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256

Table9.3 Pharmacological chaperones forMPSs.

Disorder PC IC50 or Ki Mutation

MPS II

IC50= 30.1 μM Patient fibroblasts [87]

P231L/ P231L

N63D 1.6fold at 10

L67P 3fold at 10 μM

R88H 3fold at 10 μM

Y108S 1.8fold at 10 μM

P231L 39.6fold at

MPS IIIB

IC50= 67 μM

11.4 μM

Ki=

P358L/ P358L

E153K/ E153K

Y140C/ Y140C

IC50= 374 μM

130 μM

Ki=

P358L/ P358L

E153K/ E153K

Y140C/ Y140C

MPS IIIC

Ki= 0.28 mM Patient fibroblasts [89]

L137P/ S518F

S541L/ c.234+1G.A

P283L/ R344C

S518F/ S518F

R344H/ R384X

N273K/ N273K

R344C/ R344C

S518F/ S518F

Max. activity enhancement Refe rences

1.7fold at 0.1

μM

HEK293T cells

μM

10 μM

Patient fibroblasts [88]

1.8fold at 100

μM

1.1fold at 10 and μM

100

1.1fold at 10 μM

Patient fibroblasts

1.6fold at

0.01

μM

2.4fold at 1

1.5fold at 0.1

μM

2.3fold at 14 mM

2.1fold at 14 mM

2.5fold at 7 mM

2.5fold at 7

1.2fold at 7 mM

2.1fold at 7 mM

1.7fold at 7 mM

1.5fold at 7 mM

Table9.4 Inhibitors ofIDS and GALNS (human enzymes).

9.3 Mucopolysaccharidoses

257

Compounds

IDS, IC50 (rp/n)

n.d.

13= 140 μM (rp=23; rp/n= 3)

14= 31 μM (rp=177; rp/n= 20)

GALNS, IC

(rp; rp/n) Refer ences

0.3 μM

[93] (rp; rp/n not calculated)

0.2 μM (rp; rp/n not calculated)

13= 47 μM

[94] (rp=83; rp/n= 9) 14= 85 μM

(rp=59, rp/n= 7)

n.i.

0.004 mg ml1 of Au GNPs

[corresponding to 0.52 μM of iminosugar]

(rp=7500)

n.d.

9 μg ml1 of Au GNPs

[corresponding to 13 μM of sugar]

(rp not calculated)

a) n.d.=non determined. n.i.=no inhibition at 1 mM. b) rp=IC50 of the monovalent reference/IC50 of the multivalent compound.

[95]

Data not

published

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9.4 Sphingolipidoses

9.4.1 Fabry Disease

FD is a Xlinked recessive LSD with generally more severe manifestations in males. This progressive and multisystemic disease is caused by mutations in GLA gene resulting in deficiency of the lysosomal enzyme αgalactosidaseA (αGal A), which leads to the accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in lysosomes in cells throughout the body. The prevalence of FD is estimated at 1 : 40 (9.3 : 100 severe classical form, predominantly affecting juvenile males or adult females, and a nonclassical form, more prominent in males with residual enzyme activity[96]. In classically affected patients, early symptoms include angiokeratoma, anhidrosis, neuropathic pain, gastrointestinal symptoms, and microalbuminuria, while progressive renal failure, heart failure, and stroke generally occur later in life. In nonclassically affected patients, the disease presents a more variable and smoother course. In general, the shortened life expectancy of FD patients is related to the organ damage degree[97–99].

Since 2011, Fabry patients have benefited from two ERT products: Agalsidase alfa (Replagal have the same amino acid sequence, but a different glycosylation. The latter is the only one approved by the FDA, while both are approved by the EMA. Although the initial clinical trials showed that ERT produced beneficial effects on neuropathic pain, cardiac mass, and kidney function, disease complications may still occur in patients treated with this therapy[100, 101].

In addition, due to the very short plasma halflife of the enzyme, its therapeutic activity is characterized by very short “on” peaks and long “off” activity valleys, which recently prompted the development of two secondgeneration ERTs for FD. These are recombinant αGal A derived from plants, namely mossαGal A (Phase I clinical trials) and tobaccoαGal A modified with polyethylene glycol (PEG) chains (pegunigalsidase, Phase II clinical trials), essentially aimed at reducing the costs of the treatment. More interestingly, in the last 10 able to improve ERT administration, by addressing important issues such as the protection of the naked enzyme, the increase of enzymes’ halflife in plasma, and a targeted delivery of the protein for improving cell internalization. Trimethylchitosan nanoparticles, cholesteroldipalmitoylphosphatidylcholine nanoli alized with RGD tripeptide moieties, and extracellular vesicles are only some of the exiting tools developed to improve the delivery of αGal A enzyme and recently reviewed by Schwartz and coworkers[102].

Regarding SRT for FD, glucosylceramide synthase (GCS) inhibitors have been developed to reduce the accumulation of precursors of Gb3 at the biosynthetic cascade, but not specifically the FD toxic substrate Gb3. To date, there are two products under clinical investigation: the ceramide mimetic Venglustat (Ibiglustat, Sanofi Genzyme) and the iminosugar Lucerastat (Nbutyldeoxygalactonojirimycin,

000–170 000 live births, with increased incidence in Nova Scotia

000 males) [1]. Phenotypically, FD can be distinguished into the more

, Shire HGT) and Agalsidase beta (Fabrazyme®, Genzyme Inc.), which

years, nanotechnology offered a series of platforms

posomes function-

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4, Figure9.3, Idorsia Pharmaceuticals, Switzerland). The latter is the galactose configured analog of Miglustat (3, Figure9.3), the first drug marketed as SRT for GD. Lucerastat has been proven to avoid the accumulation of Gb3in tissue and is currently under Phase 3 of clinical trials, mostly centered on patients with neuropathic pain and gastrointestinal symptomatology[103, 104].

PC studies for FD mostly used DGJ iminosugar 5 (Table 9.5), now known as

Galafold

(Migalastat, Amicus Therapeutics), which is a potent inhibitor of αGal A, but when administered at subinhibitory doses increases enzymatic activity for some GLA gene mutations[58]. Being an analog of the terminal galactose of Gb3, 5 binds and stabilizes wildtype and mutant forms of αGal A[113]. It was also proven to alleviate the Gb3 storage in mouse kidneys[114] and in recent years, the several clinical trials conducted to validate 5 as PC for FD confirmed its beneficial effects on organ function, Gb3 clearance, and αGal A activity[25, 115, 116]. Once proven to be safe and well tolerated, Galafold was approved in USA, EU, Israel, Australia, and Canada as an oral treatment for Fabry patients with amenable mutations. Patients’ eligibility for treatment with Galafold is determined using an invitro enzyme activity assay and within the eligible group (GLA mutations leading to the loss of gross structural protein domains and loss of αGal A expression are not amenable), an increase ranging from 1.2 up to 30.4fold was observed in the αGal A activity[105].

Some structural modifications of DGJ (5) resulted in an improved PC activity as reported by Ortiz Mellet and collaborators. Indeed, DGJ thioureas 17 and 18 resulted in less potent inhibitors than 5 (Table9.5) but were able to equalize its chaperoning activity (threefold increase of αGal A activity) at lower concentration (3 μM vs. 20 μM) in skin fibroblasts derived from Fabry patients homozygous for the R301G mutation. In addition, a maximal enhancement of fivefold was obtained by increasing to 30 μM the concentration of 17 and 18, which did not show cytotoxicity up to 500 μM[106]. Later, among a new family of 1deoxygalactonojirimycinaryl thiou- reas (DGJArTs), compounds 19 and 20 exhibited a significantly higher chaperoning efficiency than 5 at 30 μM in SV40mediated transformed cell lines from normal and Q279E FD fibroblasts, as well as the ability to reduce the accumulation of Gb3in FD cells. In addition, PCs 19 and 20 act in a synergistic manner with the proteostasis regulator 4phenylbutyric acid (Table9.5)[108].

Among different pyrrolidine and piperidine iminosugars isolated from the roots of Adenophora triphylla and fully characterized, the pyrrolidine iminosugar 2,5dideoxy2,5iminoaltritol (DIA, 21) showed strong competitive inhibition toward αGal A and was proven to stabilize the enzyme invitro assay of thermal denaturation (αGal A activity was lost within 60 minutes heating at 48 °C, while it remained over 70% in the presence of 100 μM 21). In addition, the treatment with 21 for three days dosedependently increased intracellular αGal A activity, with a maximal increase of 9.6fold at 500 μM (Table9.5)[109].

The only example of a multivalent PC for FD was reported in 2020 by Carmona and coworkers. In particular, the nonavalent pyrrolidine iminosugar 22 (Table9.5), a potent competitive inhibitor of αGal A, increased by 5.2fold the activity of the misfolded enzyme in R301G patient cells at 2.5 μM, demonstrating the potential use of multivalent ligands in treatment of FD[110].

259

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260

Table9.5 PCs and combined ERT/PC for Fabry disease.

PC IC50 or Ki Mutation

IC50= 60 nM (pH=5)

= 10 nM

(pH=7)

15.1 nM

Ki=

FD patients (Ph3 clinical trials) [105]

1.2 up to 30.4fold

Patient fibroblasts [106, 107]

R301G 3fold at 20 μM

R301Q 10.8fold at

IC50 (17)=4.5

R301G 3fold at 3 μM

μM (pH=5) IC

(17)=

0.2 μM (pH=7) IC

(18)=

37 μM (pH=5) IC

(18)= 5 μM

(pH=7)

IC50

Q279E 3fold at 3 μM (19)=0.34 μM (pH=5)

(19)=

0.043 μM (pH=7)

(20)=

0.074 μM (pH=5)

(20)=

μM

0.016 (pH=7)

IC50= 0.69 μM R301Q

lympho-

blasts

Max. activity enhancement Refer ences

10 μM 20fold at

μM

100

[106]

5fold at 30

μM

[108]

4fold at 30

9.6fold at

μM

[109]

500 μM

IC50= 1.2 μM Ki=

0.20 μM

R301G 5.2fold at

μM

2.5

[110]

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 μM [111]

Max. activity enhancement Refer ences

at10 mM

enhancement

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]

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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

μM) of this cotreatment study in W162X patient cell line, without any detect-

at 100 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

 

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

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

up to threefold at 30 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 μM (27) (Table9.6).

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

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) and 125–175% (3–30 μM) in fibroblasts bearing the G202R/L444P mutation and of 30–40% (0.3–1 μM) and 40–120% (3–30 μM) in fibroblasts bearing the F213I/L444P muta- 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].

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264

Table9.6 PCs forGaucher disease.

PC IC50 or Ki Mutation

IC50= 0.06 μM Ki

= 0.016 μM

= 8.4 nM)

(Ki

N370S 3fold at

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

[75]

50 μM (32)

[132]

0.5 μM

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

>5 mM N370S 3fold at 1 μM [134]

(Continued)

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15.09.202611 Мб0Книга_5940_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202611 Мб0Книга_5941_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202611 Мб0Книга_5942_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202611 Мб0Книга_5943_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202611 Мб0Книга_5944_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202610 Мб0Книга_5945_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202610 Мб0Книга_5946_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202610 Мб1Книга_5947_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.09.202610 Мб0Книга_5949_Библиотеки_им_академика_М_И_Перельмана.pdf
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30.08.202672 Мб0Книга_595_Библиотеки_им_академика_М_И_Перельмана.pdf