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Table9.5 (Continued)

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261

PC IC50 or Ki Mutation

Ki= 38.5 μM R301Q 10.8fold

PC/ERT IC

or Ki Mutation Max. activity

IC50= 0.67 μM

(pH=5)

IC50= 0.053 μM (pH=7)

Ki= 3.5 μM W162X 12fold at

N215S 9fold at 50

Max. activity enhancement Refer ences

at10

enhancement

μM [111]

100 μM

Regarding PCs not specific for the enzyme active site, Fleet and coworkers reported in 2011 that the enantiomer of 5, DGJ (23, Table9.5), was a noncompetitive inhibitor of αGal A, about 1000fold weaker than 5 (competitive). Compound 23 still behaved as chaperone with a 10.8fold activity enhancement in Fabry R301Q fibroblasts at 10

mM, which was like that observed with a 1000fold lower concentration of 5 (10 μM). When administered simultaneously, the mixture of enantiom- ers clearly showed doseresponse synergistic effects, enhancing αGal A up to 14fold, thus suggesting that the concomitant binding to two different sites might further stabilize the enzyme conformation[107].

More recently, thanks to in silico docking, an allosteric hot spot for ligand binding was identified, and 2,6dithiopurine, which preferentially bonds this site, was demonstrated to stabilize recombinant human αGal A (rhαGal A) invitro and to rescue the A230T mutant αGal A that is not responsive to 5 in a cellbased assay[117].

Regarding the ERT/PC therapy, coformulation of αGal A and DGJ (5) for treatment of FD was patented in 2014 by Khanna etal.[118].

As an example of the utility of natural productinspired combinatorial chemistry in the search for stabilizers of rhαGal A, Cheng and coworkers identified two lead compounds belonging to pyrrolidine and piperidine iminosugar families, respectively. Indeed, coadministration of 50 μM concentration of 3epiADMDP (24, Table 9.5) with rhαGal A (1 nM) in the Fabry N215S cell line was found to enhance overall

[107]

Refer ences

[112]

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

262

αGal A activity of approximately ninefold, while αGal A alone (ERT) or 24 alone (PC) are able to only enhance overall αGal A activity twofold[111]. More recently, structural modifications and bioevaluations performed on a series of C2 and C6 derived (3S,4S,5S)trihydroxylated piperidines allowed to identify derivative 25 (Table9.5), which showed the best improvement of rhαGal A (12fold increase at 100 μM) of this cotreatment study in W162X patient cell line, without any detect- able cytotoxicity toward normal lymphocytes, or inhibition of other human glycosidases[112].

9.4.2 Gaucher Disease

GD is the most common LSD with an incidence of two cases per 100 000individuals, which dramatically increases in Ashkenazi Jews (100 per 100 000individuals), owing to the socalled founder effect[6]. GD is caused by mutations in the GBA gene (chromosome: 1q2122), which encodes for the lysosomal enzyme acidβglucosidase (glucocerebrosidase or GCase). GCase catalyzes the hydrolysis of glucosylceramide (GlcCer) to glucose and ceramide in the lysosomes[119].

More than 350mutations of GBA have been reported for GD patients[120], the N370S and L144P missense mutations being the most frequent ones. Three clinical types of GD are distinguished on the basis of the age onset and the severity of the associated symptoms. Type 1, the most common form, causes liver and spleen enlargement, bone pain and fractures (broken bones), and, sometimes, lung and kidney problems. It does not affect the brain and can occur at any age. Type 2, which causes severe brain damage, appears in infants. Most children who have it die by age 2: this is the rarest and most severe form. In type 3, there may be liver and spleen enlargement, the brain is gradually affected, and it usually starts in childhood or adolescence. Recently, a pathological loop between GD patients and carriers and Parkinson’s disease emerged. Although the connection between GBA mutations and Parkinson’s development is far to be fully understood, therapeutic interventions aimed at enhancing GCase activity to treat Parkinson’s disease are already under investigation[121].

ERT is effective only for type I GD (the nonneuronopathic phenotype) and there

are three drugs available to date: Cerezyme

(imiglucerase, Sanofi Genzyme, from

1994), VPRIV (velaglucerase alfa, Shire Human Genetic Therapies, from 2010), and

Elelyso

(aliglucerase alfa, Pfizer, from 2012). Imiglucerase is a modified form of human GCase, produced by recombinant DNA technology using a mammalian CHO cell culture. Velaglucerase alfa has the nativeenzyme sequence produced in a human cell line, while taliglucerase alfa is plantcellderived and produced in an inexpensive platform[122].

Regarding SRT, the first drug developed was the iminosugarbased drug Zavesca (Miglustat, Nbutyl DNJ, 3), which is able to reversibly inhibit GCS and consequently reduce the production of GlcCer, representing an appropriate choice for type 1 GD patients.

Since 3 mechanism was first demonstrated in 1994, its safety and efficacy have been extensively investigated and nonnegligible adverse effects have been

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263

unfortunately identified, especially gastrointestinal disturbances and tremors.

In addition, Zavesca

is contraindicated in pregnancy, in anticipation of pregnancy and breastfeeding, because maternal death and infertility were observed in mouse models[97].

Later, the more selective GCS Eliglustat

(Cerdelga, Sanofi Genzyme) was

introduced and approved both by FDA (2014) and EMA (2015) as a firstline treat-

ment for adults with Type 1 GD [123]. Unfortunately, neither Zavesca

Eliglustat

can cross the BBB and cannot be applied in the treatment of neurono-

nor

pathic GD.

The compound that reached the most advanced clinical trial as PC for GD is isofagomine (IFG, 6, Figure9.3), which was unfortunately stopped at Phase II trials because it was not effective in reducing the accumulation of GlcCer in GD patients. Although being a strong competitive inhibitor of human lysosomal GCase, (Ki=

0.016 μM; IC

up to threefold at 30

= 0.06 μM)[124] 6 was found to increase mutant GCase activity

μM in fibroblasts with the N370S missense mutation, associated to Type 1 GD (Table9.6)[125]. IFG failure in clinical trials was attributed to its high hydrophilicity, which might hamper an efficient transport to the cells. For this reason, a series of alkylated iminosugars were later developed, among which the 6nonyl IFG (26) [126, 127], the nonyldeoxynojirimycin (NNDNJ, 27) [128, 129] and the α1CnonylDIX (28)[130] resulted in the most promising PCs, being able to enhance GCase activity in N370S GD fibroblasts, ranging from 1.5fold at 3 nM (26) to 2fold at 10

Moreover, bicyclic nojirimycin (NJ) analogs with structure of sp

μM (27) (Table9.6).

iminosugars were found to behave as very selective, competitive inhibitors of GCase, and compounds 29–31 also displayed a better chaperoning activity than the parent NNDNJ (27) toward some mutations involved in neuronopathic GD forms. In particular, they resulted in increases in GCase activity of 60–75% (0.3–1

μM) in fibroblasts bearing the G202R/L444P mutation and of 30–40%

(3–30 (0.3–1

μM) and 40–120% (3–30 μM) in fibroblasts bearing the F213I/L444P muta-

μM) and 125–175%

tion, while 27 showed no effect in these two cell lines (Table 9.6) [131]. More

recently, the same group reported several DNJbased sp

iminosugars incorporating an orthoester fragment, which are able to switch from hydrophobic to hydrophilic in the pH 7 to pH 5window, having a dramatic effect on the enzyme binding affinity, and thus maximizing the chaperone over the inhibitory behavior[75]. pHsensitive compounds 32, 33, and 34 showed to be better GCase ligands (1.3 to 200fold) than Ambroxol (IC

= 41.5 μM), a nonglycomimetic PC under clinical trial for GD[138],

at the neutral pH (ER), while at acidic pH (lysosome), the product 35, obtained from the hydrolysis of 32–34, was a threefold weaker ligand than Ambroxol. More interestingly, compound 32 was able to increase GCase activity by sixfold in N188S/ G193W GD fibroblasts, while a modest enhancement was obtained for the N370S mutation (1.5fold).

Among pyrrolidine iminosugars, the Ctridecyl derivative of DAB1 (1,4dideoxy 1,4 iminoarabinitol) (36) showed the same GCase activity enhancement as IFG (6) in GD fibroblasts bearing the N370S mutation, but at a 10 times lower concentration (0.5 μM)[132].

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

264

Table9.6 PCs forGaucher disease.

PC IC50 or Ki Mutation

IC50= 0.06 μM

N370S 3fold at Ki = 0.016 μM (Ki = 8.4 nM)

IC50= 0.6 nM N370S 1.5fold at

IC50= 1 μM N370S 2fold at

IC50= 6.8 nM N370S 1.8fold at

Max. activity enhancement Refer ences

[124, 125]

30 μM

1.6fold at 10 μM

[126, 127]

3 nM

[128, 129]

10 μM

[130]

10 nM

Ki (29)=5.6 μM Ki (30)=3.5 μM Ki (31)=4.0 μM

N370S 60% at

0.3–1 μM 40–165% at

3–30 μM

G202R/

L444P

60–75% at

0.3–1 μM 125–155% at

3–30 μM

F213I/

L444P

3040% at

0.3–1 μM 40–120% at

3–30 μM

[131]

Table9.6 (Continued)

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265

PC IC50 or Ki Mutation

IC50 (32)=0.20 μM

(33)=0.15 μM

(34)=32.6 μM

N188S/ G193 (32)

IC50> 1000 μM  

IC50= 0.77 μM N370S 1.5fold at

Max. activity enhancement Refer ences

6fold at 50

μM (32)

[75]

[132]

0.5 μM

IC50 = 3.9 μM N370S 62% at 30 μM [133]

>5 mM N370S 3fold at 1 μM [134]

(Continued)

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

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 Ki = 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 100 μM

2.8fold at 20 μM

500 μM

300 μM

[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].

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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 = N370S cell line by 1.6fold at 500

6.9 μM), but still able to increase GCase activity in the Gaucher 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

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

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.

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269

PC IC50 or Ki Mutation

IC50= 25 μM Mouse cell lines expressing human

R201C 5.4fold at 0.5 I51T 2.2fold at 0.5 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 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

mM mM

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

μM

1.2

[148]

IC50= 32 μM Patient fibroblasts [150]

I51T/Y316C 5.5fold at 80 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

μM

(Continued)

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

270

Table9.7 (Continued)

PC IC50 or Ki Mutation

IC50= 8 nM Patient fibroblasts [151]

R201C/R201C 15fold at 10 μM R201C/H281Y 18fold at 10 μM Q255H/K578R 20fold at 10 μM 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

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]

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