.
  • FIG 10 depicts the structure of several compounds that are computationally predicted to bind to the remote site of GCase and several compounds that are in some sense structurally similar to the compounds computationally predicted to bind to the remote site. All are candidate therapeutic agents for the treatment of Gaucher disease.
  • FIG 11 is a table providing the coordinates of all solved atoms in a complex containing human GCase bound to IFG.
  • atoms in the protein and are identified using standard PDB format and nomenclature.
  • a line reading (ATOM 901 N ARG B 120 22.563 -8.039 33.493 1.00 24.54 N) refers to Arg 120 in GCase.
  • each line includes: atom number, atom identity (e.g., C, N, O etc.) amino acid name, amino acid number, x coordinate, y coordinate, z coordinate, occupancy, thermal B factor, atom identity).
  • the amino acid numbering of the proteins in this table corresponds to that in FIG 7
  • Isofagomine acts as a chaperone for N370S mutant GCase
  • IFG acts as a chaperone for GCase.
  • IFG and recombinant human glucocerebrosidase were purchased from Toronto Research Chemicals, Inc. (Ontario, Canada) and Genzyme (Cambridge, MA), respectively.
  • Gaucher patient fibroblasts (DMN89.15; N370S/N370S) were cultured in 12 well plates and incubated with or without IFG for 5 d. Cells were washed twice with medium (5 min each, 37 °C), twice with PBS (5 min. each, 37 °C), scraped, pelleted and lysed in Mcllvaine buffer (pH 5.2) with 0.1% Triton X-000 and 0.25% sodium taurocholate.
  • GCase activity was calculated as the CBE-sensitive activity and expressed in terms of specific activity (florescence released per milligram of protein per hour). Values from lysates of IFG treated cells were compared to those of untreated control cells using a two-tailed Student's t-test assuming equal variances. Differences with P values ⁇ 0.05 were considered significant, as indicated by the asterisks. All samples were analyzed in triplicate.
  • deglycosylated Cerezyme® was concentrated to about 10 mg ml -1 in 1 mM MES, pH 6.6, 0.1 M NaCl, 0.02% NaN 3 .
  • Crystals were obtained by hanging drop vapor diffusion using a crystallization solution composed of 1 M (NH 2 ) 2 SO 4 , 0.17 M guanidine HCl, 0.02 M KCl, 0.1 M acetate, pH 4.6. Crystals were cryoprotected with a gradient of glycerol. The structure was solved as described below.
  • Neutral pH crystals of GCase were obtained by hanging drop vapor diffusion using a crystallization solution composed of 0.8 M Na/K Phosphate, 0.1 M Hepes pH 7.5. Prior to data collection, these crystals were transferred to a solution containing 2 M Li 2 SO 4 and cooled in liquid nitrogen. As described below in greater detail, the GCase in the neutral pH crystals has essentially the same structure as the IFG co-crystals described below.
  • Co-crystals with IFG were obtained by soaking GCase crystals for 10 min in a solution containing mother liquor plus 200 ⁇ M IFG Crystals were frozen with a gradient of 5%-20% glycerol in mother liquor. Data were collected at GM/CA-CAT beamline at the Advanced Photon Source (Darien, IL) at 11 ,999 eV and 100 K, using a 4k x 4k MAR CCD detector, and were processed with HKL2000 (28). The structures were solved by the method of molecular replacement with the program Molrep (29) using a monomelic search model derived from PDB code IOGS (19).
  • the atomic models were fitted into their respective electron density maps using Coot (30) and refined using Refmac5 (29). Topology and geometry restraints for IFG were obtained using PRODRG2 (31), and bound water molecules were identified using Coot (30) and Arp/Warp (32). For both native and inh, 99.8% of the residues in are in the most favored and additional allowed regions of the Ramachandran plot. Superposition of coordinates was accomplished using the Secondary Structure Matching (SSM) algorithm (33), and figures were generated using Pymol (34). Computational docking was performed using Shr ⁇ dinger (Portland, OR).
  • SSM Secondary Structure Matching
  • polypeptide backbone of inh and native GCase are very similar to each other (FIG 2A) and to the previously published structures of apo GCase (protein data bank (PDB) codes IOGS (19), 2F61 (20)) as well as GCase covalently modified with CBE (PDB code 1 Y7V) (21).
  • the root mean squared deviations for Ca are 0.6 A 2 or better, indicating that inhibitor binding does not change the overall structure of the enzyme. Both the TIM barrel and immunoglobulin domains superimpose.
  • These GCase crystals differ from those published previously (19-21) in that they belong to a lower symmetry space group and native diffracts to higher resolution.
  • our GCase native structure contains a single, well-ordered glycerol molecule in the active site (vide infra, FIG 3B), in place of the sulfate anion and two water molecules modeled by Sussman and coworkers (19) or ethylene glycol observed by Grabowski and coworkers (20). Glycerol molecules were most likely introduced upon cryoprotection of the crystals.
  • IFG is bound in the active site of two of the four copies of GCase in the asymmetric unit, while glycerol is present in the remaining two monomers.
  • IFG adopts a single, well-ordered distorted chair conformation in the active site of GCase (FIG 3A), where it is held in place by extensive hydrogen bonding interactions (FIG 3C, left).
  • the hydroxyl groups of IFG interact with residues Asp 127, Trp 179, Trp 381 , and Asn 396, and the imino group is stabilized by GIu 235 and GIu 340.
  • GIu 340 has been shown previously to act as the catalytic nucleophile and GIu 235, the general acid/base (21,23).
  • Tyr 313 is hydrogen bonded to GIu 235, acting as a cap to the active site.
  • Tyr 313 is bound to GIu 340, widening the opening for solvent molecules or substrate to reach the active site.
  • the previously unobserved role of Tyr 313 in gating access to the active site by altering its hydrogen bonding interaction to the two key active site residues is important for substrate binding (vide infra) and therefore warrants further investigation.
  • IFG did not induce these changes in TMG, where instead of loop I there are antiparallel ⁇ -strands (FIG 2B).
  • Inspection of isotropic B-factors among the copies of GCase in the inh asymmetric unit reveals that the side chains of loop 1 residues are significantly more ordered in the presence of IFG than with glycerol.
  • glycerol is bound, there is poor side chain electron density for several residues in loop 1 , whereas in inh, single conformations for all side chains are readily visible in the maps. This observation suggests IFG confers increased stability to GCase.
  • All of the molecules in FIG 10 are candidate agents for increasing the stability or proper trafficking or activity of GCase. Thus, they are all candidate therapeutic agents for the treatment of Gaucher Disease. These and other agents can be tested using any suitable assay, including those described above to determine whether it increases the stability or proper trafficking or activity of GCase in vitro, in cells or cell lysates. [0061] Use of Structural Information to Select or Design Chaperones Binding at the Active Site or the Remote Site
  • FIG 9 and FIG 11 and subsets thereof can be used to identify or design agents that bind to either the active site or the remote site of GCase.
  • Such molecules can act as chaperones to direct proper trafficking of GCase and are potential therapeutic agents for the treatment of disorders associated with mutations in GCase.
  • the structural coordinates in FIG 9 and FIG 11 and subsets thereof for example the coordinates of atoms in GCase that form the active site or that interact with inh in the active site can be used to create a model of the binding pocket for compounds that bind to the active site and can act as a chaperone for GCase. In some cases such molecules are actually inhibitors of GCase enzymatic activity in vitro, but can be used as therapeutic agents.
  • the structural coordinates provided herein define the position of various atoms relative to an arbitrary point in space. Thus, the coordinates can be manipulated by addition or subtraction, inversion, crystallographic permutation, fractionalization, or combinations thereof.
  • variant structures having an insertion, deletion or substitution (e.g., a conservative substitution) of one or more amino acids.
  • variant structures can be useful in rational drug design. For example, they can be used to design drugs targeted to variant forms of GCase characterized by mutations in the amino acid sequence of GCase.
  • a variant structure e.g., a variant GCase remote site binding pocket can be compared to the GCase remote site binding pocket defined by a subset of the structural coordinates in FIG 9 or FIG 11 using any of a number of known programs for structure comparison (e.g., ProFit, (Matin et al., University College London, on the internet at bioinf.org.uk/software), Swiss-Pdb Viewer (Guex et al., E1ectrophoresis, 18:2714-2723 (1997)), QUANTA (Molecular Simulations, Inc., San Diego, CA) and Sybyl (Tripos Associates; St.
  • ProFit ProFit
  • Swiss-Pdb Viewer Guiex et al., E1ectrophoresis, 18:2714-2723 (1997)
  • QUANTA Molecular Simulations, Inc., San Diego, CA
  • Sybyl Tripos Associates; St.
  • Three-dimensional models of all or parts of the structures describe herein can be generated using any suitable graphics program, including: QUANTA (Accelrys; San Diego, CA), RIBBONS (Carson, J. Appl. Crystallogr. 24:9589-961 (1991) Insight II, Pymol. Molmol (Koradi, R., Billeter, J., and Wuthrich, K. (1996).
  • MOLMOL a program for display and analysis of macromolecular structures. J MoI Graph 14, 51-55, 29-32) Swiss-Pdb viewer, "O", Coot, GRASP (Nicholls, A., Sharp, K.A., and Honig, B. (1991).
  • Protein folding and associate insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281-296), pymol (pymol.org) and Molscript (Kraulis, P., (1991). MOLSCRIPT: Aprogram to produce both detailed and schematic plots of protein structure. J Appl Cryst 24, 946- 950).
  • the three dimensional representation can include information regarding the characteristics of the atoms and/or residues, for example, charge, hydrophobicity, torsional and rotational degrees of freedom.
  • the remote or active site binding pocket of GCase or specific atoms or residues of either binding pocket can be used to select or design a chemical entity that is expected to bind to the binding pocket. Such binding entities are putative chaperones and/or therapeutic agents.
  • An agent entity can interact with a binding pocket via non-covalent such as hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic attraction such that the binding affinity of the agent for the complex allows it to bind with sufficient affinity to promote increased trafficking of GCase.
  • a suitable agent is pieced together from fragments that have been selected to interact with a portion of the binding pocket or have been selected to provide other desirable aspects of the binding entity.
  • CAVEAT Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems, Roberts, ed., Royal Society of Chemistry, Special Publication No. 78: pp. 182-196 (1989); Lauri et al. J. Comp. Aid. Molec. Design, 8: 51-66 (1994)).
  • CAVEAT Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems, Roberts, ed., Royal Society of Chemistry, Special Publication No. 78: pp. 182-196 (1989); Lauri et al. J. Comp. Aid. Molec. Design, 8: 51-66 (1994)).
  • Many other approaches for designing or selecting an agent that binds to a binding pocket are described in the literature (Martin,
  • GLIDE® and other products from Schr ⁇ dinger, Inc. are useful for high throughput virtual screening and for more precise ligand selection and docking.
  • APBS software can be used for analysis of diffusional processes to determine ligand-protein and protein- protein binding kinetics, calculation of solvation and binding energy to determine ligand-protein and protein-protein equilibrium binding constants and aid in rational drug design (Baker et al. 2001 E1ectrostatics of nanosystems: application to microtubules and the ribosome.
  • An agent selected for the ability to bind to a binding pocket can be structurally modified to optimize binding and/or other characteristics.
  • An example of a factor to be considered is the predicted energy of deformation required to deform the entity from the predicted lowest energy conformation in solution to the binding conformation. In many cases it is desirable for the deformation energy to be less than about 12 kcal/mole, 10 kcal/mole, 7 kcal/mol or 5 kcal/mole.
  • the chemical entity can also be modified to minimize unfavorable electrostatic interactions (e.g., repulsive charge-charge, dipole-dipole and charge-dipole interactions) with the GCase and with the surrounding water molecules.
  • the structural coordinates in FIG 9 or FIG 11 and subsets thereof can be used to obtain structural information for other crystallized complexes or protein by molecular replacement and other techniques.
  • a Fourier transform of at least a portion of the structural coordinates in FIG 9 or FIG 11 or homology model thereof can be used to analyze the X-ray diffraction pattern of a selected molecular complex (e.g., a GCase variant bound to an inhibitor or chaperone) to determine at least a portion of the structure coordinates of the selected molecular complex.
  • Molecular replacement can provide an accurate estimate of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that can not be determined directly.
  • molecular replacement can be used to obtain structural coordinates for GCase bound to another ligand, e.g., a putative chaperone, irrespective of whether the space groups of the crystals are the same or different.
  • Useful test compounds include peptides, e.g., peptides that have been stabilized in an alpha helix by an alkyl or alkenyl cross-link (see Schafrneister et al., 2000 J. Am. Chem. Soc. 122:5891; Blackwell et al. 1994 Angew Chem. Int. Ed. 37:3281; and Walensky et al. Science 305:1466).

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Abstract

L'invention concerne un procédé pour mettre au point des agents qui se lient soit au site actif soit au site distant de la glucocérébrosidase. De telles molécules peuvent agir en tant que chaperons pour diriger correctement la circulation de la glucocérébrosidase et sont des agents thérapeutiques potentiels pour le traitement de troubles associés à des mutations dans la glucocérébrosidase, par exemple, la maladie de Gaucher.
PCT/US2007/080187 2006-10-02 2007-10-02 Structure de la bêta-glucosidase acide et procédés d'identification d'agents thérapeutiques Ceased WO2008054947A2 (fr)

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JP2010057899A (ja) * 2008-07-01 2010-03-18 Regents Of The Univ Of California 磁気共鳴イメージング（ｍｒｉ）を用いる白質線維トラクトの特定
WO2010116141A3 (fr) * 2009-04-09 2011-03-31 Summit Corporation Plc Association médicamenteuse pour le traitement des maladies protéostatiques
WO2011112085A1 (fr) * 2010-03-10 2011-09-15 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Sondes basées sur l'activité (abp) interagissant avec des glycosidases
JP2012527900A (ja) * 2009-05-26 2012-11-12 アミカス セラピューティックス インコーポレイテッド 生物製剤の製造および精製を改善するための薬理学的シャペロンの利用
US9206457B2 (en) 2009-05-26 2015-12-08 Amicus Therapeutics, Inc. Utilization of pharmacological chaperones to improve manufacturing and purification of biologics
US9353117B2 (en) 2010-12-08 2016-05-31 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Substituted pyrazolopyrimidines as glucocerebrosidase activators
US9404986B2 (en) 2011-05-06 2016-08-02 The Regents Of The University Of California Measuring biological tissue parameters using diffusion magnetic resonance imaging

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US9568580B2 (en) 2008-07-01 2017-02-14 The Regents Of The University Of California Identifying white matter fiber tracts using magnetic resonance imaging (MRI)
JP2010057899A (ja) * 2008-07-01 2010-03-18 Regents Of The Univ Of California 磁気共鳴イメージング（ｍｒｉ）を用いる白質線維トラクトの特定
WO2010116141A3 (fr) * 2009-04-09 2011-03-31 Summit Corporation Plc Association médicamenteuse pour le traitement des maladies protéostatiques
US9206457B2 (en) 2009-05-26 2015-12-08 Amicus Therapeutics, Inc. Utilization of pharmacological chaperones to improve manufacturing and purification of biologics
JP2012527900A (ja) * 2009-05-26 2012-11-12 アミカス セラピューティックス インコーポレイテッド 生物製剤の製造および精製を改善するための薬理学的シャペロンの利用
EP2435459A4 (fr) * 2009-05-26 2014-01-15 Amicus Therapeutics Inc Utilisation de chaperons pharmacologiques pour améliorer la fabrication et la purification de produits biologiques
CN104164412A (zh) * 2009-05-26 2014-11-26 阿米库斯治疗学公司 利用药物分子伴侣改善生物制剂的生产和纯化
AU2010254092B2 (en) * 2009-05-26 2015-11-12 Amicus Therapeutics, Inc. Utilization of pharmacological chaperones to improve manufacturing and purification of biologics
US9056847B2 (en) 2010-03-10 2015-06-16 Academisch Medisch Centrum Bij Universiteit Van Amsterdam Activity based probes (ABPs) interacting with glycosidases
WO2011112085A1 (fr) * 2010-03-10 2011-09-15 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Sondes basées sur l'activité (abp) interagissant avec des glycosidases
US9353117B2 (en) 2010-12-08 2016-05-31 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Substituted pyrazolopyrimidines as glucocerebrosidase activators
US9974789B2 (en) 2010-12-08 2018-05-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Substituted pyrazolopyrimidines as glucocerebrosidase activators
US10925874B2 (en) 2010-12-08 2021-02-23 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Substituted pyrazolopyrimidines as glucocerebrosidase activators
US9404986B2 (en) 2011-05-06 2016-08-02 The Regents Of The University Of California Measuring biological tissue parameters using diffusion magnetic resonance imaging

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