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266

Table9.6 (Continued)

PC IC50 or Ki Mutation

IC50= 29.3 μM L444P/

Ki= 1.4 μM L444P/

Ki= 6.9 μM

IC50= 0.78 μM

= 0.40 μM

IC50= 59.6 μM Ki

= 6.87 μM

L444P

N370S 1.6fold at

N370S 2fold at 3 μM [76]

N370S 2fold at

Max. activity enhancement Refer ences

1.8fold at μM

100

2.8fold at

20 μM

500 μM

μM

300

[135, 136]

[137]

[124]

a) Noncompetitive inhibitor.

Apart from iminosugars, other carbohydratederived analogs have been studied as PCs for the treatment of GD. As a representative example, Díaz and coworkers reported on a series of pyranoidtype glycomimetics with a cis1,2fused glucopyranose 2alkylsulfanyl1,3oxazoline structure[133].

The best results of the series were obtained with compound 37, showing a GCase improvement of 62% at 30 μM in homozygous N370S mutated fibroblasts, which is superior to that observed for Ambroxol at the same concentration.

Finally, it should be noticed that Compain and coworkers contributed to this field with the only examples of multivalent PCs for GD reported to date, to the best of our knowledge, using the iminosugar 1deoxynorijimicin (DNJ) as the bioactive unit. The trivalent acetylDNJderivative (38) provided a threefold increase of GCase residual activity at 1 μM in N370S GD fibroblasts, being more active than the corre- sponding deprotected analog, thus suggesting an improved permeability and cellular uptake[134].

 

Although L444P mutation is resistant to most PCs, the recently reported 2octyl trihydroxypiperidine 39 showed a remarkable 80% activity rescue (1.8fold GCase enhancement) in fibroblasts bearing this homozygous mutation[135, 136]. A higher enhancement toward this mutation was obtained only with a much more sophisticated system that involved a nortropane iminosugar functionalized with a terminal polyfluorinated fragment in form of βcyclodextrin (βCD) complex. In particular, the fluorinated iminosugar βCD complex 40 showed 2.8fold GCase enhancement at 20 μM[137].

Due to the lack of an approved PC, the incidence of GD, and the high number of GBA mutations involved in this pathology, there are a multitude of publications reporting on potential PCs for different GD mutations. Their comprehensive review is beyond the scope of this chapter; therefore, only selected examples are cited in this chapter.

Analogously to what already observed with the enantiomeric couple DGJ (5) and DGJ (23) for FD (Section9.4.1), IFG (6) and IFG (41) are competitive and noncompetitive inhibitors of GCase, respectively, with 41 being a less potent inhibitor than 6 (Ki = 6.9 μM), but still able to increase GCase activity in the Gaucher N370S cell line by 1.6fold at 500 μM[124]. Curiously, also hybrid analogs of α1C nonylDIX (28), obtained by combining the iminosugar scaffold with triazolyl alkyl side chains by means of CuAAC click chemistry reactions, behaved as noncompetitive inhibitors of GCase and could enhance GCase activity up to twofold at 10 nM in GD fibroblasts bearing the homozygous G202R mutation[139].

More recently, further evidence of the impact of inhibitors chirality on their affinity with a target protein and of the efficacy of noncompetitive inhibitors was provided by the study on the Coctyl pyrrolidines 42 and their enantiomers 43. While 43 is a modest competitive inhibitor of GCase (IC potent noncompetitive inhibitor (IC

 = 0.78 μM). In addition, both pyrrolidines

 = 59.6 μM), 42 is a much more

were also able to enhance GCase residual activity in N370S homozygous Gaucher fibroblasts, with the noncompetitive inhibitor 42 having a chaperoning activity comparable to IFG (6) and NNDNJ (27)[76].

Regarding the ERT/PC therapy, Murray and coworkers demonstrated that preincubation of Cerezyme (imiglucerase, Sanofi Genzyme) with IFG (6) significantly increased stability of the human recombinant enzyme to heat, neutral pH, and denaturing agents invitro. Moreover, preincubation of Cerezyme with 6 prior to uptake by cultured cells resulted in increased intracellular GCase activity accompanied by an increase in enzyme protein, thus suggesting that this co incubation before infusion might improve the effectiveness of ERT for Gaucher patients[140, 141].

267

9.4.3 Niemann–Pick

NP disorders are different disorders with distinct genetic origins. Types A and B NP disorders are caused by mutations in the gene encoding the lysosomal sphingomyelin (SM)degrading enzyme acid sphingomyelinase (ASM). Common manifestations of both disease types are hepatosplenomegaly and appearance of cherryred spots in the retina whereas neurodegeneration is only manifest in patients with

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268

NPA. Type C NP disorder (NPC) is caused by mutations in the genes that encode lysosomal cholesteroltransport proteins NPC1 (95% of the cases) or NPC2. The most common symptoms of NPC include hepatosplenomegaly and neurologic deterioration with ataxia, motor pathologies, and horizontal saccadic eye movements (HSEMs)[142]. The treatment for NP disease was based on different drugs such as antiepileptics, anticholinergic, or antidepressants to alleviate symptoms, i.e. tremor, dystonia, or seizures. Miglustat (3, Zavesca), a small iminosugar molecule that reversibly inhibits glycosphingolipid synthesis, is currently available for NPC[143]. In the NPA and NPB types, current research focuses on hematopoietic cell transplantation and enzyme replacement[144].

9.4.4 GM1 Gangliosidosis and Morquio B (β-Gal)

Two lysosomal storage diseases, GM1gangliosidosis (GM1) and Morquio B disease (MBD), are caused by sequence alterations in a single gene, GLB1. They result in functional deficits of acid βgalactosidase (βGal), an enzyme that cleaves terminal βlinked galactose residues from complex carbohydrates in the lysosomal compartment. Both diseases are inherited in an autosomal recessive manner. Depending on the mutations, degradation of one or the other of the βgalactosidase substrates is more or less impaired. If degradation of sphingolipidosis GM1gangliosidosis is predominantly defective, the patients develop the symptomatology of GM1 gangliosidosis, while accumulation of KS is an indication for Morquio disease type B. GM1gangliosidosis is considered a neurodegenerative disorder and MBD is characterized by marked skeletal abnormalities, corneal clouding, cardiac involvement, and increased urinary excretion of KS but no clinical signs of storage in neural tissues[145].

At present, only symptomatic and supportive therapies are available for patients with GM1gangliosidosis and Morquio B. For GM1, only symptomatic treatment for some of the neurologic symptoms is available, which does not significantly alter the progression of the condition. For example, anticonvulsants may initially control seizures. Supportive treatments may include proper nutrition and hydration and keeping the affected individual’s airway open[146]. For Morquio B, only physical therapy and surgical procedures, such as spinal fusion, may help with scoliosis and other bone and muscle issues [147]. Therapies relying on PCs may constitute a future option for the treatment of these lysosomal diseases. 1Deoxygalactonojirimycin (DGJ, 5, Figure9.3) (IC

= 25 μM) was able to rescue the activity of mutant βGal in

mouse fibroblasts with different mutations (from two to sevenfold) after culture with 0.5 mM[148]. Several Nalkylated DGJ derivatives were synthesized to enhance the compound specificity and affinity to galactosidases, such as the as NbutylDGJ (NBDGJ, 4 Figure 9.3 and Table9.7) and NnonylDGJ (NNDGJ, 44, Table9.7) [148, 149]. A bicyclic DGJ derivative 6SNBIDGJ (5N,6SN′butyliminomethylidene)6thio1deoxygalactonojirimycin, 45, Table9.7), was evaluated as a novel PC for GLB1. This derivative inhibits human βGal with an IC

of 32 μM, and signifi-

cantly increases the thermostability of the enzyme. Treatment of GM1 patient fibroblasts with 20 and 80 μM 45 showed a significant improvement of GLB1 activity

Table9.7 PCs forGM1gangliosidosis and Morquio B disease.

 

269

PC IC50 or Ki Mutation

IC50= 25 μM Mouse cell lines expressing human

R201C 5.4fold at 0.5 mM

I51T 2.2fold at 0.5 mM

R201H 2.6fold at 0.5 mM

R457Q 6.0fold at 0.5 mM

W273L 1.8fold at 0.5 mM

Y83H 1.7fold at 0.5 mM

IC50= 3.5 μM Mouse cell lines expressing human

R201C 4.8fold at 0.5 mM

I51T 6.1fold at 0.5 mM

R201H 2.1fold at 0.5 mM

R457Q 5.4fold at 0.5 mM

W273L 1.8fold at 0.5 mM

Y83H 1.1fold at 0.5 mM

IC50= 0.12 μM Patient fibroblasts [149]

R148S/D332N 4.1fold at 1.2 μM

R148S/R482H 4.9fold at 1.2 μM

R201H/ IVS142A>G

Max. Activity Enhancement Refer ences

β‐galactosidase

4fold at 1.2 μM

7.8fold at 1.2 μM

7.3fold at 1.2 μM

13.8fold at

1.2 μM

[148]

IC50= 32 μM Patient fibroblasts [150]

I51T/Y316C 5.5fold at 80 μM

I51T/R457Q 4.9fold at 80 μM

R201C/ R201C 4.9fold at 80 μM

COS7 cells

Y444C 2.8fold at 80 μM

R201H 2.5fold at 80 μM

R590H 2fold at 80 μM

(Continued)

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270

Table9.7 (Continued)

PC IC50 or Ki Mutation

IC50= 8 nM Patient fibroblasts [151]

R201C/R201C 15fold at 10

R201C/H281Y 18fold at 10

Q255H/K578R 20fold at 10

H281Y/splicing 35fold at 10 μM

R457Q/R457Q 7.3fold at 10 μM

S191N/ R351Term

W273L/ R482H

W273L/ W509C

IC50= 0.4 nM Patient fibroblasts [152]

Halfmaximal recovery of mutant βgal activity at 0.01 μM in fibroblast of GM1gangliosidosis patient

IC50= 75 μM Patient fibroblasts [153]

R201H/ IVS142A>G)

Max. Activity Enhancement Refer ences

μM

11fold at 10 μM

1.5fold at 10

μM

6.2fold at 394 μM

IC50= 44 μM Patient fibroblasts

R201H/

2fold at 100

μM

IVS142A>G)

IC50= 0.2 μM Mouse cell lines expressing human

β‐galactosidase

R201C/ R201C 5.1 at 0.2

μM

R201H/ R201H 4.50 at 0.2 μM

R457Q/R457Q 2.4 at 0.2 μM

W273L/

2.2 at 0.2 μM

W273L

Y83H/ Y83H 2.0 at 0.2 μM

[154]

Table9.7 (Continued)

 

271

PC IC50 or Ki Mutation

IC50= 6 μM Patient fibroblasts [155]

R201H/H281Y 12.5fold

R201H/S149F 12.3fold

W273L/ W273L

IC50= 0.47 μM Patient fibroblasts [156]

R201C/ R201C 3.5fold at 2 μM

a) NNDGJ one dose over 5 days. b) NNDGJ three doses over 15 days. Note: the mutations indicated in bold are associated with an MPS IVB phenotype.

Max. Activity Enhancement Refer ences

1.3fold

(between 2 and 5.9fold) for human fibroblasts carrying different mutations and on 24 (27%) out of 88mutated GLB1 enzymes expressed in COS7 cells[150].

In addition, several Calkylated azasugars displayed a better activity in terms of inhibition and chaperoning activity. Demotz and coworkers identified Cpentyl 4epiisofagomine (46, Table9.7) as a highly potent and selective inhibitor of human lysosomal βGal able to increase the enzyme activity in 56% of the evaluated mutations, which ranged from 1.5 to 35fold. Specifically, MPS IVB fibroblasts showed a 1.5fold increase in the GLB1 activity at 10 μM[151, 152]. The nonyl analogous 47 (Table9.7) was a potent inhibitor of lysosomal βGal (IC

= 0.4 nM) and was more

active than the pentyl derivative as a PC (halfmaximal recovery of βGal activity was reached at 0.01 μM concentration), but it was a very potent inhibitor of lysosomal βglucosidase (GCase, IC

= 40 nM), an activity, which may cause undesirable side

effects[152]. Moreover, the “allcis” trihydroxypiperidines 48 and 49 (Table9.7) were good inhibitors of lysosomal βGal and were able to increase βGal activity in GM1gangliosidosis patient fibroblasts up to two to sixfold (at <100 μM concentration)[153]. The valienamine derivative NOEV (Noctyl4epibetavalienamine, 50, Table9.7) NOEV has an IC

of 0.2 μM against human GLB1 and increased the enzyme activ-

ity between 2.0 and 5.1fold in mouse fibroblasts expressing GLB1 carrying GM1 gangliosidosis (p.R201C, p.R201H, and p.R457Q) or MPS IVB (p.W273L and p.Y83H) mutations. Similar results were observed in human fibroblasts from GM1 gangliosidosis patients. In vivo evaluation of NOEV was performed in a model mouse of juvenile GM1gangliosidosis, expressing a mutant enzyme protein R201C. Oral administration of NOEV led to a significant increase in GLB1 activity, which resulted in significant enhancement of the enzyme activity in the brain and other tissues[154].

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272

A significant contribution also came from the Graz group, starting with the compound coined as DLHEXDGJ (51, Table9.7), which showed significant activity enhancements (1.3 at 12.5fold) in GM1gangliosidosis and MPS IVB patient fibroblasts with 20–500

μM[155]. In parallel to IFG, valienamine, and deoxygalactono- jirimycin derivatives, highly functionalized cyclopentane derivatives (“carbasugars”) were also investigated as potential PCs. The dansyl aminohexyl derivative 52 was found to be one of the more promising PCs of this series (Table9.7)[156].

9.4.5 GM2 Gangliosidosis (β-Hexosaminidase)

GM2gangliosides are catabolized by the lysosomal hydrolases βhexosaminidases (HEX) through the hydrolysis of the Nacetylgalactosamine residues. HEX are a subset of isozymes formed by the dimerization of α and β subunits: HEXA (αβ), HEXB (ββ), and HEXS (αα). In addition, GM2gangliosides degradation involves the GM2 activator protein (GM2AP), which presents the gangliosides to α subunit of HEXA. Mutations in the genes encoding for α (HEXA), β (HEXB), or GM2AP (GM2A) proteins affect the lysosomal degradation of GM2ganglioside and other glycolipids, causing their accumulation into the lysosome and the GM2 gangliosidoses Tay–Sachs disease (TSD), SD, or GM2activator protein deficiency (AB variant), respectively[157].

CNS dysfunction is the main characteristic of GM2gangliosidoses patients that includes neurodevelopmental alterations, neuroinflammation, and neuronal apoptosis. Currently, there is no approved therapy for GM2 gangliosidoses, but several clinical trials with different therapeutic strategies including HSCT, ERT, and gene therapy are ongoing. The BBB presents a developmental challenge for therapeutic agents for these disorders. In this sense, alternative routes of administration of recombinant enzymes (e.g. intrathecal or intracerebroventricular) were evaluated, as were the delivery systems that allow the transport of proteins to the CNS. Yet, none of these tricks has materially altered the course of the disease[158].

A potential approach is SRT using inhibitors of GCS to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Miglustat (3, Figure9.3) increased lifespan, improved clinical features, and reduced ganglioside storage in murine models of SD[159] and TSD[50]. While in a clinical trial of lateonset TSD patients, the drug did not meet its efficacy endpoint target[160], Miglustat completed the Phase 3 of clinical trial on patients with acute infantileonset GM2gangliosidosis (NCT00672022).

PCs have been identified for treatment of GM2gangliosidoses. Tropak etal. reported that both adult TSD and SD fibroblasts grown in culture medium containing some HEX inhibitors including the NAcetylgalactosamine (GalNAc, 53), NAcetylglucosaminethiazoline (NGT, 54), 6Acetamido6deoxycastanosper- mine (ACAS, 55), 2Acetamido2deoxynojirimycin (ADNJ, 56), 2Acetamido1,2 dideoxynojirimycin (AdDNJ, 57) showed increase of HEX activity, between 2.6 and 5.8fold, above untreated fibroblasts (Table9.8)[161].

Table9.8 PCs forGM2gangliosides.

 

273

PC IC50 or Ki Mutation

Ki= 1.9 mM

a,c

Patient fibroblasts [161]

αG269S/ αG269S

Ki= 300 nM

a,c

Patient fibroblasts

αG269S/ αG269S

βP504

S/16kb 5′

16kb/16kb (HexB)

IC50= 500 nM

a,c

Patient fibroblasts

αG269S/ αG269S

Max. activity enhancement Refer ences

2.9fold (total Hex) at 270 mM

2.6fold (total Hex) at 0.9 mM

5.8fold (HexA and S)

6.1fold (total Hex) at 0.9 mM

4.3fold (HexA and S)

2.7fold (total Hex) at 0.9 mM

3.6fold (total Hex) at 0.18 mM

Ki= 5 nM

Ki= 700 nM

Patient fibroblasts

αG269S/ αG269S

Patient fibroblasts

αG269S/ αG269S

2.8fold (total Hex) at 0.2mM

3.3fold (total Hex) at 0.5 mM

(Continued)

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274

Table9.8 (Continued)

PC IC50 or Ki Mutation

Ki= 15 μM

Ki= 3.7 μM

n.d.

Patient fibroblasts

αG269S/ αG269S

Ki= 180 μMan.d.

Ki= 0.041 μM Patient fibroblasts [163]

αG269S/ αG269S

Max. activity enhancement Refer ences

[162]

1.8fold (HexA and S)

1.8fold (total Hex) at 50

μM

14.8fold (HexA) at 100 μM

a) Value determined using human placental Hex.

Value determined using Jack Bean Hex.

b) c) Compound is able to stabilize HexA under thermal denaturation. d) n.d.=non determined.

Fleet and coworkers reported 2acetamido analogs of DAB (1,4dideoxy1,4 iminoarabinitol) and LAB (1,2,4trideoxy1,4iminoarabinitol). In particular, compounds 58, 59, and 60 showed modest to good inhibitory activity toward HEX (3.7–180 μM), and only compound 59 (the best inhibitor of the series) was tested as PC providing HEX activity enhancement up to 1.8fold at 50 μM. The authors did not exclude a potential activity enhancement by compounds 58 and 60 despite their lower inhibition strength, which often results in beneficial for the PC activity (Table 9.8) [162]. In order to identify potential PCs for GM2 gangliosidoses, a molecular docking and dynamics simulation study identified the pyrrolidine 2,5 dideoxy2,5iminomannitol (DMDP, 60) amide as the strongest competitive inhibitor of HEXA. DMDP amide improved the intracellular activity of HEXA up to

14.8fold at 100 μM in TSD fibroblasts patients (Table9.8)[163].

   

The pyrimethamine, a noncarbohydrate derivative, is the most promising PC for GM2 Gangliosidosis, the compound completed the Phase 2 of trial clinic (NCT01102686)[164–168].

9.4.6 Krabbe

KD (also called globoid cell leukodystrophy) is a severe neurological condition caused by defects in the enzyme βgalactocerebrosidase (GALC). It is part of a group of disorders known as leukodystrophies, which result from the loss of myelin (demyelination) in the nervous system. GALC is required for the hydrolysis of galactosphingolipids, including the major lipid component of myelin βgalactocerebroside (GalCer) required for lipid turnover and maintenance of the myelin sheath that surrounds and protects neurons. Histological signs of disease include the widespread loss of myelin in the central and peripheral nervous systems, profound neuroinflammation, and axonal degeneration. Patients suffering from KD also display neurological deterioration. The only approved and available treatment option for KD is HSCT. However, combination therapies (HSCT, ERT, gene therapy, and SRT [ cycloserine]) that target different pathogenic mechanisms/pathways have been more effective at reducing histological signs of disease, delaying disease onset, prolonging life span, and improving behavioral/cognitive functions in animal models of KD [169, 170]. Several recent studies have identified GALC inhibitors able to stabilize the enzyme under thermal denaturation, which may have great potential for future PC and ERT/PC therapies for KD (compounds 5 and 62–68, Table9.9)[171–173].

275

9.5 Glycogen Storage Disorders

9.5.1 Pompe Disease

The glycogen storage disease type II (GSDII), or Pompe disease (PD), is due to the deficit of lysosomal glycogen degradation enzyme acid αglu characterized by progressive accumulation of lysosomal glycogen in heart and skeletal muscles. Symptoms include muscle weakness, fatigue, dysphagia, respiratory insufficiency, and enlarged liver. ERT is the approved treatment for PD (Table9.10). A major shortcoming of the current standard of care is the inability of Lumizyme dase alfa; Sanofi) Genzyme to reach skeletal muscle efficiently[174]. This limitation of ERT motivated the scientific community to develop the next generation of therapies for PD based on combined ER/PC therapy, gene therapy, and nanotechnology systems functionalized with receptorbinding molecules, thus promoting longtime circulation and controlled release in muscles of recombinant enzyme. These new approaches have already progressed to the clinic[175].

In recent years, PC treatment with 1deoxynojirimycin (DNJ, 69) has become a potential therapeutic treatment for patients with PD. In vitro studies have shown

(alglucosi-

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