Glucocerebrosidase (GCase) activity modulation by 2-alkyl trihydroxypiperidines: Inhibition and pharmacological chaperoning
Graphical abstract
Introduction
Lysosomal storage diseases (LSDs) are a group of inherited metabolic pathologies characterized by lysosomal dysfunction due to gene mutations that encode for enzymes, membrane integral proteins and lysosomal localization transport proteins. These gene mutations affect the function of the encoded protein, resulting in lysosomal malfunction and gradual accumulation of substrates inside the lysosomes, which ultimately leads to cell dysfunction and cell death. To date, more than 70 different LSDs have been described [1], [2]. LSDs are genetically and clinically heterogeneous disorders characterized by the variable association of visceral, ocular, haematological, skeletal and neurological manifestations, which are, in most cases, responsible for physical and neurological handicaps. These disorders collectively affect 1 in 5,000 live births [1]. Gaucher disease (GD) is the most common LSD of glycosphingolipids. It occurs at a frequency of between 1 in 40,000 to 1 in 60,000 in the general population, and of 1 in 500 to 1 in 1,000 among Ashkenazi Jews [3]. GD is caused by mutations in the GBA gene (mapped on chromosome: 1q21-22), which encodes for the lysosomal enzyme acid β-glucosidase (glucocerebrosidase, also known as GCase, EC 3.2.1.45, MIM*606463) [4].
GCase hydrolyses the β-glucosyl linkage of glucosylceramide (GlcCer (1), Fig. 1) in the lysosomes and requires the coordinated action of saposin C and negatively-charged lipids for maximal activity [5]. The active site of the enzyme contains at least three domains with differing specificities: (i) the catalytic site, a hydrophilic pocket that recognizes glucosyl moieties; (ii) an aglycon binding site that is hydrophobic and has affinity for the alkyl chains of GlcCer; and (iii) a hydrophobic domain that interacts with negatively charged lipids to increase hydrolytic rates [6]. Symptoms associated with GD are due to the progressive accumulation of GlcCer in various organs. Thus, GD is a multisystemic disorder with clinical manifestations at all ages dependent on the subtype of GD. Three clinical forms of GD can be distinguished depending on the degree of neurological involvement. Type 1, the most common form (OMIM#230800), which was considered non-neuronopathic until recent discoveries; Type 2, acute neuronopathic (OMIM#230900), the rarest and most severe form; Type 3, subacute chronic neuronopathic (OMIM#231000), with later onset and a slower progressive course [7]. At present, more than 500 GBA gene mutations have been reported in Gaucher patients (data from Human Gene Mutation Database (HGMD®professional) 2019.2; http://www.hgmd.cf.ac.uk/ac).
Recently, GCase mutations were shown to be a major risk factor for Parkinson’s disease (PD), the second most common neurological disorder [8]. Individuals carrying heterozygous mutations in the gene coding for GBA do not develop Gaucher disease but have a remarkable increased risk for developing PD. Even more interestingly, recent studies highlighted that the loss of glucocerebrosidase function contributes to the pathogenesis of Parkinson’s disease [9] and that both the mutation and the enzyme activity should be considered when developing GBA-targeted PD therapy [10]. Although additional studies are required for understanding the molecular basis connecting Gaucher disease to Parkinson disease, the modulation of GCase activity is emerging as the key therapeutic target for both pathologies [11].
During the past two decades, the research in the field of LSDs has made significant progress, particularly in the development of a variety of innovative therapeutic approaches. These include strategies aimed at increasing the residual activity of the missing enzyme by infusing a recombinant enzyme (the enzyme replacement therapy, ERT), or reducing the synthesis of the stored substrate (GlcCer) by inhibiting its biosynthesis with small compounds (the so-called substrate reduction therapy, SRT) [4]. Gaucher patients affected by the non-neuronopathic form of Gaucher disease are treated with enzyme replacement therapy (ERT) through infusion of the recombinant enzyme [e.g. Imiglucerase (Cerezyme®), Genzyme; velaglucerase alfa (VPRIV®), Shire and taliglucerase alfa (Elelyso®), Pfizer]. However, this therapy has a high cost, requires the patients’ frequent hospitalization and is not effective for the neuronopathic forms of the disease. An alternative option for GD is the Substrate Reduction Therapy (SRT) by treatment of patients with drugs [such as miglustat (ZavescaTM), Actelion and the most recently approved eliglustat (CerdelgaTM), Sanofi/Genzyme] [12], able to inhibit the enzyme which produces the glycosphingolipid that accumulates during the disease. The main drawbacks of the SRT are the side effects induced by the inhibitor and that it is limited to treating the type 1 form of the disease. More recently, an alternative therapeutic strategy for missense mutations has emerged, namely the pharmacological chaperone therapy (PCT). PCT is based on the use of inhibitors of the deficient enzymes able to enhance their residual hydrolytic activity when they are employed at sub-inhibitory concentrations. Pharmacological chaperones (PCs) induce or stabilize the proper conformation of the defective enzyme, improving its stability in the endoplasmic reticulum (ER), and preventing its aggregation and premature degradation [13], [14].
In principle PCs may provide several advantages over the previously mentioned therapies, they can be administered orally and have the ability to cross the blood brain barrier (BBB) and therefore can potentially treat the neuronopathic forms of the disease. The pharmacological chaperone approach could be applied to a whole range of diseases related to protein misfolding, such as Alzheimer, Parkinson, Huntington, or amyotrophic lateral sclerosis [15], [16].
Structurally, the most promising PCs for LSDs are glycomimetics with a nitrogen atom replacing the endocyclic oxygen (such as deoxynojirimycin, DNJ (2), Fig. 1) or the anomeric carbon of sugars (e.g. isofagomine, IFG (3) or 1,5-dideoxy-1,5-iminoxylitol, DIX (4), Fig. 1), also known as iminosugars, or azasugars, respectively [17].) As inhibitors of carbohydrate-processing enzymes, imino- or azasugars and other substrate analogues which behave as competitive glycosidase inhibitors are good PC candidates [18]. The first oral PC drug has recently been commercially developed for the treatment of Fabry disease (another LSD) in Europe (Migalastat, Galafold, Amicus Therapeutics), but no PC for Gaucher disease has yet reached the market. Considering the potential benefits of discovering an effective chaperone for GCase not only for GD but also for GD-related PD and for the sporadic forms of PD that do not have the GBA mutation, there is an urgent need to develop new small compounds with the ability of modulating the GCase enzyme activity. Isofagomine (Fig. 1) reached the most advanced stage of development (Phase III clinical trials in a combined therapy with recombinant GCase). IFG showed remarkable in vivo effects in a neuronopathic Gaucher disease mouse [19], and in a PD mouse model of synucleinopathy [20]. However, it was not effective in reducing the accumulation of lipid substrates; it is believed that its high hydrophilicity hampers an efficient transport into the ER and the lysosome [18]. For this reason, several alkylated imino- or azasugars were synthesized and investigated as PCs for GD, showing better metabolic properties [18].
The position of the alkyl chain seems to be crucial for the activity. Indeed, while for DNJ analogues the N-alkylation gave good PCs for GCase (e.g. compound 5, Fig. 1) [21], in the case of azasugars the alkylation of the carbon adjacent to nitrogen furnished better PCs for GD. This is shown by the relevant examples reported by Withers and co-workers (compound 6 [22], Fig. 1) and by Compain, Martin and co-workers, who synthesized one of the most potent mutant GCase enhancer ever reported (compound 7, Fig. 1, which displayed 1.8-fold activity increase in N370S mutated fibroblasts at a concentration as low as 10 nM) [23].
Following our longstanding interest in the total synthesis of polyhydroxylated nitrogen heterocycles and their non-natural analogues as glycosidases inhibitors [24], [25], [26], [27], [28], we found that the peculiar configuration of trihydroxypiperidine 8 [29], [30], [31], the enantiomer of a natural product, imparted inhibitory properties to commercial α-l-fucosidase (IC50 = 90.3 μM) [32]. On N-alkylation with an eight carbon atom chain (as in compound 9, Fig. 1), an interesting 50% inhibition at 1 mM concentration was gained towards β-glucosidase from almonds. This prompted us to test compound 9 on the GCase enzyme on a pool of leukocytes from healthy donors, resulting in an interesting IC50 = 30 μM [33]. Even more intriguingly, compound 9 behaves as modest PC for GCase, rescuing the enzyme activity of 1.25-fold when tested at a concentration of 100 μM [33]. Based on the observation of the subtle role played by the alkyl chain in the biological activity, as shown by the previously mentioned examples on IFG and DIX derivatives, we decided to synthesize trihydroxypiperidines 10 and 11 alkylated at C-2 with chains of different lengths, in order to investigate in detail the role of the length, position and configuration of the alkyl chain on this differently configured piperidine skeleton.
We report herein full details and the completion of our study [34], which includes the synthesis, biological evaluation towards commercial and human lysosomal enzymes, the ex-vivo activity on cell lines, and a molecular docking investigation.
Section snippets
Chemistry: synthesis and structural assignment of the trihydroxypiperidines alkylated at C-2
The retrosynthetic strategy employed for the preparation of the compounds assayed in this work is shown in Scheme 1. We previously observed that the addition of MeMgBr and EtMgBr to the carbohydrate-derived aldehyde 15, obtained from d-mannose, and to nitrone 14 derived thereof [35] proceeded with opposite diastereofacial preference. However, the addition of a suitable Lewis Acid, while having little effect with aldehyde 15 resulting in only a slight decrease in diastereoselectivity, was able
Conclusions
In conclusion, we report the synthesis of two series of epimeric trihydroxypiperidines 10 and 11 alkylated at C-2 with both configurations, through a stereodivergent approach that relies on the stereoselective addition reaction of Grignard reagents to the d-mannose-derived nitrone 14. The absence or the presence of a suitable Lewis acid allowed reversal of the selectivity of the addition, giving access to both diastereoisomers of the formed hydroxylamines 12 and 13, respectively. An efficient
Chemistry
General methods: Commercial reagents were used as received. All reactions were carried out under magnetic stirring and monitored by TLC on 0.25 mm silica gel plates (Merck F254). Column chromatographies were carried out on Silica Gel 60 (32–63 μm) or on silica gel (230–400 mesh, Merck). Yields refer to spectroscopically and analytically pure compounds unless otherwise stated. 1H NMR spectra were recorded on a Varian Gemini 200 MHz, a Varian Mercury 400 MHz or on a Varian INOVA 400 MHz
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
We thank Fondazione Cassa di Risparmio di Pistoia e Pescia (Bando Giovani@Ricerca scientifica 2017) for a fellowship to C.M. and financial support. We thank MIUR-Italy (“Progetto Dipartimenti di Eccellenza 2018−2022” allocated to the Department of Chemistry “Ugo Schiff”), Università di Firenze and Fondazione CR Firenze for financial support (Bando congiunto per il finanziamento di progetti competitivi sulle malattie neurodegenerative. Project: A multidisciplinary approach to target Parkinson’s
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