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WO2009049421A1 - Compositions et procédés permettant d'améliorer l'activité enzymatique dans la maladie de gaucher, la gangliosidose à gm1, la maladie de morquio de type b et la maladie de parkinson - Google Patents

Compositions et procédés permettant d'améliorer l'activité enzymatique dans la maladie de gaucher, la gangliosidose à gm1, la maladie de morquio de type b et la maladie de parkinson Download PDF

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WO2009049421A1
WO2009049421A1 PCT/CA2008/001843 CA2008001843W WO2009049421A1 WO 2009049421 A1 WO2009049421 A1 WO 2009049421A1 CA 2008001843 W CA2008001843 W CA 2008001843W WO 2009049421 A1 WO2009049421 A1 WO 2009049421A1
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formula
lysosomal storage
disease
storage disorder
pyridinyl
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PCT/CA2008/001843
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Don J. Mahuran
Michael B. Tropak
Justin D. Buttner
Jan E. Blanchard
Eric D. Brown
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The Hospital For Sick Children
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Publication of WO2009049421A1 publication Critical patent/WO2009049421A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • GAUCHER GM1-GANGLIOSIDOSIS/MORQUIO B DISEASE.
  • the present invention relates generally to therapeutic compositions and methods for treatment of Gaucher disease, GM1 -gangliosidosis, Morquio B disease, and Parkinson's disease.
  • LSD lysosomal storage disorders
  • a deficiency in the enzyme beta-glucocerebrosidase (referred to interchangeably herein as "GCase” or “Gcc”), which cleaves the terminal glucose residue from lactosylceramide, results in the LSD Gaucher disease.
  • the severity of these disorders is correlated with relative levels of remaining enzyme activity and the degree of accumulation of the substrate.
  • Gaucher patients are treated using an expensive enzyme replacement therapy at a cost of about USD$300,000 per year per patient, or using nonspecific substrate reduction therapy wherein the enzyme deficiency is not treated but rather the accumulation of substrate is treated by reducing the synthetic levels of all gangliosides.
  • lysosome is generally associated with the degradation of proteins and other macromolecules endocytosed from the extracellular environment, it also plays a major role in degrading intracellular organelles (mitochondria and peroxisomes) (1 , 2), long lived cytosolic proteins (general/ structural) and misfolded aggregated protein from both the cytosol; for example, Huntingtin (poly-Gin expansion mutants), a-Synuclein (Parkinson's); and the ER; for example, a 1 -antitrypsin (Z mutation, Glu342Lys); through the macroautophagy-lysosomal system (3).
  • UPS cytosolic ubiquitin-proteasome system
  • Endoplasmic reticulum (ER)-stress is a factor in the pathobiology of Gaucher and other lysosomal storage diseases (LSDs).
  • LSDs lysosomal storage diseases
  • Baf inhibits lysosomal acidification and thus the fusion between macroautophagic vesicles (autophagosomes) and lysosomes, which produces the autolysosome where the process of cargo begins, for example ⁇ -Synuclein, degradation, autophagic "flux".
  • the inhibition of autolysosome formation results in an increase in the number of autophagosomes; but this is due to a decrease in flux, not an increase in autophagy. Decreased flux results in a build up of undegraded, defective, polyubiquitinated proteins and dysfunctional mitochondria, which in turn can initiate apoptosis (8, 9).
  • a lysosomal storage disorder such as Gaucher disease, GM1- gangliosidosis/Morquio B disease, or Parkinson's disease.
  • composition comprising a therapeutically effective amount of a beta-glucocerebrosidase inhibitor together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder.
  • composition comprising a therapeutically effective amount of an N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • a composition comprising a therapeutically effective amount of (a)
  • a method for treating a lysosomal storage disorder comprising administration of a therapeutically effective amount of an N-(4-pyridinyl)-2-furamide derivative to a subject in need thereof.
  • a method of treating a lysosomal storage disorder comprises administration of a therapeutically effective amount of (a) 5[(4-chlorophenyl) thio] quinazoline-2,4-diamine) according to formula 2 or a derivative thereof, wherein the derivative comprises substitution of an amine group at position 2 or 4; (b) 4-amino-1H-1 ,5-benzodiazepine-3-carbonitrile hydrochloride according to formula 4; (c) 4-(2-chloro-6-fluorostyryl) benzyl (4-fluoroanilino) methanimidothioate hydrobromide) according to formula 5; (d) 4-[phenyl ( ⁇ 2-[(phenyl ⁇ 2,4,5- trioxo-1-[4-(trifluoromethyl) phenyl]tetrahydro-1 H-pyrrol-3-yliden ⁇ methyl) amino] ethyl ⁇ amino) methylidene]-1-[4-(trifluoromethyl
  • (4-pyridinyl)-2-furamide derivative for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof.
  • the invention provides a use of a therapeutically effective amount of:
  • Figures 1 to 14 show GCase activity in Gaucher patient fibroblasts homozygous for the N370S mutation at escalating doses of various GCase inhibitory compounds identified following a screen of the Maybridge library (see Table 1).
  • Figure 15 shows increase in activity levels of GCase in Gaucher patient fibroblasts homozygous for the N370S mutation treated with increasing concentrations of
  • Figure 16 shows an increase in GCase activity in Gaucher patient fibroblasts homozygous for the N370S mutation at escalating doses of the GCase inhibitory compounds MWP01127 identified following a screen of the Maybridge library (see Table 1).
  • Figure 17 is a schematic illustration of the docked pose of MWP 00127 of
  • Figure 18 shows a relative increase in beta-galactosidase activity in the presence of N-n-DGJ for fibroblasts from a cat with GM1 gangliosidosis. Hex activity levels remain unchanged.
  • Figure 19 shows a relative increase in beta-galactosidase activity in the presence of budenoside (a beta-galactosidase inhibitor, see Table 3) for fibroblasts from a cat with GM1 gangliosidosis.
  • budenoside a beta-galactosidase inhibitor, see Table 3
  • Figure 20 shows the activity of purified GCase in the presence of increasing concentrations of HTS 01898. There is a dose dependent increase in GCase activity relative to DMSO containing enzyme.
  • Figure 21 shows changes in Gcc and Hex activity in GC patient fibroblasts using compounds outlined in Example 6.
  • Figure 22 shows inhibitory function of MWP and MAC compounds outlined in
  • Figure 23 shows the Effect of compound MWP and MAC on trafficking Gcc from the ER to lysosomes in GD patient fibroblasts.
  • Figure 24 is a summary of perturbations in H/D-exchange from selected regions of GCC in the absence versus presence of lignads.
  • Figure 25A illustrates data obtained in a primary screening of compounds relating to an inhibition assay, to confirm and determine the IC50 value in the presence of 0.8 mM MUGIc.
  • Figure 25B illustrates further data obtained in a primary screening of compounds relating to an inhibition assay, to confirm and determine the IC50 value in the presence of 0.8 mM MUGIc.
  • Figure 26A illustrates data obtained in a primary screening of compounds relating to heat denaturation attenuation assay, i.e. remaining Gcc activity in the presence of the compound following heating to 50 0 C for 20 min.
  • Figure 26B illustrates further data obtained in a primary screening of compounds relating to heat denaturation attenuation assay, i.e. remaining Gcc activity in the presence of the compound following heating to 50 0 C for 20 min.
  • Figure 27A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Gcc in GD patient fibroblasts (N370S/N370S) after cells were treated for five days with the indicated concentration of test compound.
  • Figure 27B illustrates further data obtained in a primary screening of compounds relating to changes in intracellular levels of Gcc in GD patient fibroblasts
  • Figure 28A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts
  • Figure 28B illustrates further data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts
  • PC pharmacological chaperones
  • small molecules typically functioning as inhibitors that have been shown to enhance the levels of mutant protein that can be transported to the lysosome.
  • These compounds may be used as therapeutics for lysosomal storage disorders that are characterized by a mutant enzyme showing reduced residual activity in the lysosome due to retention of the mutant protein in the endoplasmic reticulum.
  • glucocerebrosidase or beta-galactosidase inhibitory activity are described herein. These inhibitors can behave as pharmacological chaperones for mutants of glucocerebrosidase or beta-galactosidase enzymes, respectively. Inhibitors that stabilize the enzyme against thermal denaturation act as pharmacological chaperones by increasing the levels of the active enzyme in human and/or animal derived GM1 gangliosidosis/Morquio B or Gaucher cells.
  • a composition comprising a therapeutically effective amount of an N-(4-pyridinyl)-2-furamide derivative together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder.
  • the N-(4-pyridinyl)-2- furamide derivative may comprise a phenoxy substituted moiety at the 5- position of an n-(4- pyridinyl)-2-furamide; or an n-pyridinyl-4-acrylamide group.
  • An exemplary N-(4-pyridinyl)-2- furamide derivative comprises 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3:
  • the lysosomal storage disorder for treatment may be Gaucher disease, for example: late-onset Gaucher disease, GM1-gangliosidosis, or Morquio B disease.
  • the lysosomal storage disorder may be Parkinson's disease associated with Gaucher disease.
  • a method of treating a lysosomal storage disorder comprising administration of a therapeutically effective amount of an N-(4-pyridinyl)-2-furamide derivative to a subject in need thereof.
  • the N-(4-pyridinyl)-2-furamide derivative may comprise a phenoxy substituted moiety at the 5- position of an n-(4-pyridinyl)-2-furamide; or an n-pyridinyl-4-acrylamide group.
  • the N-(4-pyridinyl)-2-furamide derivative may comprise 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3.
  • the lysosomal storage disorder may be Gaucher disease, such as late-onset Gaucher disease, GM1 -gangliosidosis, or Morquio B disease. Further the lysosomal storage disorder may be Parkinson's disease associated with Gaucher disease.
  • Te N-(4-pyridinyl)-2-furamide derivative may comprise a phenoxy substituted moiety at the 5- position of an n-(4-pyridinyl)- 2-furamide; or an n-pyridinyl-4-acrylamide group, such as the compound according to Formula 3.
  • the lysosomal storage disorder is Gaucher disease, for example, late-onset Gaucher disease, GM1 -gangliosidosis, or Morquio B disease. Further, the use may be for Parkinson's disease associated with Gaucher disease.
  • composition comprising a therapeutically effective amount of:
  • the lysosomal storage disorder is Gaucher disease, such as late-onset
  • Gaucher disease GM1 -gangliosidosis, or Morquio B disease.
  • the lysosomal storage disorder may be Parkinson's disease associated with Gaucher disease.
  • the method of treating a lysosomal storage disorder described herein comprises administration of a therapeutically effective amount of: (a) 5[(4-chlorophenyl) thio] quinazoline-2,4-diamine) according to Formula 2, or a derivative thereof wherein the derivative comprises substitution of an amine group at position 2 or 4; (b) 4-amino-1 H-1 ,5- benzodiazepine-3-carbonitrile hydrochloride according to Formula 4; (c) 4-(2-chloro-6- fluorostyryl) benzyl (4-fluoroanilino) methanimidothioate hydrobromide) according to Formula 5; (d) 4-[phenyl ( ⁇ 2-[(phenyl ⁇ 2,4,5-trioxo-1-[4-(trifluoromethyl)phenyl]tetrahydro-1 H-pyrrol-3- yliden ⁇ methyl) amino] ethyl ⁇ amino) methylidene]-1-[4-(
  • Formula 2 or a derivative thereof wherein the derivative comprises substitution of an amine group at position 2 or 4; (b) a compound according to Formula 4; (c) a compound according to Formula 5; (d) a compound according to Formula 6; (e) a compound according to Formula 7; or (f) a compound according to Formula 7A for treatment of a subject or for preparation of a medicament for treatment of a subject having a lysosomal storage disorder is described herein.
  • GCase activity was measured by release of 4-methylumbelliferyl fluorophore from 4-methylumbelliferyl-b-D-glucopyranoside (MUbGIc). Total Assay volume was 50 ⁇ l_, For enzyme activity monitored continuously, reactions were initiated with 1mM MUbGLc at room temperature and monitored for 7 min using 330 nm and 450 nm excitation and emission filters, respectively.
  • Table 1 provides a list of GCase inhibitors identified by screening the
  • Maybridge library These inhibitors may function as pharmacological chaperones for mutants of GCase. GCase activity against the full Maybridge library was evaluated with a cut-off of 30% residual activity.
  • the Maybridge library is available at www.maybridge.com which is part of Thermo Fisher Scientific.
  • Table 1 provides the following category headings which apply to the columns shown. However, in some columns these headings are truncated due to space constraints.
  • Column 1 shows the molecule identification number.
  • Column 2 depicts the chemical structure of each molecule.
  • Column 3 gives the chemical formula of each molecule.
  • Colum 4 provides the molecular weight of each molecule.
  • Column 5 provides a code representing a molecule's designation code.
  • Column 6 provides an exemplary IUPAC name for each molecule.
  • Column 7 (Cell min ) provides a measurement of relative increase in cells (minimum value), while column 8 (Cell max ) provides a measurement of relative increase in cells (maximum value).
  • Table 1 illustrates exemplary compounds, many of which show a clear dose dependent increase in GCase activity in patient cells. Significantly, this effect is specific the activity of another lysosomal enzyme Hexosaminidase remained unchanged at all concentrations of the compounds relative to mock treated cells. These different inhibitory small molecules, acting as pharmacological chaperones, fell into different classes of compounds. Exemplary compounds are outlined below.
  • each of these compounds increased GCase activity almost two-fold relative to untreated cells, they differed in inhibitory activity by almost two-orders of magnitude.
  • the inhibitory strength did not necessarily correlate with chaperoning efficacy.
  • compound MWP 01127 has three-fold lower lc50 relative to compound HTS 02324.
  • MWP 01127 increases GCase to a similar extent as compound HTS 02324 but at a three-fold lower concentration.
  • Table 1 illustrates formula 1 , showing molecule "60", or MWP 01127 (also referenced as 5[(4-methylphenyl) thio] quinazoline-2,4-diamine), a diamino quinozoline.
  • MWP 01127 showed inhibitory activity and is an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • the inhibitory activity may be maintained in derivatives of this molecule.
  • Such quinazoline derivatives would have similarity to Formula 1 in that at least one pendant amino group would be present, of the two currently found at positions 2 and 4. Further the nitrogen atom at position 3 would be present.
  • functional derivatives may have one or more hydrophobic moiety at positions 6 or 7. Such hydrophobic moieties may comprise aromatic rings or pendant alkyl chains.
  • Table 1 illustrates formula 2, showing molecule "61 " or MWP 01128 (also referenced as 5[(4-chlorophenyl) thio] quinazoline-2,4-diamine), a diamino quinozoline.
  • MWP 01128 showed inhibitory activity and is an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • the inhibitory activity may be maintained in derivatives of this molecule.
  • Such quinazoline derivatives would have similarity to Formula 2 in that at least one pendant amino group would be present, of the two currently found at positions 2 and 4. Further the nitrogen atom at position 3 would be present.
  • functional derivatives may have one or more hydrophobic moiety at positions 6 or 7. Such hydrophobic moieties may comprise aromatic rings or pendant alkyl chains.
  • Table 1 illustrates formula 3, showing Molecule "31" or HTS 02324 (also referenced as 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide), a pyridinyl-2-furamide.
  • HTS 02324 showed inhibitory activity and is thus an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • Table 1 illustrates formula 4, showing molecule "4" or BTB 03346 (also referenced as 4-amino-1 H-1 ,5-benzodiazepine-3-carbonitrile hydrochloride), a benzodiazepine.
  • BTB 03346 showed inhibitory activity and is thus an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • Table 1 illustrates formula 5, showing molecule "77 " or RJF 01159 (also referenced as 4-(2-chloro-6-fluorostyryl) benzyl (4-fluoroanilino) methanimidothioate hydrobromide), an anilinomethanimidothioate.
  • RJF 01159 showed inhibitory activity and is thus an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • Table 1 illustrates formula 6 showing molecule "73" or RJC 02132 (also referenced as 4-[phenyl ( ⁇ 2-[(phenyl ⁇ 2 I 4,5-trioxo-1-[4-(trifluoromethyl)phenyl]tetrahydro-1 H- pyrrol-3-yliden ⁇ methyl) amino] ethyl ⁇ amino) methylidene]-1-[4-(trifluoromethyl) phenyl] pyrrolidine-2,3,5-trione), which is a pyrrolidine-2, 3, 5 trione.
  • RJC 02132 showed inhibitory activity and is thus an exemplary pharmacological chaperone for a therapeutic composition and method of treatment.
  • Figure 1 to Figure 14 illustrate the effect of escalating doses of GCase inhibitors on GCase activity in Gaucher (N370S/N370S) patient fibroblasts. Varying concentrations (X-axis) of compounds listed in Table 1 are tested. Activity is shown (Y-axis). An increase in GCase activity following treatment with different hits from the Maybridge library is illustrated, relative to DMSO treated cells.
  • Figure 1 illustrates the activity of RJC 01351 , CD 02284, CD 00240, and RH
  • Figure 2 illustrates BTB 03449, MWP 01127 (molecule 60); BTB 11331 ; JFD
  • Figure 3 illustrates JFD 00243; CD 00466; RH 01061 ; JFD 03132; SP 00756;
  • Figure 4 illustrates CD 01259; RH 02106; KM 04397; KM 04984; HTS 09832;
  • Figure 5 illustrates BTB 14755; HTS 01243; MWP 01128 (molecule 61); HTS
  • Figure 6 illustrates BTB 03346; SP 00991 ; SPB 04204; BTB 07496; MWP
  • Figure 7 illustrates SPB 00915; JFD 03571 ; RJC 00357; RJC 02270; HTS
  • Figure 8 illustrates XAX 00155; JFD 02816; CD 03798; HTS 07133; KM
  • Figure 9 illustrates KM 04549; RJC 00100; S 15348; RH 00644; SEW 06186;
  • FIG. 10 illustrates BTB 06478; HTS 07424; BTB 12892; HTS 04241 ; BTB
  • Figure 11 illustrates BTB 08066; BTB 11015; BTB 11334; SP 00752; BTB
  • Figure 12 illustrates RDR 01439; SEW 00556; RJC 02132 (molecule 73);
  • Figure 13 illustrates BTB 13370; BTB 14731 ; SEW 04707; KM 04550; S
  • Figure 14 illustrates BTB 10392; MBE 00151 ; BTB 13368; CD 11038; HTS
  • Figure 15 shows lsofagomine concentrations versus activity of GCase.
  • N370S/N370S N370S/N370S
  • Lysates from treated cells were prepared and GCase and Hex activity determined using the appropriate substrates, MU-bGlcNAC(squares) and MU-bGlc (diamonds).
  • the lc50 of lsofagomine is 60 nM; however -1000 fold concentrations are required to increase GCase in patient fibroblasts.
  • Figure 16 shows relative enzyme activity in the presence of decreasing concentrations of MWP 01127, versus activity in the presence of decreasing concentrations of isofagomine.
  • the optimal concentration of MWP 01127 in terms of chaperoning efficacy (2.5 fold at 15 ⁇ M) more closely matches the IC50 values (7 ⁇ M).
  • MWP 01127 is able to more readily enter the cell (and the endoplasmic reticulum) in comparison to Isofagomine.
  • MWP 01127 is able to chaperone other GCase mutants, such as the F213I mutation predominantly found in the oriental population of late-onset Gaucher patients (not shown).
  • Figure 17 schematically illustrates the docked pose of MWP 01127 of
  • Formula 1 in an active site, consistent with H-D exchange derived from Gcase:MWP complex. Pi-Pi interaction (indicated by the central gray tube) between the aromatic ring of ligand and Tyr313 of Gcase. This docked pose structure supports the presence of an aromatic ring at position 5. Aromatic rings and/or alkyl chains at positions 5, 6, 7 or 8 may serve a similar function.
  • GIu 235 Hydrophobic interaction is occurring at Tyr 313 and Trp 381. Pi-Pi interaction may be occurring at Tyr 313. Other interacting residues may include Ser 237, Phe 246, Leu 314, and GIu 340. These interactions between MWP 01127 and selected residues of the active site of Gcase may be responsible for observed activity.
  • the carboxylate group of GIu 235 and hydroxyl groups of Tyr 244 are proposed to be involved in hydrogen bonding interactions with the amine (C2 of quinazoline) and nitrogen (position 3 quinazoline), respectively of MWP 01127. Other derivatives having similar interactions in the docked pose may show comparable activity.
  • Table 2 illustrates N-nonyl deoxygalactonojirimycin (or N-n-DGJ), a known beta-galactosidase inhibitor, and is specifically a 59 nM competitive inhibitor of human lysosomal beta-galactosidase (bGal).
  • N-alkylated (adjacent to C1 position) deoxygalactonojirimycin derivatives would function as inhibitors of beta-galactosidase and are therefore candidate pharmacological chaperones for treatment of GM1 gangliosidosis.
  • Table 2 also illustrates such an N-alkylated compound: N-butyl deoxygalactonojirimycin, also known as N-butyl DGJ.
  • GM1 -gangliosidosis animals were treated with a decreasing dose of the compound for three days. Relative to mock treated cells, bGal activity was increased between five and nine fold at a inhibitor concentration of 0.5 uM.
  • Figure 18 shows beta-galactosidase is increased in N-n-DGJ treated fibroblasts from a cat with GM1 gangliosidosis. By comparison, Hex activity remained unaffected. B-gal or Hex activity was measured in lysates prepared from cat fibroblasts treated for 3 days with increasing concentrations of N-n-DGJ using MU-bGal or MU-b- GIcNAc substrates. At higher concentrations bGal activity was inhibited. [00107] N-n-DGJ and its derivatives would be efficacious pharmacological chaperones capable of enhancing enzyme activity of mutant bGal in organs such as liver and kidney. Alkylated DGJ derivatives that are capable of more efficiently traversing the blood brain barrier may more readily or effectively increase bGal activity in the central nervous system.
  • Table 3 shows a panel of bGal inhibitors isolated by screening the NINDS library of compounds for small molecules that decrease the activity of bGal (Residual Activity enclosed in brackets).
  • Ambroxol is an inhibitor of bGal, (as well as GCase) the dramatically increased IC50 (100 mM for bGgal versus 29 uM for GCase) combined with the toxicity of the compound at these higher mM levels reduces potential for practical use at the levels required for bGal inhibition.
  • Figure 19 shows the relative increase in beta-galactosidase activity in the presence of Budenoside for fibroblasts from a cat with GM1 gangliosidosis.
  • Beta- galactosidase was measured in lysates prepared from cat fibroblasts treated for 3 days with increasing concentrations of Budenoside using MU-bGal substrate. Hex activity levels remain unchanged.
  • Compounds enhancing GCase activity may be distinct from pharmacological chaperones. Also described herein is a class of non-inhibitory compounds that activate the purified enzyme in vitro.
  • the prototypical compound enhancing the activity of beta- glucocerebrosidase is HTS-01898, a benzothiophene. Although the mechanism by which they activate beta- glucocerebrosidase is not known, they do not appear to act as pharmacological chaperones.
  • Previously described compounds such as phosphatidylserine or taurodeoxycholate increase the activity of the purified beta-glucocerebrosidase. Although the detailed mechanism by which these compounds increase GCase activity is not known, it is proposed that they bind to sites on the enzyme and induce conformational changes that enhance the activity of the enzyme.
  • Formula 7 shows molecule HTS 01898 (also referenced as ethyl 2-[(2- morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate), a benzothiophene.
  • Formula 7A illustrates a related activator, having a structure similar to Formula 7 (also noted as HTS 1896).
  • Formula 7B (also noted as HTS 1897) is not an activator, but is an analog of HTS 1898.
  • Formula 7B has a nitrogen atom in the piperidine group that could act as a hydrogen donor.
  • Formula 7 and 7A activate Gcase at a pH between 5.5 and 7, but demonstrate less activation at pH 4-4.5.
  • the compound of formula 7 increases GCase activity in vitro (purified protein) and following treatment of patient cells for 5 days by 40-60% relative to DMSO treated protein or cells.
  • This compound does not act as an inhibitor, but does increase the Vmax of the GCase (cerezyme) when the activity of the enzyme is examined at different concentrations of the compound and substrate.
  • the compound does not stabilize the enzyme against thermal denaturation at 50 ° C.
  • the compound may activate the activity of the enzyme in a manner similar to SapC or other activators such as taurodeoxycholate or phosphatidylserine which differ structurally from compound HTS 01898.
  • Compound HTS 01898 is a benzothiophene.
  • benzothiophene compounds that contain a modified morpholino moiety attached to the 2 position and an ethyl carboxylate at the 3 position may function in a similar manner.
  • Other substitutions at positions 2 and 3 can be envisioned, fulfilling the same role as the morpholino or ethylcarboxylate substituents, could also result in an active compound.
  • Figure 20 illustrates that molecule HTS 01898 increases the activity of purified GCase in vitro.
  • HTS 01898 concentration in uM is shown on the X axis, while GCase activity is shown on the Y axis, illustrating a proportional relationship between concentration and activity.
  • Gcc ⁇ -glucocerebrosidase
  • GD Gaucher Disease
  • Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs) increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), allowing increased lysosomal transport of the enzyme.
  • high throughput screen of the 50,000 compound Maybridge library identified non- carbohydrate based inhibitory molecules referred to as MWP 01127, a 2,4 diamino 5- substituted quinazoline (IC50 5 ⁇ M) and a 5-substituted pyridinyl-2-furamide referred to as HTS 02324 (IC50 8 ⁇ M).
  • MWP 01127 and HTS 02324 are further assessed. Each molecule was found to raise the levels of functional Gcc 1.5-2.5 fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of Gcc in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium exchange mass spectrometry, identified a domain III active site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of MWP 01127 or HTS 02324, demonstrating a common effect on pharmacological chaperone enhancement of intracellular transport.
  • Gaucher Disease is the most common of the approximately 70 lysosomal storage diseases known. It is an autosomal recessive multisystem disorder with a high level of morbidity, and in severe cases is fatal at an early age.
  • the biochemical hallmark of GD is the storage of glucocerebroside (GlcCer), the precursor of 95% of all cellular glycosphingolipids, primarily in the tissues of the reticuloendothelial system and the brain arising from deficiency of lysosomal ⁇ -Glucocerebrosidase (Gcc, EC 3.2.1.45) encoded by the GBA gene.
  • Type I nonneuronopathic
  • II acute neuronopathic
  • III subacute neuronopathic
  • Type I GD incidence 1/40,000-1/60,000
  • the highest carrier frequency of Type I GD occurs amongst Ashkenazi Jewish adults (1/400 to 1/600) with about 90% of these individuals carrying one of just four alleles, specifically: N370S, F213I, L444P, G202R.
  • Type I GD patients (N370S heterozygotes/ homozygotes) have residual enzyme activity levels that are about 5-20% of normal, which closely matches the critical threshold level of 11-15% of normal activity, determined using a murine macrophage cell line treated with conduritol-B-epoxide (CBE) as a model of Type I GD, necessary to prevent the storage of GlcCer.
  • CBE conduritol-B-epoxide
  • Type I and to a lesser extent Type Il and III forms of GD currently benefit from two existing therapeutic approaches. These are: 1 ) enzyme replacement therapy (ERT) which ameliorates many manifestations of GD, and is both a safe and effective treatment. However, it is very costly; and 2) substrate reduction therapy (SRT) which attempts to limit the storage of GlcCer by using small molecules to inhibit its synthesis in vivo.
  • ERT enzyme replacement therapy
  • SRT substrate reduction therapy
  • N-butyl-DNJ N-butyl-DNJ (MiglustatTM or ZavescaTM) which inhibits the first step in glycolipid synthesis, and has shown some promise in treating GD Type I. However, it is not as effective as ERT, and the treatment is associated with unpleasant side effects, such as severe diarrhea.
  • EET enzyme enhancement therapy
  • PCs small molecule "pharmacological chaperones"
  • ER-QC ER quality control system
  • ESD ER associated degradation system
  • EET has shown promising preclinical results in at least four lysosomal enzyme deficiencies and could be applied to other lysosomal storage disorder.
  • successful PCs have also been competitive inhibitors of their target enzymes.
  • ERT has been successfully used to treat Type I GD patients there are benefits to considering other therapeutic modalities such as SRT or EET. These could be used in lieu of or in combination with ERT. Small molecules are less expensive, can be given orally and usually cross the blood-brain barrier, opening up the possibility of treating Type Il and III GD patients. As EET augments transit of the mutant Gcc from the ER, it also has the potential to attenuate the unfolded protein response and prevent ER stress that can lead to apoptosis and other inflammatory responses. Recently, components of the ER-QC system have been implicated as factors involved in determining the clinical impact of Gcc mutations.
  • the degree to which the different Gcc PCs enhance intracellular enzyme levels depends on the nature of the particular mutation.
  • the Gcc PC, N-octyl valienamine chaperones the F213I mutation better than the N370S mutation.
  • Overall the G202R substitution is most responsive to chaperoning, whereas the L444P mutation, associated with the neuronopathic form of GD in the homozygous form, thus far remains refractile to EET.
  • the intracellular activity of the L444P mutation can be increased by growing patients cells at a decreased temperature of 30° C.
  • Gaucher PC consist of glucose-based azasugars either with an alkylated side chain, e.g. N-butyl-deoxynojirimycin (NB-DNJ) or N-nonyl-deoxynojirimycin (NN-DNJ) and derivatives thereof, or without an alkylated side chain, e.g. lsofagomine (IFG).
  • alkylated side chain e.g. N-butyl-deoxynojirimycin (NB-DNJ) or N-nonyl-deoxynojirimycin (NN-DNJ) and derivatives thereof, or without an alkylated side chain, e.g. lsofagomine (IFG).
  • IFG is currently undergoing a phase I/I I clinical trial sponsored by Amicus Therapeutics.
  • IFG is a nanomolar inhibitor
  • Gcc activity is increased more than two-fold when GD type I patient fibroblasts are treated with 10-100 ⁇ M concentration of the compound.
  • Example 1 outlines the screening for Gcc inhibitors in a library of small drug- like molecules, the 50,000 compound Maybhdge library, for inhibitors of purified Gcc.
  • the availability of additional frameworks for Gcc inhibitors that also function as PCs could potentially increase the repertoire of GBA mutations responding to EET.
  • the examination of the effects on protein dynamics that occurs upon Gcc-binding of such non-carbohydrate based pharmacological chaperones could also be helpful in identifying the relevant region(s) of Gcc that when stabilized, increase its intracellular transport efficiency.
  • Example 1 As outlined in Example 1 , utilizing a high throughput screening (HTS) strategy, Gcc inhibitors that functioned as PCs in cell-based assays were identified in the Maybridge library. Their effect on the conformational dynamics of wild-type Gcc was determined by H/D-Ex which revealed a single common region in Gcc that was stabilized upon binding of IFG or either of these other two compounds. In this example, two compounds and their derivatives are discussed in more detail.
  • HTS high throughput screening
  • Non- carbohydrate based PCs for Gcc mutants were indirectly identified by first screening for inhibitors, i.e. reduction in the hydrolysis of the substrate, methyl-umbelliferyl ⁇ -D glucopyranoside (MUGIc), to the fluorogenic product methylumbelliferone (MU) by purified Gcc , in a primary HTS of the small molecule, drug-like library from Maybridge.
  • MUGIc methyl-umbelliferyl ⁇ -D glucopyranoside
  • MU fluorogenic product methylumbelliferone
  • the Z' statistic based on the activity of the enzyme in the presence the compound diluent, DMSO (High control), as compared to a known inhibitor, castanospermine (Low control), was calculated and resulted in value of 0.75, i.e. a very good separation of the high and low controls.
  • 680 hits were obtained based on a cut-off of 3 standard deviations from the mean of the activity. To facilitate screening of the hits in a secondary screen the hit zone was empirically lowered to 30% of the mean, resulting in 108 hits.
  • HTS 02324 (interchangeably referred to as "MAC” in this example)
  • MWP 01127 (interchangeably referred to as "MWP” in this example).
  • Table 4 illustrates that, using the colourimetric substrate p-nitrophenyl- ⁇ -D- glucopyranoside (pNPGIc), MWP and MAC were found to be low micromolar inhibitors of Gcc with IC50s of 7.8 and 4.7 ⁇ M, respectively.
  • IFG carbohydrate-based Gcc inhibitor
  • MAC ⁇ -galactosidase
  • ⁇ -GIc ⁇ -glucosidase
  • Hex Hex
  • MWP and IFG show detectable activity against these enzymes, albeit at concentrations more than a 100-fold higher. Both MWP and IFG also showed activity against human cytosolic ⁇ - glucosidase that also hydrolyses glucosylceramide and whose catalytic domain also consists of a ( ⁇ / ⁇ )8 TIM barrel.
  • Figure 21 shows changes in Gcc and Hex activity in GC patient fibroblasts using the MWP and MAC compounds.
  • GD patient cells carrying the N370S/N370S alleles were treated with MAC or MWP for five days.
  • Activity levels are relative to cells treated with solvent only (DMSO).
  • a Y-axis value of 1 indicates no change.
  • part C relative changes in Gcc (Black bars) and Hex (Gray bars) activity following treatment of either N370S/N370S or F213I/L444P GD cells with IFG (25 ⁇ M), MWP (12.5 ⁇ M) or MAC (12.5 ⁇ M) are shown.
  • Part D shows that compounds MWP and MAC increase levels of lysosomal Gcc in N370S/N370S Gaucher patient fibroblasts.
  • the iron-dextran colloid method was used to prepare a lysosome enriched fraction from N370S/N370S GD cells treated with compounds MWP (12.5 ⁇ M) or MAC (12.5 ⁇ M) or vehicle (0.1% DMSO).
  • Gcc upper panel
  • the lysosomal marker Lamp-2 lower panel
  • MWP and MAC are mixed-type inhibitors of Gcc and are most efficient at neutral pH.
  • the changes in apparent Km and Vmax values of Gcc for MUGIc were determined at 5-7 different concentrations of MAC or MWP.
  • Figure 22 illustrates that MWP and MAC are mixed type of inhibitors that function optimally at a neutral pH.
  • MWP square symbols
  • MAC round symbols
  • inhibitory compounds acting as PC would be most active at the neutral pH found in the ER (where their binding increases the stability of the mutant enzyme, offsetting some of the destabilizing effects of the mutation) and least active in the lysosome (where they could continue to inhibit the activity of the cognate enzyme). Consequently, the inhibitory activity of each compound was evaluated over a pH range of 4.5 to 7. Both MWP and MAC are most active as inhibitors at neutral pH ( Figure 22, part C)).
  • MWP-Ia simple quinazoline derivative
  • MWP-Ib diethoxy quinozoline derivative
  • Treatment of GD patient cells with MWP or MAC changes the intracellular localization of Gcc.
  • the intracellular localization of mutant Gcc before and after treatment with MWP or MAC was probed using indirect fluorescent immunostaining.
  • Cells were co-stained with IgGs against Gcc and either a marker for lysosomes, Lamp-1 , or an ER marker, protein disulfide isomerase (PDI).
  • PDI protein disulfide isomerase
  • Figure 23 shows the effect of compound MWP and MAC on trafficking Gcc from the ER to lysosomes in GD patient fibroblasts.
  • Cells, N370S/N370S (top panel) and L444P/F213I (bottom panel) were treated with DMSO, MWP (10 ⁇ M) or MAC (12 ⁇ M) (labels running down sides of panels).
  • DMSO DMSO
  • MWP 10 ⁇ M
  • MAC (12 ⁇ M)
  • Scale bars (white line) from left to right are 10 ⁇ m (DMSO), 13 ⁇ m (MWP) and 16 ⁇ m (MAC).
  • DMSO DMSO
  • MFP 13 ⁇ m
  • MAC 16 ⁇ m
  • MWP or MAC was probed using indirect fluorescent immunostaining.
  • Cells were co-stained with IgGs against Gcc and either a marker for lysosomes, Lamp-1 , or an ER marker, protein disulfide isomerase (PDI).
  • PDI protein disulfide isomerase
  • Gcc staining was diffuse and distinct from the punctate staining pattern of Lamp-1 ( Figure 23, part A) top and bottom panels labelled N370S/N370S and F213I/L444P). Instead Gcc staining colocalized with the ER marker PDI ( Figure 23, part B), as shown in top and bottom panels labelled N370S/N370S and F213I/L444P).
  • Figure 24 is a summary of perturbations in H/D-exchange from selected regions of Gcc in the absence versus the presence of ligands.
  • Deuterium buildups over time (30s to 3000s) for different regions of Gcc ⁇ ligand were mapped onto the sequence of human Gcc. Segments showing significant perturbations in the presence of IFG, MAC or MWP relative to the apo enzyme are superimposed upon the cartoon ribbon diagram representation of the IFG bound Gcc X-ray crystal structure (2NSX).
  • Surrounding the cartoon are representations of deuterium buildup curves for Gcc-segments 243-249, 187- 197, 310-312, 315-336, 130-134, and 386-396.
  • Deuterium buildup curves are shown for selected segments of Gcc in the absence of ligand (cross symbol), or in the presence of excess IFG (triangular symbol), MWP (square symbol) or MAC (circle symbol). Illustration generated with PyMOLTM (DeLano Scientific) and KaleidagraphTM (Synergy Software).
  • Figure 24 illustrates that in the presence of any of these compounds, there was a decrease in the deuteration of peptides surrounding the active site relative to the unliganded control.
  • IFG- binding perturbed the largest area of Gcc (16/26 of peptides), compared to MWP (6/26) and MAC that affected only one region, encompassed by peptide 243-248. Surprisingly this is the only region that is most clearly and strongly perturbed by all three ligands. When examined over time, each of the regions that were affected by ligand binding was perturbed to different degrees by the three compounds.
  • MWP and MAC function as PC using three independent approaches. Firstly
  • MWP and MAC increase Gcc activity 1.5-2.5 fold in GD patient cells bearing either the N370S or F213I alleles. Secondly, they specifically increase the levels of Gcc in lysosomes of these cells by more than two-fold.
  • Lamp-1 Lysosomal Associated Protein-1
  • PDI Protein Disulfide lsomerase
  • Gcc active site it is only inhibited by MWP at a ten-fold higher concentration relative to Gcc. Consequently, the activity levels of the neutral cytosolic ⁇ -glucosidase would not be expected to be affected by the concentration of MWP (12 ⁇ M) that was shown to enhance Gcc activity. It is interesting that IFG like MWP also inhibits neutral ⁇ -glucosidase, suggesting that the two compounds may interact with the active site in a similar manner.
  • the quinazoline framework in MWP is found in drugs such as trimetrexate, antifungal and antineoplastic agents that function as dihydrofolate reductase (DHFR) inhibitors, as well as doxazocin, a selective ⁇ -1- adrenergic blocker. Both these compounds do not inhibit Gcc activity (data not shown), possibly due to substitutions on the amino groups or the 6-position of quinazoline.
  • DHFR dihydrofolate reductase
  • doxazocin a selective ⁇ -1- adrenergic blocker. Both these compounds do not inhibit Gcc activity (data not shown), possibly due to substitutions on the amino groups or the 6-position of quinazoline.
  • the selectivity of MWP for Gcc over DHFR could be increased by modifying the substituents on the 5-position of quinazoline.
  • IFG lacks a hydrophobic group that could have such an effect on residues 235-252.
  • the two conformers of the enzyme may relate to conformational changes that Gcc is proposed to undergo following activation via its interaction with SapC.
  • Maybridge collection (Maybridge PLC, UK) were used in the initial screen. Compounds evaluated in the secondary screen and their derivatives were re-ordered from Maybridge PLC (UK) or Chembridge (USA) and solubilized using DMSO or water.
  • Human Gcc (cerezyme) was purchased from Genzyme (USA).
  • Genzyme USA
  • a Concanavalin A-binding fraction of human placental lysate was used as a source for lysosomal enzymes ⁇ -Gal and ⁇ -Glc.
  • Human Hex was purified from placenta. Almond ⁇ -glucosidase was purchased from Sigma (USA). Human neutral ⁇ -glucosidase kindly provided by N.
  • the colourimetric substrate p- nitrophenyl- ⁇ -D-glucopyranoside (SIGMA, USA) was also used to monitor Gcc, human cytosolic ⁇ -glucosidase and Almond ⁇ -glucosidase activity.
  • N370S fibroblast cell line from a patient diagnosed with the Type I Gaucher disease homozygous for the N370S mutation (Molecular Diagnostics Laboratory, SickKids, Toronto, Ont, Canada); "F213I” fibroblast cell line from a patient diagnosed with type I Gaucher bearing the F213I/L444P alleles (kindly provided by F. Choy, University of Victoria, Canada). All cell lines were grown in ⁇ -minimal essential media ( ⁇ -MEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA), and antibiotics Penicillin/ Streptomycin (Invitrogen, USA) at 37°C in a humidified CO 2 incubator.
  • ⁇ -MEM ⁇ -minimal essential media
  • FBS fetal bovine serum
  • Penicillin/ Streptomycin Invitrogen, USA
  • Reaction rates were calculated as the slope of the data of the second to ninth data point, inclusive.
  • Each reaction consisted of Gcc (72 ⁇ g/mL), Taurodeoxycholate (TdC, 0.24%), Human Serum Albumin (0.1%), and MUGIc substrate (625 ⁇ M) and compounds in 20 mM Citrate-Phosphate (CP) buffer.
  • Library compounds dissolved in DMSO were added to a final concentration of 20 ⁇ M.
  • Each 80 compound set from the library was analyzed in duplicate using two quadrants of the 384 well plate. Eight replicate High (2% DMSO) and Low controls (2% DMSO 1 Castanospermine (45 ⁇ M)) were included in each quadrant of the 384 well plate.
  • the residual activity (RA) of the enzyme in the presence of each of the compounds was determined.
  • 8 replicate High and Low controls were used to generate a Z' statistic, which measures the variability of the rate values for Gcc.
  • a Z' statistic of 0.75 was obtained for the primary Gcc screen i.e. a very good separation of the high and low controls.
  • Gcc and other glycosidase Activity Assays were measured by release of 4-methylumbelliferyl fluorophore from MUGIc. Assays (50 ⁇ L) contained Citrate - Phosphate buffer (CP) (20 mM, pH 5.5) TdC (0.2%) and MUGIc (0.8 mM ). For the endpoint assay, the reaction at 37 0 C was terminated by raising the pH to 10.5, above the pKa of 4-MU by adding 2-amino 2-methyl 1-propanol (0.1 M, 200 ⁇ L).
  • Mass Spectrometry The mass of selected secondary hits was confirmed by the Advanced Proteomic Centre at Sickkids (Toronto, Canada) using a QToF mass spectrometer (Waters/Micromass, Manchester, UK)
  • Indirect Immunofluorescence and confocal microscopy imaging were performed as follows. In brief, cells were seeded onto 18 mm diameter coverslips for 16-2Oh, then washed and fixed with paraformaldehyde (2.5%)(EMS) in PBS (pH 7.2), for 20 min at 37°C. Blocking and permeabilization was performed for 1h at room temperature with saponin (0.2%) (Sigma) and 10% of either goat or horse normal serum (Wisent Inc.) in phosphate-buffered saline (SS-PBS).
  • EMS paraformaldehyde
  • PBS pH 7.2
  • Blocking and permeabilization was performed for 1h at room temperature with saponin (0.2%) (Sigma) and 10% of either goat or horse normal serum (Wisent Inc.) in phosphate-buffered saline (SS-PBS).
  • Primary antibodies used were rabbit polyclonal IgG anti-human Gcc (raised by us against purified recombinant Gcc), mouse monoclonal IgGI anti-human LAMP-1 (DHSB, Iowa) and anti-rat Protein Disulfide lsomerase (PDI) (Stressgen Bioreagents, Canada). Secondary antibodies were Alexa fluor 488 chicken anti-rabbit IgG and Alexa fluor 594 chicken anti-mouse IgG (Molecular Probes) at a 1/200 dilution in SS- PBS solution.
  • Samples were analyzed using a Zeiss AxiovertTM confocal laser microscope equipped with a 63 x 1.4 numerical aperture Apochromat objective (Zeiss) and LSM 510 software; DAPI-stained nuclei were detected on the same system with a Chameleon two- photon laser. Confocal images were imported and contrast/ brightness adjusted using VolocityTM 4 program (Improvision inc.). Intensity settings were not changed when recording the images of Gcc or PDI staining between the same treated and untreated cell lines.
  • a Gcc stock (80 ⁇ M) was prepared by dissolving Cerezyme powder (31 mg) into H 2 O (500 ⁇ l_) .
  • Stocks (47 mM) of IFG, MAC or MWP were prepared in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • a 59:1 molar ratio of IFG, MAC or MWP to Gcc was prepared by combining the Gcc stock (50 ⁇ l_) with the compound stock (5 ⁇ l_).
  • a DMSO containing "no-ligand" control was prepared by combining the Gcc stock (50 ⁇ l_) with DMSO (5 ⁇ L).
  • the quenched solution was immediately pumped at 200 ⁇ L/min over an immobilized porcine pepsin column (104 ⁇ L bed volume) with trifluoroacetic acid (TFA) (0.05%) for three minutes with contemporaneous collection of proteolytic products by way of a trap column (4 ⁇ L bed volume).
  • Pepsin was immobilized on Poros 20 AL media (30 mg/mL, Applied Biosystems) as per the manufacturer's instructions.
  • the software program SEQUESTTM (Thermo Fisher Scientific, San Jose, CA) was used to tentatively identify the sequence of dynamically-selected parent-peptide ions. This tentative peptide identification was verified by visual confirmation of the parent ion charge state. These peptides were then further examined to determine if the quality of the measured isotopic envelope was of sufficient quality to allow an accurate geometric centroid determination. Centroid values were then determined using a proprietary program developed in collaboration with Sierra Analytics. Back-exchange corrections and deuteration level calculations were implemented.
  • FIG. 25A illustrates data obtained in a primary screening of compounds relating to an inhibition assay, to confirm and determine the IC50 value in the presence of 0.8 mM MUGIc.
  • Figure 25B illustrates further data obtained in a primary screening of compounds relating to an inhibition assay, to confirm and determine the IC50 value in the presence of 0.8 mM MUGIc.
  • Figure 26A illustrates data obtained in a primary screening of compounds relating to heat denaturation attenuation assay, i.e. remaining Gcc activity in the presence of the compound following heating to 50 0 C for 20 min.
  • Figure 26B illustrates further data obtained in a primary screening of compounds relating to heat denaturation attenuation assay, i.e. remaining Gcc activity in the presence of the compound following heating to 50 0 C for 20 min.
  • Figure 27A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Gcc in GD patient fibroblasts (N370S/N370S) after cells were treated for five days with the indicated concentration of test compound.
  • Figure 27B illustrates further data obtained in a primary screening of compounds relating to changes in intracellular levels of Gcc in GD patient fibroblasts
  • Figure 28A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts
  • Figure 28B illustrates further data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts
  • Table 5 provides a list of the 108 primary hits that reduced Gcc residual activity below 30%. Shown are the Maybridge Identification Code for the compound and directly beneath the code is proivided the IC50 value (in ⁇ M) determined in the secondary screen (column labelled "Code”) and the 2D structure of the compound is shown beside each (column labelled "Structure”). Table 5

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Abstract

La présente invention concerne des compositions thérapeutiques et des procédés de traitement de maladies lysosomales, telles que la maladie de Gaucher, la gangliosidose à GM1, la maladie de Morquio de type B et la maladie de Parkinson. Lesdites compositions comprennent des composés qui présentent une activité inhibitrice des glucocérébrosidases et des bêta-galactosidases, destinés à être utilisés en tant que chaperons pharmacologiques pour les formes mutantes de l'enzyme. L'invention concerne également des procédés de traitement impliquant l'administration de quantités thérapeutiquement efficaces de ces composés aux sujets concernés.
PCT/CA2008/001843 2007-10-18 2008-10-17 Compositions et procédés permettant d'améliorer l'activité enzymatique dans la maladie de gaucher, la gangliosidose à gm1, la maladie de morquio de type b et la maladie de parkinson WO2009049421A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
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WO2010116141A3 (fr) * 2009-04-09 2011-03-31 Summit Corporation Plc Association médicamenteuse pour le traitement des maladies protéostatiques
US8536148B2 (en) 2009-09-04 2013-09-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Disabling autophagy as a treatment for lysosomal storage diseases
JP2014515001A (ja) * 2011-04-20 2014-06-26 塩野義製薬株式会社 Ｔｒｐｖ４阻害活性を有する芳香族複素環誘導体
WO2014145512A3 (fr) * 2013-03-15 2014-12-31 President And Fellows Of Harvard College Inhibiteurs de l'autophagie à petites molécules, efficaces, et leurs procédés d'utilisation
WO2016035934A1 (fr) * 2014-09-05 2016-03-10 한국생명공학연구원 Modèle cellulaire humain de gangliosidose à gm1 et utilisation de ce dernier
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WO2016120808A1 (fr) 2015-01-28 2016-08-04 Minoryx Therapeutics S.L. Hétéroarylaminoisoquinolines, procédés pour les préparer et leurs utilisations thérapeutiques
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WO2020023390A1 (fr) 2018-07-25 2020-01-30 Modernatx, Inc. Traitement enzymatique substitutif basé sur l'arnm combiné à un chaperon pharmacologique pour le traitement de troubles du stockage lysosomal
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JP2014515001A (ja) * 2011-04-20 2014-06-26 塩野義製薬株式会社 Ｔｒｐｖ４阻害活性を有する芳香族複素環誘導体
US9340500B2 (en) 2011-04-20 2016-05-17 Shionogi & Co., Ltd. Aromatic heterocyclic derivative having TRPV4-inhibiting activity
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WO2016035934A1 (fr) * 2014-09-05 2016-03-10 한국생명공학연구원 Modèle cellulaire humain de gangliosidose à gm1 et utilisation de ce dernier
US10035830B2 (en) 2014-09-05 2018-07-31 Korea Research Institute Of Bioscience And Biotechnology Method for treatment of GM1 gangliosidosis
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KR101916901B1 (ko) * 2016-01-29 2018-11-08 한양대학교 에리카산학협력단 2―아릴카보닐하이드라진카보티오아마이드 유도체 화합물을 포함하는 소포체 스트레스 관련 질환의 예방 또는 치료용 약제학적 조성물
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US11440898B2 (en) 2016-12-28 2022-09-13 Minoryx Therapeutics S.L. Isoquinoline compounds, methods for their preparation, and therapeutic uses thereof in conditions associated with the alteration of the activity of beta galactosidase
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