WO2009049421A1 - Compositions and methods for enhancing enzyme activity in gaucher, gm1-gangliosidosis/morquio b disease, and parkinson's disease - Google Patents

Abstract

Therapeutic compositions and methods for treatment of lysosomal storage disorders, such as Gaucher, GM1-gangliosidosis/Morquio B disease and Parkinson's disease are described herein. The compositions comprise compounds having glucocerebrosidase and beta-galactosidase inhibitory activity for use as pharmacological chaperones for mutant forms of the enzyme. Methods of treatment involve providing therapeutically effective amounts of such compounds to subjects in need thereof.

Description

COMPOSITIONS AND METHODS FOR ENHANCING ENZYME ACTIVITY IN

GAUCHER. GM1-GANGLIOSIDOSIS/MORQUIO B DISEASE.

AND PARKINSON'S DISEASE

FIELD OF THE INVENTION

[0001] The present invention relates generally to therapeutic compositions and methods for treatment of Gaucher disease, GM1 -gangliosidosis, Morquio B disease, and Parkinson's disease.

BACKGROUND OF THE INVENTION

[0002] In lysosomal storage disorders (LSD), substrates accumulate to pathological levels in the lysosome as a result of a mutation in a catabolic enzyme. Both enzymes beta- glucocerebrosidase and beta-galactosidase are involved in the catabolism of glycosphingolipids.

[0003] Individuals with a mutation in the enzyme beta-galactosidase have a reduced ability to hydrolyse the terminal galactose residue from GM1 ganglioside or keratan sulphate resulting in accumulation of this substrate in neuronal lysosomes, as seen in GM1- gangliosidosis and Morquio B patients. There is presently no treatment available for GM1 gangliosidosis/Morquio B patients.

[0004] 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. Currently, 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.

[0005] In some instances of Parkinson's disease, where a mutant allele is present, a treatment involving correction of lipid storage is desirable. In these Parkinson's patients, a mutant enzyme form results is dysfunctional due to improper folding. [0006] Although the 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). Short-lived (functional) proteins in the cytosol and soluble, but misfolded proteins in the ER are degraded by the cytosolic ubiquitin-proteasome system (UPS). The two degradation systems interact and macroautophagy (referred to as autophagy hereafter) can partially compensate when the UPS is inhibited/ overloaded. Importantly both systems can be up-regulated in response to ER-Stress resulting from the unfolded protein response (UPR) (4).

[0007] Endoplasmic reticulum (ER)-stress is a factor in the pathobiology of Gaucher and other lysosomal storage diseases (LSDs). Earlier reports associating ER-stress with the pathophysiology of LSDs focused on the stored substrate's (glucocerebroside in Gaucher disease) ability to inhibit Ca²⁺ uptake by the ER as the primary source of stress, resulting in apoptosis and neurodegeneration, e.g. (5). However Wei et al. (6) have recently shown that cell death by apoptosis is mediated by ER- and oxidative- stresses in both neurodegenerative and non-neurodegenerative LSDs, including in fibroblasts from Gaucher patients (7). They also found that chemical disruption of lysosomal homeostasis induces ER- stress in normal fibroblasts, suggesting the existence of a cross-talk between the lysosomes and the ER, for example, an abnormality in one organelle can adversely affect the other. They further demonstrated that alleviation of ER- and oxidative- stresses by non-specific chemical chaperones, such as trimethylamine-N-oxide, protects LSD cells from apoptosis. Finally they reported that LSD fibroblasts showed sensitivity to brefeldin-A (Baf)-induced apoptosis. 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 α-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). [0008] Importantly, the increased risk of developing Parkinson disease and other synucleinopathies documented for even carriers of Gaucher disease (10, 11 ) could be explained by inhibited autophagic flux due to increased steady state levels of glucocerebroside in lysosomes and/or misfolded glucocerebrosidase protein in the ER, resulting from the mutant allele. Pharmacological chaperones target both of these potential mechanisms and could be used to reverse or prevent the progression of Parkinson's diseases in newly diagnosed patients found also to be carriers of Gaucher disease.

[0009] It is, therefore, desirable to provide therapeutic compositions and methods for treatment of these conditions.

SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to provide a therapeutic composition or method for treatment of a lysosomal storage disorder such as Gaucher disease, GM1- gangliosidosis/Morquio B disease, or Parkinson's disease.

[0011] In a first aspect, there is provided a composition comprising a therapeutically effective amount of a beta-glucocerebrosidase inhibitor together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder.

[0012] Further, 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 is provided.

[0013] A composition is provided comprising 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-1H-pyrrol-3-yliden} methyl) amino] ethyl} amino) methylidene]-1-[4- (trifluoromethyl) phenyl] pyrrolidine-2,3,5-trione) according to formula 6; (e) ethyl 2-[(2- morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1 -benzothiophene-3-carboxylate) according to formula 7; or (f) the compound according to formula 7A, together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder. [0014] In another aspect, there is provided a method for treating a lysosomal storage disorder comprising administering a therapeutically effective amount of a beta- glucocerebrosidase inhibitor to a subject in need thereof.

[0015] A method is provided 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.

[0016] 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) phenyl] pyrrolidine-2,3,5-trione) according to formula 6; (e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3- carboxylate) according to formula 7; or (f) the compound according to formula 7A, to a subject in need thereof.

[0017] Further, there is provided a use of a therapeutically effective amount of an N-

(4-pyridinyl)-2-furamide derivative for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof.

[0018] Further, the invention provides a use 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-1H-pyrrol-3-yliden} methyl) amino] ethyl} amino) methylidene]-1-[4- (trifluoromethyl) phenyl] pyrrolidine-2,3,5-trione) according to formula 6; (e) ethyl 2-[(2- morpholinoacetyl)amino]-4,5, 6, 7-tetrahydro-1-benzothiophene-3-carboxylate) according to formula 7; or (f) the compound according to formula 7A, for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof. [0019] Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures.

[0021] 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).

[0022] Figure 15 shows increase in activity levels of GCase in Gaucher patient fibroblasts homozygous for the N370S mutation treated with increasing concentrations of

Isofagomine.

[0023] 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).

[0024] Figure 17 is a schematic illustration of the docked pose of MWP 00127 of

Formula 1 in an active site.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Figure 21 shows changes in Gcc and Hex activity in GC patient fibroblasts using compounds outlined in Example 6.

[0029] Figure 22 shows inhibitory function of MWP and MAC compounds outlined in

Example 6. [0030] Figure 23 shows the Effect of compound MWP and MAC on trafficking Gcc from the ER to lysosomes in GD patient fibroblasts.

[0031] Figure 24 is a summary of perturbations in H/D-exchange from selected regions of GCC in the absence versus presence of lignads.

[0032] 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.

[0033] 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.

[0034] 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 50⁰C for 20 min.

[0035] 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 50⁰C for 20 min.

[0036] 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.

[0037] Figure 27B illustrates further 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.

[0038] Figure 28A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts

(N370S/N370S) after cells were treated for five days with the indicated concentration of test compound.

[0039] 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

(N370S/N370S) after cells were treated for five days with the indicated concentration of test compound. DETAILED DESCRIPTION

[0040] Described herein are pharmacological chaperones (PC), 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.

[0041] Compounds having 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.

[0042] Because there is currently no treatment for GM1 gangliosidosis/Morquio B patients, and current Gaucher therapies are expensive, the compounds described herein for use as pharmacological chaperones represent an opportunity to treat patients at a reduced costs and with increased specificity.

[0043] A composition is described herein 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:

Formula 3. [0044] The lysosomal storage disorder for treatment may be Gaucher disease, for example: late-onset Gaucher disease, GM1-gangliosidosis, or Morquio B disease.

[0045] Further, the lysosomal storage disorder may be Parkinson's disease associated with Gaucher disease.

[0046] A method of treating a lysosomal storage disorder is provided 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. Further, the N-(4-pyridinyl)-2-furamide derivative may comprise 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3.

[0047] In this method, 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.

[0048] Further, there is defined herein the use of a therapeutically effective amount of an N-(4-pyridinyl)-2-furamide derivative for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof. 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.

[0049] A composition is provided, comprising a therapeutically effective amount of:

(a) 5[(4-chlorophenyl) thio] quinazoline-2,4-diamine) according to Formula 2:

Formula 2 [0050] or a derivative of this compound comprising substitution of an amine group at position 2 or 4 may be used.; (b) 4-amino-1 H-1 ,5-benzodiazepine-3-carbonitrile hydrochloride according to Formula 4:

HCI Formula 4

[0051] or a derivative of this compound; (c) 4-(2-chloro-6-fluorostyryl) benzyl (4- fluoroanilino) methanimidothioate hydrobromide) according to Formula 5:

[0052] (d) 4-[phenyl ({2-[(phenyl {2,4,5-thoxo-i -[4-(trifluoromethyl)phenyl]tetrahydro-

1 H-pyrrol-3-yliden} methyl) amino] ethyl} amino) rnethylidene]-1-[4-(thfluoromethyl) phenyl] pyrrolidine-2,3,5-trione) according to Formula 6:

Formula 6;

[0053] (e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-

3-carboxylate) according to Formula 7:

[0054]

Formula 7;

[0055] or the compound according to Formula 7A:

[0056] Formula 7 A;

[0057] together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder.

[0058] The lysosomal storage disorder is 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.

[0059] 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-(trifluoromethyl) phenyl] pyrrolidine- 2,3,5-trione) according to Formula 6; (e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7- tetrahydro-i-benzothiophene-3-carboxylate) according to Formula 7; or (f) the compound according to Formula 7A to a subject in need thereof.

[0060] Use of a therapeutically effective amount of (a) a compound according to

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.

[0061] EXAMPLE 1

[0062] Determining GCase Activity of Molecules from Maybridge Library.

[0063] Compounds having GCase inhibitory activity were identified following a screen of the Maybridge library.

[0064] Reactions were set-up in a black-walled 384 well plates using SAGIAN Core

System (Beckman Coulter, Inc. Fullerton, CA, USA) equipped with an ORCA arm for labware transportation, a Biomek FX with a 96-channel head for liquid handling, an Analyst HT (Molecular Devices Corp., Sunnyvale, CA, USA) for fluorescence detection. All reactions were performed in duplicate (separate plates). High controls (64 wells) consisted of Gcase (cerezyme) (400 ng/ml ) in Citrate Phosphate (CP) buffer containing pH 5.5 and 0.25% Taurocholate and 0.5% DMSO. Low controls (64 wells) additionally contained castanospermine at 1 μM. Test wells contained 10 mM of each library compound

[0065] GCase activity was measured by release of 4-methylumbelliferyl fluorophore from 4-methylumbelliferyl-b-D-glucopyranoside (MUbGIc). Total Assay volume was 50 μl_, 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.

[0066] 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.

[0067] 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). Column 9 (Heat _min) shows remaining activity following thermal denaturation (minimum value), while column 10 (Heat _max) shows remaining activity following thermal denaturation (maximum value). Column 11 provides lc50 value (wherein the value = 20 +8 stated value in micromolar). Column 12 provides the lc50 correlation coefficient, representing how well the dose response fits to a sigmoidal curve (a value of 1 = perfect fit).

Table 1 - GCase Inhibitors

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

[0068] 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.

[0069] Although 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. However, the inhibitory strength did not necessarily correlate with chaperoning efficacy. For example, compound MWP 01127 has three-fold lower lc50 relative to compound HTS 02324. Yet MWP 01127 increases GCase to a similar extent as compound HTS 02324 but at a three-fold lower concentration.

[0070] 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.

[0071] In Formula 1 (or MWP 01127) 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. As well, 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.

Formula 1 [0072] 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.

[0073] In Formula 2 (or MWP 01128) 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. As well, 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.

Formula 2

[0074] 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.

Formula 3

[0075] 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.

HCI Formula 4

[0076] 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.

Formula 5

[0077] Table 1 illustrates formula 6 showing molecule "73" or RJC 02132 (also referenced as 4-[phenyl ({2-[(phenyl {2_I4,5-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.

Formula 6 [0078] 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.

[0079] Figure 1 illustrates the activity of RJC 01351 , CD 02284, CD 00240, and RH

00783.

[0080] Figure 2 illustrates BTB 03449, MWP 01127 (molecule 60); BTB 11331 ; JFD

03146; RH 01716; BTB 02998; RJC 02479; and SB 01940. Notably, the activity increased with increasing concentrations of MWP 01127.

[0081] Figure 3 illustrates JFD 00243; CD 00466; RH 01061 ; JFD 03132; SP 00756;

SP 00531 ; BLT 00154; BTB 07350..

[0082] Figure 4 illustrates CD 01259; RH 02106; KM 04397; KM 04984; HTS 09832;

MBE 00148; BTB 01437; and JFD 02972.

[0083] Figure 5 illustrates BTB 14755; HTS 01243; MWP 01128 (molecule 61); HTS

06789; BTB 02328; RJF 01158; SP 00526; KM 03455. Notably, the activity increased with increasing concentrations of MWP 01128.

[0084] Figure 6 illustrates BTB 03346; SP 00991 ; SPB 04204; BTB 07496; MWP

00649; JP 00649; JP 00639; SP 00867; and BTB 13814.

[0085] Figure 7 illustrates SPB 00915; JFD 03571 ; RJC 00357; RJC 02270; HTS

04578; SP 00859; RH 00643; and SPB 00968.

[0086] Figure 8 illustrates XAX 00155; JFD 02816; CD 03798; HTS 07133; KM

07661 ; SPB 07453; BTB 13902; and RJF 00257..

[0087] Figure 9 illustrates KM 04549; RJC 00100; S 15348; RH 00644; SEW 06186;

NH 00306; MBE 00145; and CD 00469.

[0088] Figure 10 illustrates BTB 06478; HTS 07424; BTB 12892; HTS 04241 ; BTB

11079; BTB 13089; BTB 14729; and MBE 00142. [0089] Figure 11 illustrates BTB 08066; BTB 11015; BTB 11334; SP 00752; BTB

15232; HTS 07780; SEW 02957; and SEW 04852.

[0090] Figure 12 illustrates RDR 01439; SEW 00556; RJC 02132 (molecule 73);

BTB 05211 ; ML 00213; SP 01065; RJF 01159; and CD 00502. Notably, the activity increased with increasing concentrations of RJC 02132.

[0091] Figure 13 illustrates BTB 13370; BTB 14731 ; SEW 04707; KM 04550; S

13592; S 05133; MBE 00152; and RJC 01843.

[0092] Figure 14 illustrates BTB 10392; MBE 00151 ; BTB 13368; CD 11038; HTS

02324; SEW 05565; HTS 06793; and SPB 02638.

[0093] EXAMPLE 2

[0094] MWP 01127 as a Pharmacological Chaperone

[0095] Figure 15 shows lsofagomine concentrations versus activity of GCase. There is an increased activity levels of GCase in Gaucher patient fibroblasts homozygous for the N370S mutation (N370S/N370S) treated with increasing concentrations of lsofagomine, grown over a five day period. 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.

[0096] 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. In contrast, the optimal concentration of MWP 01127 in terms of chaperoning efficacy (2.5 fold at 15 μM) more closely matches the IC50 values (7μM). These data indicate that MWP 01127 is able to more readily enter the cell (and the endoplasmic reticulum) in comparison to Isofagomine. On the basis of the behavior of Isofagomine, it appears that 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). [0097] 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.

[0098] In the docked pose, hydrogen bonding is suspected to occur at Tyr 244 and

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.

[0099] EXAMPLE 3

[00100] Chaperoning beta-Galactosidase Mutants (GM1 gangliosidosis) by N- nonyldeoxygalactonojirimycin

[00101] 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).

[00102] Other 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.

[00103] Table 2 also illustrates such an N-alkylated compound: N-butyl deoxygalactonojirimycin, also known as N-butyl DGJ.

[00104] These two structures of two known beta-galactosidase inhibitors. Shown are the compound name, the IC50 in micromolar (in parentheses) using 0.3 mM MUbGaI substrate, and the structures.

[00105] To test the chaperoning activity of N-n-DGJ, immortalized cat fibroblasts from

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.

[00106] 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.

[00108] EXAMPLE 4

[00109] 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). Although 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.

[00110] 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. These results indicate that Budenoside functions as a pharmacological chaperone for mutant bGal in cat fibroblasts.

[00111] EXAMPLE S

[00112] Activators of GCase Activity.

[00113] 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.

[00114] 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.

HTS 1898 HTS 1896 HTS 1897

Formula 7 Formula 7A Formula 7B

[00115] 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.

[00116] Compound HTS 01898, is a benzothiophene. Thus other 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.

[00117] 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.

[00118] EXAMPLE 6

[00119] Point mutations in β-glucocerebrosidase (Gcc) can result in a deficiency of both Gcc activity and protein in lysosomes causing Gaucher Disease (GD). 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. As described in Example 1 , above, 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 μM) and a 5-substituted pyridinyl-2-furamide referred to as HTS 02324 (IC50 8 μM).

[00120] In this example, 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.

[00121] 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 β-Glucocerebrosidase (Gcc, EC 3.2.1.45) encoded by the GBA gene. Although the disorder represents a broad and continuous spectrum of clinical involvement, three main clinical phenotypes are generally recognized: Type I, nonneuronopathic; II, acute neuronopathic: and III, subacute neuronopathic. Type I GD (incidence 1/40,000-1/60,000) accounts for the bulk of the patients, who are generally mildly affected. 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.

[00122] 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. Thus, like other lysosomal storage disorders, it appears that only a relatively small increase in Gcc activity is necessary to prevent and/ or reverse the clinical progression of the disease.

[00123] 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. Currently the only FDA-approved SRT-agent is N-butyl-DNJ (Miglustat™ or Zavesca™) 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. Currently, a new therapeutic strategy, enzyme enhancement therapy (EET), is being evaluated in Phase I/I I clinical trials. EET uses small molecule "pharmacological chaperones" (PCs) to stabilize the native conformation of a mutant enzyme as it folds in the endoplasmic reticulum (ER)₁ allowing it to pass the ER quality control system (ER-QC), avoiding the ER associated degradation system (ERAD), and be transported to the lysosome. EET has shown promising preclinical results in at least four lysosomal enzyme deficiencies and could be applied to other lysosomal storage disorder. To date successful PCs have also been competitive inhibitors of their target enzymes. It is believed that once the PC-enzyme complex reaches the lysosome, the large amounts of stored substrate(s) will displace the PC and continue to stabilize the enzyme. However, it is desirable to identify PCs that inhibit best at the neutral pH of the ER in order to optimize its binding strength and thus its ability to stabilize the folding process, and minimize its inhibitory properties once the complex enters the acidic environment of the lysosome, where stored substrate should continue to stabilize the enzyme.

[00124] Although 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.

[00125] The degree to which the different Gcc PCs enhance intracellular enzyme levels depends on the nature of the particular mutation. For example, 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. However, the intracellular activity of the L444P mutation can be increased by growing patients cells at a decreased temperature of 30° C.

[00126] To date most 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).

IFG is currently undergoing a phase I/I I clinical trial sponsored by Amicus Therapeutics. Although IFG is a nanomolar inhibitor, Gcc activity is increased more than two-fold when GD type I patient fibroblasts are treated with 10-100μM concentration of the compound. Other more potent and selective Gcc inhibitors, alpha-1-C-nonyl iminoxlitol and 6-C-nonyl isofagomine, with Ki values, 2nM and 0.6nM, respectively, have been described that also more than double Gcc residual activity in Gaucher patients fibroblasts but act at nanomolar concentrations.

[00127] The mechanism by which NB-DNJ, NN-DNJ or IFG-binding stabilizes the wild- type enzyme has been explored by x-ray crystallography at acidic and/or neutral pH. The general consensus is that residues from three loops (residues 311-319, 342-354 and 393- 396), surrounding the substrate-binding pocket are stabilized upon binding of the glycone moiety of these PCs. The most striking finding of the crystallization studies was that PC- binding preferentially stabilizes a helical-turn conformation within a loop region located at the mouth of the active site (residues 311-319). It is proposed that the helical-like conformation is important for the chaperoning activity of IFG. However, crystal contacts, interchain or intermolecular contacts, which occur solely as the result of protein crystallization, could have obscured the identification of additional regions of importance in chaperone-enhanced intracellular transport.

[00128] Hydrogen/ deuterium exchange coupled with mass spectrometry (H/D-Ex) has been used to probe protein dynamics in solution in the presence and absence of ligand. This procedure has been used to map ligand binding sites and to detect ligand-induced conformational and/or dynamic changes of a protein. Using this approach, changes have been detected in protein dynamics in several regions of Gcc following IFG binding. Five of these regions, 119-127, 177-184, 230-240, 310-312 and 386-400, are consistent with the locations of residues involved in PC binding as determined by crystallography. However, additional perturbations observed in regions 187-197, 243-249 and 414-417 were not previously seen. These results highlight the importance of examining the structural dynamic properties of Gcc-PC complexes in solution.

[00129] 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. In this example, 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.

[00130] 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.

[00131] Results

[00132] Primary screen for identification of Gcc inhibitors. The following methodology was used in Example 1, and is described in more detail here. 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 β-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. To evaluate the signal to noise ratio, 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. Following screening of 49,586 compounds for activity against Gcc, 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.

[00133] Secondary screen to validate PC activity of each hit. Three distinct characteristics of each hit were evaluated in the secondary screens using 4 assays and six different concentrations of the candidate compound. The characteristics evaluated for each hit were: 1 ) Their IC50; each of the 108 hits from the primary screen showed a dose response curve yielding from single digit to more than 100 μM IC50 values (26 compounds with values ranging from 0.7 - 9.9 μM; 49 with values ranging between 10 - 50 μM; 16 hits with values between 50 and 100 μM and 17 compounds with values greater than 100 μM). 2) Their ability to attenuate heat denaturation. Only 49 of the 108 hits attenuated the thermal denaturation of Gcc to varying degrees. The remaining 59 hits were excluded as candidate PCs because 46 of the compounds had no effect and 13 resulted in increased thermal denaturation. 3) Their ability to function as specific, nontoxic PCs in GD cells. Changes in activity levels of both mutant intracellular Gcc and wild type Hex, to control for specificity and toxicity, were monitored following treatment of GD cells with varying concentrations of each hit. After treating GD (N370S/N370S) cells for five days with each of the remaining hits, approximately 20% produced a ≥1.4 fold increase in Gcc activity relative to cells treated with DMSO. Of these 21 compounds, certain compounds, showed increased Gcc activity over two concentration ranges and did not affect the activity of Hex at the corresponding concentration.

[00134] In this example, two lead compounds are discussed, referred to in this example as HTS 02324 (interchangeably referred to as "MAC" in this example), and MWP 01127 (interchangeably referred to as "MWP" in this example).

[00135] Structure and selectivity of two Gcc inhibitory compounds MWP and

MAC. 7he two compounds, MWP and MAC, were found to be a 5-[((4-methylphenyl)thio) quinazoline-2,4-diamine and a 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide, respectively, shown as Formula 2 and Formula 3, respectively. 5-Substituted 2,4- diaminoquinazolines and 5-substituted pyridinyl-2-furamides, have not been previously described as Gcc inhibitors.

[00136] Table 4 illustrates that, using the colourimetric substrate p-nitrophenyl- β-D- glucopyranoside (pNPGIc), MWP and MAC were found to be low micromolar inhibitors of Gcc with IC50s of 7.8 and 4.7 μM, respectively.

Table 4

Specificity of Gcc inhibitory compounds.

Enzyme/Compound MWP^lal MAC^[bl IFG

Human β-Gcc^|d] _{7 8}[c] 4.7 0.030

Human cytosolic β- ₅₁ [hj >400 1.0 glucosidase^[dI

Human β-Gal^[el 570 >1150 180

Human α-Glc^lf] 1300 >1150 290

Almond β-Glc^[dl 190 >1150 0.026 Human Hex¹⁹¹ >700^[" NI(1150) NI(IOOO)

[a] MWP - MWP 01127, See Formula 2;

[b] MAC - HTS 02324, See Figure 3;

[c] IC₅₀ μM;

[d] Enzyme activity evaluated using the substrate pNPGIc (1.6mM);

[e] MUGaI; (0.25 mM);

[f] MUaGIc (0.5 mM);

[g] MUG (0.4mM);

[h] Full Dose response curve could not be generated; estimated IC₅₀;

[i] NI - non-inhibitory at highest concentration evaluated

[00137] By comparison IFG, a known carbohydrate-based Gcc inhibitor, was found to have an IC50 of 30 nM using pNPGIc. Whereas MAC shows no inhibition towards other lysosomal enzymes such as human β-galactosidase (β-Gal), α-glucosidase (α-GIc) and 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 β- glucosidase that also hydrolyses glucosylceramide and whose catalytic domain also consists of a (β/α)8 TIM barrel. While both compounds MWP and IFG also enhanced Gcc activity in N370S/N370S patient cells at 12.5 μM, this value is below the 50 μM IC50 of MWP for neutral β-glucosidase but greater than the corresponding 1 μM IC50 value of IFG for this enzyme. Additionally both IFG and MWP inhibit almond β-glucosidase. However, while the IC50 of IFG towards the almond enzyme is nearly identical to that of the human enzyme, the IC50 of MWP for almond β-glucosidase is increased 24-fold relative to human Gcc. In contrast MAC is virtually non-inhibitory towards either the almond enzyme or the neutral β- glucosidase (Table 4). Thus, unlike MAC, MWP and IFG have similar inhibitory profiles.

[00138] Compounds MWP and MAC increase Gcc protein levels in the lysosomes of GD cells. The effect of MWP and MAC on Gcc and Hex levels in N370S/N370S patient fibroblasts was examined over a larger range of concentrations.

[00139] Figure 21 shows changes in Gcc and Hex activity in GC patient fibroblasts using the MWP and MAC compounds.

[00140] In parts A) and B), 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. Hex activity levels serve as a control for toxicity. Standard deviation (n=3) is shown for each point. In 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 μM), MWP (12.5 μM) or MAC (12.5 μM) 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 μM) or MAC (12.5 μM) or vehicle (0.1% DMSO). Gcc (upper panel) or the lysosomal marker Lamp-2 (lower panel) were visualized by Western blotting.

[00141] Both MWP and MAC compounds showed signs of toxicity at concentrations greater than 30 μM, as indicated by the parallel decrease in intracellular Gcc and Hex activities (Figure 21 , parts A) and B)). Although maximum increase in Gcc activity in patient cells was seen at a concentration of 12.5 μM for both compounds, MWP (Figure 21, parts A) and C)) treatment resulted in a 2.5 fold increase in enzyme activity in comparison to the 1.5 fold rise seen with MAC (Figure 21, parts B) and C)). In order to compare the efficacy of the compounds in patient fibroblasts with different Gcc mutant alleles, the effect of IFG (25 μM), MWP (12.5 μM) or MAC (12.5 μM) on the F213I allele, more commonly found in GD patients of Asian descent, was examined. In these cell lines the efficacy of the two compounds was reversed relative to similarly treated GD cells expressing the N370S Gcc allele. MAC- treatment resulted in a 2.4 fold increase in Gcc activity as compared to a 1.6 fold elevation observed in MWP-treated cells (part C)). The cellular localization of the enhanced Gcc activity observed in MWP- or MAC-treated GD cells, was probed by preparing lysosome- enriched fractions and examining their Gcc and Lamp-2 levels by Western blotting. A clear enrichment in Gcc protein with little change in Lamp-2 levels was observed in the treated versus untreated cells (part D)).

[00142] 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.

[00143] Figure 22, parts A) and B) show that , unlike IFG which is a classic competitive inhibitor, the Vmax decreased along with an apparent increase in Km with increasing dose of either MWP or MAC. These data are consistent with a mixed-type of inhibition, which has also been reported for other non-carbohydrate-based inhibitors of Gcc. [00144] Figure 22 illustrates that MWP and MAC are mixed type of inhibitors that function optimally at a neutral pH. In part A) MWP (square symbols) and part B) MAC (round symbols) were tested at 5 concentrations, two above and below their IC50 values, each in the presence of 7 different concentrations of the substrate (MUGIc). The resultant apparent Km (mM) (Right Y-axis, hollow symbols) and Vmax (Relative Fluorescence Units (RFU)/hr) (Left Y-axis, filled symbols) values for each inhibitor concentration (X-axis, μM) are shown as linear graphics. In part C), the relative inhibitory activity of MWP (square symbols) and MAC (round symbols) were determined at different pH values.

[00145] Ideally, 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)).

[00146] Active Derivatives of Compounds MWP and MAC. To evaluate the structure versus IC50, toxicity and PC-activity relationships of the compounds, a simple quinazoline derivative (referred to as MWP-Ia, shown below as formula 8) (2,4,-diamino-6- nitroquinazoline), lacking any pendant hydrophobic groups) and a diethoxy quinozoline derivative (referred to as MWP-Ib, shown below as formula 9) (7,8-diethoxy-quinazoline 2,4- diamine) were examined.

Formula 8 or MWP-Ia

Formula 9 or MWP-I b [00147] The former compound (MWP-Ia) exhibited a 10-fold reduction, the latter derivative (MWP-Ib) showed a 2-3 fold reduction in inhibitory activity. These results suggest the importance of the size and identity of the hydrophobic group at the 4 and 5 positions. Although, in cultured cells MWP resulted in cell death at concentrations >17μM, 1b showed no significant toxicity even at 800μM. In the structure of Formula 8, MWP-Ia, having IC50 of 61 μM, charged moieties are present at position 6 of the quinazoline ring, resulting in reduced inhibitory activity relative to Formula 1.

[00148] In the structure of Formula 9, MWP-Ib, having IC50 or 22 μM, linear alkyl chains are tolerated at positions 7 and 8 of the quinazoline backbone without a significant reduction observed in inhibitory activity, relative to Formula 1.

[00149] Whereas substitution of the phenoxy-furamide group in MAC with a phenyl ring, forming 2-methyl-N-pyridin-4-yl-benzamide (MAC-2a, shown below as formula 10) reduced its inhibitory activity more than 10-fold, substitution with an alkyl group forming 2,2- dimethyl-N-(4-pyridinyl)propanamide (MAC-2b, shown below as formula 11 ) produced an essentially non-inhibitory compound. The MWP derivative MWP-Ia was able to enhance Gcc activity 1.5 fold in patient fibroblasts bearing either the N370S or F213I allele. On the other hand, compound MAC-2a, did not significantly increase Gcc activity in either of these cell lines. Thus the parental compounds identified through HTS have lower IC50 values and greater PC-activity in patient cells than the derivatives so far evaluated.

Formula 10

Formula 11

[00150] 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).

[00151] 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 μM) or MAC (12 μM) (labels running down sides of panels). In part A), the primary IgGs against Gcc or the lysosomal marker Lamp-1 are visualized as green or red, respectively. In merged images yellow denote colocalization in lysosomes. Scale bars (white line) from left to right are 10 μm (DMSO), 13 μm (MWP) and 16 μm (MAC). In part B) The primary IgGs against Gcc or the ER marker PDI are visualized. In merged images, colocalization in the ER is visualized.

[00152] 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). In untreated cells (DMSO only), 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).

[00153] However, when N370S or F213I patient cells were treated with either MWP

(Figure 23, part A, 2nd row top/bottom panels) or MAC (Figure 23, part A, 3rd rows top/bottom panels), their Gcc staining pattern increased in fluorescence intensity, became more punctate and exhibited a greater co-localization with Lamp-1 (Figure 22, part A 3rd column in each panel). Furthermore there was a notable decrease in the overlap between Gcc and PDI staining of N370S and F213I cells treated with MWP. In the case of MWP- treated N370S and F213I cells, there was also an observable decrease in the overall intensity level of PDI staining, suggesting a decrease in ER stress, for which PDI is also a marker, as compared to untreated cells (Figure 23, part B, 2nd column, 2nd row in all panels).

[00154] Profiling changes in protein dynamics within the Gcc molecule upon PC- binding. Since PCs are proposed to stabilize mutant proteins by affecting their conformational dynamics, we used hydrogen/deuterium exchange mass spectrometry (H/D- Ex) to examine and map such changes within the Gcc molecule. These experiments were performed in solution, in the absence or presence of a 59-fold molar excess of either ligands IFG, MWP or MAC. The degree and rate of deuteration of 26 distinct Gcc peptides generated post D₂O exposure, were determined.

[00155] 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 PyMOL™ (DeLano Scientific) and Kaleidagraph™ (Synergy Software).

[00156] 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. The number of regions (16/26 ,6/26 and 1/26) perturbed by each of the ligands (IFG, MWP, MAC) parallels the maximal enhancement in Gcc activity levels observed in N370S/N370S patient cells following treatment with the corresponding compounds (3.9 fold, 2.4 fold and 1.5 fold, shown in Figure 21, part D).

[00157] Discussion. By screening a library of drug-like compounds we identified 108 novel Gcc inhibitors with IC50 values ranging from 6 μM to greater than 100 μM. Both MWP and MAC function best as PCs at 12 μM, very close to their IC50 values. Currently all confirmed PCs for lysosomal storage diseases have been demonstrated to be inhibitory molecules that can stabilize the enzyme against thermal denaturation. However, the current results illustrate that every inhibitory compound that does this, does not necessarily also function as a PC in patient cells.

[00158] 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. Lastly, using immunofluorescence it was shown that treatment of both sets of patient cells resulted in increased colocalization of Gcc with Lysosomal Associated Protein-1 (Lamp-1), a lysosomal marker, and a corresponding decrease in Gcc colocalization with the ER marker Protein Disulfide lsomerase (PDI). Additionally both compounds inhibit Gcc best at the neutral pH of the ER. These data indicate that these compounds enhance the folding and thus the intracellular transport of the Gcc mutants from the ER to the lysosome.

[00159] Although cytosolic β-glucosidase shares some of the residues found in the

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 β-glucosidase would not be expected to be affected by the concentration of MWP (12 μM) that was shown to enhance Gcc activity. It is interesting that IFG like MWP also inhibits neutral β-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 α-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.

[00160] The regions in the wild-type Gcc structure that are stabilized by the two PCs in this example were identified by H/D-Ex experiments and compared to those regions affected by IFG. At a 59-fold molar excess of MWP, MAC or IFG, specific regions of the enzyme were rigidified (reduced the extent of H/D exchange). Although the stabilizing effects of the compounds were examined using wild-type enzyme, these results likely extend to the mutant enzyme. [00161] Although IFG binding induced the greatest degree of perturbation in the three loops, MWP had a greater overall impact on the rate of hydrogen/deuterium exchange than MAC. The only region demonstrating a significant reduction in the rate of hydrogen/deuterium exchange by all three PCs was the segment encompassing residues 243-249. This is a rather surprising observation given that none of the crystal structures of Gcc:ligand complexes have shown any residues in this region making a direct contact with the bound glycone moiety. However, this region does contain Leu241 which forms a hydrophobic contact with the alkyl chain present in Gcc:N-n-DNJ complex. Furthermore computational docking studies of a truncated glucocerebroside ligand derivative into the active site of the enzyme (2NSX) suggested that the alkyl chain would lie in a shallow hydrophobic groove between residues 311-317 and 235-252. Although one could envision a hydrophobic group such as the 4-methylphenylthiol in MWP or the 3,5-dichlorophenoxy in MAC lying in this groove, IFG lacks a hydrophobic group that could have such an effect on residues 235-252.

[00162] The rigidification observed in residues 235-252 may arise indirectly as a result of the concerted movement brought about by the direct interaction of the ligands with one of these loops. Alternatively, differences between the two experimentally derived binding profiles may be due to the constraints placed on the breathing motions of the protein monomers in the crystal lattice versus the protein in solution. The conformational differences in loop 341-350 observed in the first two crystallographically derived structures of Gcc are attributed to crystal contact differences. It is interesting to note that in all structures to date with the exception of 2NSX, residues Trp348 (2NT1 , 2J25, 2V3E, 2V3D) or Asp353 (2NT0) in loop 342-354, make crystal contacts via a hydrogen-bond with Ser242 that is part of loop 235-242 in the adjacent monomer in the crystal lattice. Thus one could speculate that the lack of observed differences in these regions upon ligand binding in the crystal structures may be due to the constraints imposed by the crystal contacts between the two loops in adjacent monomers.

[00163] The degree to which each of the three PCs (IFG, MWP or MAC) are able to enhance Gcc activity in GD patient cells with the N370S mutation appears to most closely correlate with perturbation effects on H/D-Ex in loop 243-248. The importance of this loop in the formation of a functional Gcc is also indicated by the fact that its residues are conserved to greater degree across a wider phylogenetic distance (Tetropods, Fugu, Honey Bee and C. elegans) than residues found in loop 311-319. Our H/D-Ex data on this limited set of PCs are consistent with the crystallographic data correlating an enhancement in global stability with the rigidification of loop 311-319 and/or loop 342-354. However, it is difficult to reconcile whether the conformational changes observed in these loops are more relevant to allosteric control of Gcc activity, if conformational fluctuations lead to recognition by the ER-QC machinery and ER retention. The two conformers of the enzyme may relate to conformational changes that Gcc is proposed to undergo following activation via its interaction with SapC.

[00164] The H/D-Ex experiments on Gcc in the presence and absence of IFG, MWP or MAC underscore the importance of other regions of Gcc, such as loop 235-252, in mediating the transport enhancing effects of PCs on N370S and F213I mutants. As a technique H/D-Ex serves to highlight structural regions of Gcc undergoing important conformational changes.

[00165] Experimental Methods

[00166] Chemicals and Reagents: A total of 50,000 drug-like compounds from the

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). A Concanavalin A-binding fraction of human placental lysate was used as a source for lysosomal enzymes β-Gal and α-Glc. Human Hex was purified from placenta. Almond β-glucosidase was purchased from Sigma (USA). Human neutral β-glucosidase kindly provided by N. Juge (Biosciences FRE-3005- CNRS Universite Paul Cezanne Aix Marseille III, France) was expressed and purified from Pichia pastoris. Fluorogenic substrates purchased from SIGMA (USA) included; 4- methylumbelliferyl-β-D-glucopyranoside (MUGIc), Gcc; 4-methylumbelliferyl-β-D- galactopyranoside (MUGaI), β-Gal; 4-methylumbelliferyl-α-D-glucopyranoside (MU-α-GIc), α- GIc; 4-methylumbelliferyl-β-N-acetylglucosamine (MUG), Hex. The colourimetric substrate p- nitrophenyl- β-D-glucopyranoside (pNPGIc) (SIGMA, USA) was also used to monitor Gcc, human cytosolic β-glucosidase and Almond β-glucosidase activity.

[00167] The following cell lines were used: "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 α-minimal essential media (α-MEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA), and antibiotics Penicillin/ Streptomycin (Invitrogen, USA) at 37°C in a humidified CO₂ incubator.

[00168] Primary Screening. Human Gcc was screened against the 50,000 compound library of drug-like small molecules (Maybridge pic, Cornwall, UK) in duplicate in 384-well plate format. The screen was fully automated on a SAIGAIN core system (Beckman-Coulter Inc., Fullerton, CA) with an ORCA arm for labware transportation, a Biomek FX for liquid handling, and an Analyst HT (Molecular Devices Corp., Sunnyvale, CA) for fluorescence detection (λex = 330 nm; λem = 460 nm). All liquid handling and activity detection was done at room temperature. Each 384-well assay plate was read 9 times, with 105 s between each read. Reaction rates (RFU/sec) were calculated as the slope of the data of the second to ninth data point, inclusive. Each reaction consisted of Gcc (72 μg/mL), Taurodeoxycholate (TdC, 0.24%), Human Serum Albumin (0.1%), and MUGIc substrate (625 μM) and compounds in 20 mM Citrate-Phosphate (CP) buffer. Library compounds dissolved in DMSO were added to a final concentration of 20 μM. 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% DMSO₁ Castanospermine (45 μM)) 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. To obtain an estimate of the variability of the assay, 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.

[00169] Secondary Screening. The dose-response curves of the 108 hits selected from the primary screen were determined by the endpoint Gcc assay, in the presence of seven concentrations (0.1-100 μM) of the putative inhibitor diluted in DMSO. IC50 values were determined. Compounds exhibiting sigmoidal dose response curves were selected as bona fide inhibitors.

[00170] Gcc and other glycosidase Activity Assays. Gcc activity was measured by release of 4-methylumbelliferyl fluorophore from MUGIc. Assays (50 μL) 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 37⁰C 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 μL). The increase in fluorescence was measured using a Spectramax Gemini EM MAX™ (Molecular Devices Corp, Sunnyvale, CA) fluorometer and detected at excitation and emission wavelengths set to 365 nm and 450 nm, respectively. For inhibition studies using the enzymes other than Gcc, the following buffeπsubstrate combinations were used: β-Gal: MUGaI (1.6 mM in 20 mM CP 5.5), almond β-glucosidase: pNPGIc (1.6 mM in 20 mM CP pH 5.5:), human cytosolic β-glucosidase: pNPGIc (0.25 mM in 20 mM CP pH 5.5), α- MUaGIc GIc (0.5 mM in 20 mM CP pH 5.5), Hex: MUG (0.4 in 20 mM CP pH 4.5: mM). All reactions were performed at 37° C as an endpoint assay as described for Gcc above.

[00171] To control for compounds that were either fluorescent or quenchers near the emission maxima of MU the inhibitory activity of compounds was also confirmed using the colourimetric substrate pNPGIc under conditions described for the MUGIc endpoint assay, except that absorbance was measured at 405 nm and for kinetic analyses 1.6 mM pNPGIc was used. Kinetic parameters (Km, Vmax) and IC50 values were fitted to a non-linear curve fitting of the data to the Sigmoidal Dose Response equation (described above) or Michaelis- Menten equation using Prism™ 4.0 (Graphpad Software).

[00172] Heat inactivation assay. Heat inactivation experiments were performed using

Cerezyme powder (Gcc) (13 mg/mL) in CP buffer (20 mM), diluted a further 1/200-1/400 in CP buffer (20 mM, pH 5.5) containing TdC (0.2%). Diluted samples of Gcc containing inhibitors or DMSO, were split into two aliquots, one was left on ice, and the other heat- treated at 50° C. Heat-treated enzyme samples at each time point were cooled on ice until completion of the time series. For enzyme activity, samples were pre-equilibrated to room temperature for 10 min, followed by addition of MUGIc (0.8 mM final) substrate and incubated at room temperature for a further 20 min. Remaining activity was expressed as a ratio of Gcc activity in the presence of the test compound following incubation at 50° C versus activity of the corresponding aliquot in the presence of the test compound held at 4°C.

[00173] Determination ofpH dependence of inhibitory activity of MAC and MWP towards Gcc. Gcc (100 ng/mL) was diluted into 20 mM CP buffer of pH ranging from 4.5 - 7 in steps of 0.5 units. Compound MAC (12 μM final), MWP (10μM final) or DMSO (1% final) was added to the enzyme mix and equilibrated for 15 min. at room temperature. Following addition of an equal volume of MUGIc (0.8 mM final), an endpoint assay was performed at 37° C as described above. Residual activity was expressed as a ratio of Gcc activity at a given pH in the presence of the test compounds (MWP or MAC), versus activity at the corresponding pH in the presence of DMSO.

[00174] Evaluating Chaperoning Activity of Compounds in Cell Culture. Gaucher patient fibroblasts (10,000-50,000 cells per well) were seeded onto 24 well plates at (ca. 50% confluence). The next day, the medium was replaced with fresh α-MEM-FBS with or without a test compound (1/100 dilution). Test compounds were dissolved in DMSO. Mock or compound-treated cells were evaluated in triplicate after growth for 5 days at 37° C in a CO₂ humidified incubator.

[00175] To measure Gcc activity in treated Gaucher fibroblasts, media was removed, cells were washed twice with PBS and subsequently lysed by the addition of Triton X-100 (0.4%) and TdC (0.4%) in CP (20 mM, pH 5.5). An aliquot (25 μl_) of the lysate was mixed with an equal volume of MUGIc (10 mM final) and assayed for total Gcc activity. To control for variability in cell numbers between replicate wells, the remaining aliquot of the lysate, was used to assay for lysosomal Hex, with the substrate MUG using the endpoint assay described above.

[00176] Purification of Iron-dextran-labeled Lysosomes. An enriched lysosomal fraction was prepared from Gaucher patient N370S/N370S fibroblasts treated with either DMSO (0.1%), MWP (12.5 μM) or MAC (12.5 μM) for five days, followed by labeling with Iron-dextran colloid and subsequent purification by magnetic chromatography. Lysosomal Gcc was monitored fluorometrically using the substrate MUGIc.

[00177] Western Blotting. The enriched lysosomal fractions (1 μg) from treated and untreated Gaucher patient cells were subjected to SDS-PAGE on a bis-acrylamide gel (10%), and the separated proteins were transferred to nitrocellulose. A rabbit polyclonal IgG against human Gcc or mouse monoclonal Lamp-2 antibody were used. Blots were developed using chemiluminescent substrate according to the manufacturers protocol (Amersham, Bioscences, UK). Bands were visualized and optical density quantitated using a high sensitivity gel documentation system (Fluorchem 8000) consisting of a cooled CCD camera coupled with Alpha lnnotech software (Alpha lnnotech Corp., USA). [00178] 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)

[00179] Indirect Immunofluorescence and confocal microscopy imaging. Indirect immunolabeling was 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). Primary and secondary antibodies were diluted in SS-PBS solution and overlaid on the coverslips for 1 hour at room temperature; secondary antibodies were also overlaid for 1 hour, at room temperature and in the dark; extensive washes with PBS were performed after primary and secondary antibody incubations. Nuclear staining was done with DAPI (Molecular Probes) at 1/50,000 in PBS. Coverslips were mounted onto glass slides using fluorescent mounting medium (DakoCytomaton). Primary antibodies used were rabbit polyclonal IgG anti-human Gcc (raised by ourselves 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 Axiovert™ 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 Volocity™ 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.

[00180] Hydrogen/Deuterium Exchange Mass spectrometry Experiments. A Gcc stock (80 μM) was prepared by dissolving Cerezyme powder (31 mg) into H₂O (500 μl_) . Stocks (47 mM) of IFG, MAC or MWP were prepared in dimethyl sulfoxide (DMSO). A 59:1 molar ratio of IFG, MAC or MWP to Gcc was prepared by combining the Gcc stock (50 μl_) with the compound stock (5 μl_). A DMSO containing "no-ligand" control was prepared by combining the Gcc stock (50 μl_) with DMSO (5 μL). An exchange reaction was initiated by diluting each mixture (5 μL) of with Tris (15 μL , 50 mM) to give a final pH of 7.8, and allowed to proceed at 23° C for a series of predetermined time periods (30, 100, 300, 1000 and 3000 s). The exchange was quenched by lowering the reaction temperature to 1° C and by dropping the pH of the reaction to 2.5 by the addition of a pre-chilled solution (30 μl_, 1 ° C) containing urea (2M) and tris(2-carboxyethyl)phosphine (TCEP) (1M). The quenched solution was immediately pumped at 200 μL/min over an immobilized porcine pepsin column (104 μL bed volume) with trifluoroacetic acid (TFA) (0.05%) for three minutes with contemporaneous collection of proteolytic products by way of a trap column (4 μL bed volume). Pepsin was immobilized on Poros 20 AL media (30 mg/mL, Applied Biosystems) as per the manufacturer's instructions. Peptide fragments were eluted from the trap column and separated by C18 column (Magic C18, Michrom BioResources, Inc) with a linear gradient of solvent B (13%) to solvent B (40%) over 23 minutes (solvent A, 0.05% TFA in water; solvent B, 95% acetonitrile, 5% water, 0.0025% TFA; flow rate 5 μL/min - 10 μL/min). Mass spectrometric analyses were carried out with a Thermo Finnigan LCQ™ mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with a capillary temperature of 200° C. Spectral data were acquired in data-dependent MS/MS mode with dynamic exclusion. The software program SEQUEST™ (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.

[00181] EXAMPLE 7

[00182] The data obtained in a screen of Maybridge library hits is outlined below.

Briefly, all 108 hits from the primary screen were evaluated for three characteristics using 4 assays: A) inhibition assay, to confirm and determine the IC50 value in the presence of 0.8 mM MUGIc ;B) heat denaturation attenuation assay, i.e. remaining Gcc activity in the presence of the compound following heating to 50⁰C for 20 min.; changes in intracellular levels C) Gcc and D) Hex activity in GD patient fibroblasts (N370S/N370S) after cells were treated for five days with the indicated concentration of test compound. [00183] 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.

[00184] 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.

[00185] 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 50⁰C for 20 min.

[00186] 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 50⁰C for 20 min.

[00187] 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.

[00188] Figure 27B illustrates further 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.

[00189] Figure 28A illustrates data obtained in a primary screening of compounds relating to changes in intracellular levels of Hex activity in GD patient fibroblasts

(N370S/N370S) after cells were treated for five days with the indicated concentration of test compound.

[00190] 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

(N370S/N370S) after cells were treated for five days with the indicated concentration of test compound.

[00191] Table 5, below 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 μM) determined in the secondary screen (column labelled "Code") and the 2D structure of the compound is shown beside each (column labelled "Structure"). Table 5

Structures and IC50 (μM) values

[00192] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments of the invention. However, it will be apparent to one skilled in the art that these specific details are not required in order to practice the invention.

[00193] The above-described embodiments of the invention are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto. All documents referred to herein are incorporated by reference.

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Claims

1. 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.

2. The composition of claim 1 wherein the N-(4-pyhdinyl)-2-furamide derivative comprises a phenoxy substituted moiety at the 5- position of an N-(4-pyridinyl)-2-furamide; or an n-pyridinyl-4-acrylamide group.

3. The composition of claim 1 wherein the N-(4-pyridinyl)-2-furamide derivative comprises 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3:

4. The composition of claim 1 , claim 2, or claim 3, wherein the lysosomal storage disorder is Gaucher disease, GM1 -gangliosidosis, or Morquio B disease.

5. The composition of claim 4, wherein the lysosomal storage disorder is late-onset Gaucher disease.

6. The composition of claim 1 , claim 2, or claim 3, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.

7. 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.

8. The method of claim 7 wherein the N-(4-pyridinyl)-2-furamide derivative comprises a phenoxy substituted moiety at the 5- position of an N-(4-pyridinyl)-2-furamide; or an N- pyridinyl-4-acrylamide group.

9. The method of claim 7 wherein the N-(4-pyridinyl)-2-furamide derivative comprises 5- (3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3:

10. The method of claim 7, claim 8, or claim 9, wherein the lysosomal storage disorder is Gaucher disease, GM1 -gangliosidosis, or Morquio B disease.

11. The method according to claim 10, wherein the lysosomal storage disorder is late- onset Gaucher disease.

12. The method according to claim 7, claim 8 or claim 9, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.

13. Use of a therapeutically effective amount of an N-(4-pyridinyl)-2-furamide derivative for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof.

14. The use of claim 13, wherein the N-(4-pyridinyl)-2-furamide derivative comprises a phenoxy substituted moiety at the 5- position of an N-(4-pyridinyl)-2-furamide; or an N- pyridinyl-4-acrylamide group.

15. The use of claim 13, wherein the N-(4-pyridinyl)-2-furamide comprises 5-(3,5- dichlorophenoxy)-N-(4-pyridinyl)-2-furamide) according to Formula 3:

16. The use of claim 13, claim 14, or claim 15, wherein the lysosomal storage disorder is Gaucher disease, GM1 -gangliosidosis, or Morquio B disease.

17. The use according to claim 16, wherein the lysosomal storage disorder is late-onset Gaucher disease.

18. The use according to claim 13, claim 14, or claim 15, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.

19. A composition comprising a therapeutically effective amount of:

(a) 5[(4-chlorophenyl) thro] 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:

HCI (c) 4-(2-chloro-6-fluorostyryl) benzyl (4-fluoroanilino) methanimidothioate hydrobromide) according to formula 5:

(d) 4-[phenyl ({2-[(phenyl {2,4_l5-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) according to formula 6:

(e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3- carboxylate) according to formula 7:

(f) the compound according to formula 7A:

together with a pharmaceutically acceptable excipient for treatment of a lysosomal storage disorder.

20. The composition of claim 19, wherein the lysosomal storage disorder is Gaucher disease, GM1 -gangliosidosis, or Morquio B disease.

21. The composition of claim 20, wherein the lysosomal storage disorder is late-onset Gaucher disease.

22. The composition of claim 19, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.

23. A method of treating a lysosomal storage disorder comprising 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) phenyl] pyrrolidine-2,3,5-trione) according to formula 6:

(e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3- carboxylate) according to formula 7:

(f) the compound according to formula 7A:

to a subject in need thereof.

24. The method of claim 23 wherein the lysosomal storage disorder is Gaucher disease, GM 1 -gangliosidosis, or Morquio B disease.

25. The method of claim 24, wherein the lysosomal storage disorder is late-onset Gaucher disease.

26. The method of claim 23, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.

27. Use 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:

HCI

(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-(thfluoromethyl)phenyl]tetrahydro-1 H- pyrrol-3-yliden} methyl) amino] ethyl} amino) methylidene]-1-[4-(trifluoromethyl) phenyl] pyrrolidine-2,3,5-trione) according to formula 6:

(e) ethyl 2-[(2-morpholinoacetyl)amino]-4,5,6,7-tetrahydro-1 -benzothiophene-3- carboxylate) according to formula 7:

(f) the compound according to formula 7A:

for preparation of a medicament for treating a lysosomal storage disorder in a subject in need thereof.

28. The use of claim 27, wherein the lysosomal storage disorder is Gaucher disease, GM1 -gangliosidosis, or Morquio B disease.

29. The use of claim 28, wherein the lysosomal storage disorder is late-onset Gaucher disease.

30. The use of claim 27, wherein the lysosomal storage disorder is Parkinson's disease associated with Gaucher disease.