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Table9.7 (Continued)

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 

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 (Table9.7) was a potent inhibitor of lysosomal βGal (IC

μM[151, 152]. The nonyl analogous 47

= 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].

 

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 deoxygalactonojirimycin 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.

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 

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

5.8fold (HexA and S)

6.1fold (total Hex) at 0.9

4.3fold (HexA and S)

2.7fold (total Hex) at 0.9

3.6fold (total Hex) at 0.18

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

(Continued)

 

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. b) Value determined using Jack Bean Hex. 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].

   

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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-

 

276

Table9.9 Inhibitors ofGALC.

Compounds GALC, Ki References

190 μM

380 nM

630 nM

52 μM

[171]

130 μM

2.3 mM

7.0μM

450 μM

a) Compound is able to stabilize GALC under thermal denaturation. b) Good inhibitor also for lysosomal βgalactosidase.

[172]

[173]

Table9.10 ERT forPompe disease.

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9.6 Glycoproteinoses

277

Enzyme

Disorder Gene

Pompe GAA αglucosidase Lumizyme® (alglucosidase alfa; Sanofi

deficiency ERT

Genzyme, Cambridge, MA, USA) available since 2010

Nexviazyme® (avalglucosidase alfa ngpt; Sanofi Genzyme, Cambridge, MA, USA) available since 2021

Current indication

Infantileonset Pompe disease

Lateonset Pompe disease

that 69 could significantly increase enzyme stability under thermal denaturation, increased enzyme activity and protein levels for different αglu mutants in patient derived fibroblasts and in transiently transfected COS7 cells (the best results are reported in Table 9.11) [176]. Studies on animal models confirmed that DNJ increased the specific activity and lysosomal delivery of mutant αglu and promoted glycogen reduction in tissues[179]. A clinical trial on DNJ showed that total αglu activity and protein in plasma were increased 1.2 to 2.8fold compared to ERT alone in Pompe patients. Moreover, muscle αglu activity was also increased[180]. Unfortunately, based on the serious adverse events of the latest clinical trial (NCT00688597) the administration of 69 on Pompe patients was terminated. Moreover, the DNJ alkylated derivative (Nbutyldeoxynojirimycin, NB DNJ, 3) was also effective in enhancing αglu residual activity in fibroblasts from PD patient carrying specific mutations and in HEK293T cells overexpressing mutated GAA gene (Table9.11)[177]. Even more remarkably, the coincubation of Pompe fibroblasts with recombinant human αgluc and the chaperone 3 resulted in more efficient stabilization of enzyme activity. Improved enzyme correction was also found invivo in a PD mouse model and PD patients treated with coadministra- tion of infusions of recombinant human αgluc and oral NBDNJ[181, 182].  NBDNJ, the unnatural enantiomer of the iminosugar 3, showed αgluc activity rescue, either when administered singularly (1.5fold at 20

μM) in PD fibroblasts bearing L552P/L552P mutation (Table9.11) or when coincubated with the recombinant human αgluc. In addition, different from its NBDNJ, NBDNJ (70) did not act as a glucosidase inhibitor. The lack of inhibition of the deficient enzyme and of other glycosidases further increases the potential of 70, especially compared with its enantiomer[178].

9.6 Glycoproteinoses

9.6.1 Fucosidosis

Fucosidosis is caused by mutations of the αfucosidase (FUCA1) gene, resulting in deficiency of the αlfucosidase enzyme. As a result of the hydrolytic enzyme deficiency, incomplete catabolism of N and Oglycosylproteins results in the accumula- tion of fucosecontaining glycolipids and glycoproteins in various tissues and urine.

 

278

Table9.11 PCs forPompe disease.

PC IC50 or Ki Mutation

IC50= 1.3 μM, Ki= 530 nM

IVS8+1G>A/ M519V

P545L/P545L 6.4fold L552P/L552P 17.8fold L552P/r.spl? 5.1fold L552P/A445P 4.3fold G54R/r.0 4.3fold

P545L 4.2fold L552P 3.9fold Y575S 8.5fold E579K 3.5fold A610V 5.8fold H612Q 4.5fold

n.d.

L552P/L552P 5.6fold at 20 μM L552P/abn splic 2.7fold at 20 μM L552P/A445P 1.8fold at 20 μM G549R/abn splic 3.7fold at 20 μM

L552P 4fold at 20 μM G549R 16fold at 20 μM



L552P/L552P 1.5fold at 20 μM

Max. activity enhancement References

Patient fibroblasts

[176]

6.6fold

COS‐7 cells

Patient fibroblasts [177]

HEK293T cells

Patient fibroblasts [178]

a) Compound is able to stabilize αglu under thermal denaturation; b) Cell lines were tested at least three times with DNJ concentrations ranging from 50 nM to 1 mM; c) Cells were treated with 100 μM DNJ; d) n.d.=not determined; e) Compound did not act as a glycosidase inhibitor.

9.7 Conclusions

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The clinical features of fucosidosis are progressive mental retardation and neurological deterioration, coarse facies, growth retardation, recurrent infections, dysostosis multiplex, and angiokeratoma. The treatment of fucosidosis is directed toward the specific symptoms that are apparent in each individual[183]. Correction of the enzymatic deficiency by allogeneic bone marrow transplantation has been first experienced on an animal model[184, 185] and then on patients with an amelioration of the clinical signs[186–188]. PCs for fucosidase have not been proposed to date, but several iminosugar derivatives have proved to be effective inhibitors. The discussion of fucosidase inhibitors is not the aim of this work, but a report of the main compounds is reported in the articles[189, 190].

9.6.2 α-Mannosidosis

αmannosidosis is an ultrarare autosomal recessive genetic disorder caused by mutations in the MAN2B1 gene encoding for αmannosidase, a lysosomal enzyme involved in glycoprotein catabolism. The result of αmannosidase deficiency is blockage of the degradation of glycoproteins, leading to an accumulation of mannoserich oligosaccharides in all tissues[191]. Accumulation of mannoserich oligosaccharides manifests in a broad variety of symptoms including skeletal abnormalities, motor function impairment, intellectual disability, hearing loss, respiratory dysfunction, recurrent infections, and cellular and humoral immune defects usually presenting in early childhood[192, 193].

Currently, intravenous ERT, Lamzede (Velmanase alfa, Chiesi Italia S.p.A.), is available since 2021 for the treatment of mild–moderate forms of αmannosidosis in adults, adolescents, and children, but it is not effective treatment for neurological involvement. Preliminary studies demonstrated the ability of HSCT to partially preserve neurocognitive function, stabilize skeletal abnormalities, and prevent early death[194–196].

Recently, a series of PCs combining the 5N,6Ooxomethylidenemannonojirimycin (OMJ) in either mono (71–76, Table9.12) or multivalent (βcyclodextrins as scaffold) fashion (77–79, Table9.12) were reported. Multivalent derivatives exhibited potent enzyme inhibition that prevailed over the chaperone effect. On the contrary, monovalent OMJ derivatives proved effective as activity enhancers for several mutant alfaman forms in patient fibroblasts and/or transfected MAN2B1KO cells[197].

279

9.7 Conclusions

The chapter summarizes the invitro screening, preclinical, and clinical results of carbohydratebased compounds in the currently available therapeutic approaches (ERT, SRT, and PCT) for LSDs, organized accordingly to LSD classification into MPS, sphingolipidoses, glycogen storage disorders, and glycoproteinoses.

 

280

Table9.12 PCs forα-mannosidosis

PC IC

IC50=

26.8 μM

IC50= 116 μM

IC50=

19.3 μM

IC50=

30.7 μM

IC50=

17.3 μM

Mutation

Max. activity enhancement

Patient fibroblasts

H72L/H72L 8.2fold at 20 μM P356R/P356R 6.4fold at 2 μM R750W/

1.35fold at 20 μM

R750W

Patient fibroblasts

H72L/H72L 8.0fold at 20 μM P356R/P356R 4.5fold at 20 μM R750W/

1.3fold at 20 μM

R750W

Patient fibroblasts

H72L/H72L 7.8fold at 20 μM P356R/P356R 5.7fold at 20 μM R750W/

1.35fold at 20 μM

R750W

Patient fibroblasts

H72L/H72L 11fold at 20 μM P356R/P356R 5fold at 20 μM R750W/

1.6fold at 2 μM

R750W

MAN21B‐KO HAP1 cells

C55F 51% increase at 2 μM H71L 34% increase at 2 μM L352P 8% increase at 2 μM L565P 14% increase at 2 μM R916C 26% increase at 2 μM

Patient fibroblasts

H72L/H72L 4fold at 0.2 μM R750W/

R750W

1.7fold at 20 and 2 nM

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