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10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
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296
Glucose
EXTRACELLULAR MEDIUM
CYTOPLASM
Hexosamine
biosynthesis pathway
HO O
GLUT
UDP
UDP-
GIcNAc
Protein Protein
(Ser/Thr)
OGT
OGA
Figure10.2 Illustration of O-GlcNAc modification of proteins via O-GlcNAc transferase
(OGT) and the hydrolase O-GlcNAcase (OGA).
the CAZy classification system[20]. The O-GlcNAc modification is particularly
abundant in the brain controlling a number of processes, including memory formation[21], and seems to be essential for the normal function of the mammalian nervous system [22–24]. Dysregulation of O-GlcNAcylation is associated with
neurodegenerative diseases. As the O-GlcNAcylation of proteins is regulated by the
intracellular glucose metabolism, which is reduced in the brain of AD patients, their
O-GlcNAc levels are nearly 50% lower than in control brains[25, 26].
The role of O-GlcNAcylation in neurodegeneration has been extensively
reviewed[6]. The expression and activities of OGT and OGA in the brain are agedependent and more than four thousand O-GlcNAc protein targets have been identified as playing critical roles in many cellular processes[6, 27]. In the mammalian
brain, O-GlcNAc modification of Tau, which is naturally O-GlcNAcylated [28],
decreases its phosphorylation and toxicity, suggesting a neuroprotective role of
brain O-GlcNAcylation for AD treatment[6].
10.2.1 O-GlcNAc Cycling as a Therapeutic Target Against
Alzheimer’s Amyloid Plaques and Neurofibrillary Tangles
Approximately 2–5% of all glucose entering the cell is channeled into the hexosamine biosynthetic pathway to generate UDP-GlcNAc [6] (Figure 10.2). Because
O-GlcNAcylation depends on the availability of UDP-GlcNAc, and in turn, intracellular UDP-GlcNAc level determines OGT activity, O-GlcNAcylation is considered a
valuable intracellular sensor of glucose metabolism that can be directly regulated in
a glucose-responsive manner[29].
Positron Emission Tomography (PET) studies [30], supported by experiments
with transgenic mice[31, 32] have shown that brain glucose metabolism declines
with age and is more impaired in AD patients. Glucose metabolism impairment
may lead to lower UDP-GlcNAc levels and consequently lower O-GlcNAcylation,
resulting in impaired GlcNAc cycling. But how does O-GlcNAcylation affect the
main pathological hallmarks of AD, i.e. formation of extracellular Aβ amyloid
plaques and intracellular NFT that is accompanied by synaptic loss and dementia?
The APP is an O-GlcNAc-modified protein processed by three different proteases,
namely the α-, the β-, and the γ-secretase. APP cleavage by α-secretase in the extracellular domain is non-amyloidogenic, generating a soluble fragment sAPPα and a
membrane-bound C-terminal fragment (APP-CTFα) (Figure 10.3). The latter is
cleaved by γ-secretase in the transmembrane region to afford the non- amyloidogenic
fragment p3 and the β-amyloid precursor protein intracellular domain (AICD). APP
β-secretase (BACE1) cleavage generates the soluble sAPPβ fragment and the
membrane-bound fragment APP-CTFβ, which is also cleaved by γ-secretase generating amyloid-β peptide monomers (Aβ1–40 and Aβ1–42), together with the
AICD[6, 33]. This abnormal cleavage of APP is an important event leading to the
overproduction and aggregation of Aβ species. Approximately, 90% of all Aβ fragments generated are Aβ1–40 and a smaller fraction corresponds to the toxic amyloidogenic, Aβ1–42, observed in amyloid plaques in the brain. Therefore, among the
29710.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
O
γ-Secretase
p3
AICD
Non-amyloidogenic processing
sAPPα
CTFα
UDP
UDP-GIcNAc
α-Secretase
OGT
O-GlcNAcylation
O
APP
sAPPβ
BACE-1
CTFβ
Amyloidogenic processing
γ-Secretase
Aβ Monomers
EXTRACELLULAR
AICD
MEDIUM
CYTOPLASM
Figure10.3 Illustration of amyloidogenic and non-amyloidogenic cleavage of APP.
Source: Adapted from Wani etal.[6].
10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
COOH
HO
R
GIcNAcstatin
Streptozotocin
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298
products generated by the amyloidogenic (initially cleaved by the BACE1) and nonamyloidogenic (initially cleaved by α-secretase) pathways, only the amyloidogenic
products are thought to lead to AD pathology[34].
Interestingly, it was demonstrated that by increasing O-GlcNAcylation, either
through pharmacological or genetic interventions, processing by α-secretase is also
increased, resulting in enhanced neuroprotective sAPPα levels and decreased Aβ
formation. In addition, hyper O-GlcNAcylation, produced in a 5XFAD Aβ mouse
model treated with the OGA inhibitor 1,2-dideoxy-2′-propyl-α--glucopyranoso-
2
[2,1-d]-Δ
′-thiazoline (NButGT Figure10.4), decreased Aβ1–40 and Aβ1–42levels,
reduced Aβ plaque formation, and improved cognition[26]. These findings suggest
that O-GlcNAc cycling is indeed a target for the identification of therapeutics for AD.
After the discovery that human brain Tau is O-GlcNAcylated, scientists have
focused on finding a link with neurodegeneration, as Tau hyperphosphorylation
leads to the formation of NFT [28]. Hence, the relationship between protein
O-GlcNAcylation and phosphorylation is key for the investigation of the molecular
mechanism leading to brain tauopathies, including AD[35]. Liu etal.[36] found
that decreased O-GlcNAc levels and increased Tau phosphorylation occur in mice
brain obtained from mice sacrificed after injection into the left ventricle of the brain
of 6-diazo-5-oxonorleucine (DON), an inhibitor of glutamine:fructose-6-P, the ratelimiting enzyme of hexosamine biosynthesis pathway. The analysis of human brain
HO
HO
HO
HO
OH
O
S
HO
OH
OH
N
CH
2CH2CH3
NButGT
O
N
NHAc NHAc
PUGNAc
N
N
NHCOCH(CH
H
O
N
O
3)2
R
2
HO
Thiamet-G R
MK 8719 R
HO
HO
Figure10.4 Structure of selected OGA inhibitors.
HO
OH
HO
Nagstatin
OH
HO
1
O
N
= OH, R2 = H
1
= R2 = F
1
N
O
NHCON
S
N
OH
N
H
CH
3
N
O
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
tissue showed that hyperphosphorylated Tau has up to four-fold lower O-GlcNAc
levels. Interestingly, the reduction of protein O-GlcNAcylation in AD and other
Tau-driven pathologies seems to vary in brain regions, as reported by Frenkel–Pinter
etal.[37]. They found an increase in O-GlcNAcylation in the hippocampus of AD
patients, suggesting that this complex disorder involves lesions in various brain
regions, which should be separately investigated for a comprehensive study of AD
etiology. Nonetheless, the research carried out so far supports that targeting
O-GlcNAcylation represents one of the most promising therapeutic opportunities to
control AD neurodegeneration by decreasing Tau hyperphosphorylation and by
recovering brain dysmetabolism, leading to reduced brain damage and improved
cognition[29].
10.2.2 OGA Inhibitors
The structure of some of the most studied OGA inhibitors is depicted in Figure10.4.
These carbohydrate-based compounds contain either a thiazoline ring, such as
NButGT, Thiamet-G, and MK8719, or an imidazole ring fused to the carbohydrate
moiety as GlcNAcstatins, which structure was inspired by that of nagstatin.
PUGNAc, one of the first OGA inhibitors discovered, shows a different structure as
the sugar is linked to a carbamate functionality. Around 2000, PUGNAc and streptozotocin (STZ) were used to increase cellular O-GlcNAc levels for the study of
related cellular processes, but STZ is a poor OGA inhibitor and is highly toxic to
pancreatic β-cells, being used to induce type 1 diabetes in animals[38].
Human O-GlcNAcase (hOGA) is expressed in two isoforms. The larger variant
OGA, localized in the nucleus and cytosol, is more active than the other isoform,
expressed only during embryo formation[27]. The crucial role of OGA for maintaining or decreasing O-GlcNAc levels in the brain is, indeed, key for brain neurodegeneration, encouraging the development of OGA inhibitors. In 2005, Vocadlo and
coworkers were able to elucidate, for the first time, the catalytic mechanism of
hOGA[39]. They described a two-step mechanism involving the nucleophilic participation of the 2-acetamido group, with the formation of an oxazole or oxazolinium intermediate. Later on, in 2016, Vocadlo’s group, supported by the experimental
work carried out with thiazoline inhibitors (see Section10.2.2.3), proposed that the
intermediate is an oxazoline rather than an oxazolinium ion (Figure10.5a)[40]. A
molecule of water attacks the anomeric center of the intermediate, breaking the
oxazole ring and leading to the hemiacetal with retention of configuration. In 2017,
the crystal structures of hOGA and its substrates were determined[41], providing
important information for further investigation of the mechanism of hOGA.
Recently, Xiong and Xu confirmed the existence of the oxazoline intermediate by
studying the whole catalytic process with hOGA using a Quantum Mechanics/
Molecular Mechanics approach[42]. Based on the calculated free energy profiles,
they showed that the intramolecular cyclization by the 2-acetamido group leading
to an intermediate bicyclic oxazolinium ion (I1) is the rate-limiting step, and their
simulations further suggest the formation of the oxazoline ring intermediate I2,
299
(a)
(b)
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10 Carbohydrates and Carbohydrate-Based Therapeutics in Alzheimer’s Disease
300
Figure10.5 Mechanism for hOGA inhibition. (a) Mechanism proposed by Macauley
etal.[39] and Cekic etal.[40] (b) Energy profiles (Kcal mol
(2020)[41] for the formation of the oxazoline intermediate by proton transfer of the
oxazolinium intermediate to the residue D
as deduced by computational simulations.
174
−1
) adapted from Xiong and Xu
resulting from proton transfer from the 2-acetamido group to residue D174
(Figure 10.5b). A relatively small barrier was found to connect EI1 and EI2
1
(5.43
kcal mol
in SCC-DFTB/MM and 1.93 kcal mol1 in DFT calculations), which
might contribute to accelerating the overall reaction rate.
The authors also made mutagenesis simulations of D174A and D175A, which
indicate that these catalytic residues mainly affect the observed reaction rate by
affecting the stability of the intermediate[42]. These results seem to be in full agreement with the experimental results of Vocadlo’s group, who developed aminothiazoline inhibitors (see Section 10.2.2.3). Their potency is strongly pK
leading to picomolar binding. The authors described them as genuine transition
-dependent,
a
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
state analogs[40]. This substrate-assisted catalytic mechanism is followed by both
hOGA and human lysosomal β-hexosaminidases, which are hydrolases of family 20
(GH20) [43], cleaving GlcNAc from terminal glycoproteins/glycosphingolipids.
Nonetheless, Vocadlo’s group experiments, in 2005[39], showed that the active site
of hOGA tolerates bulkier acetamido substituents than the one of lysosomal
β-hexosaminidase, which encouraged them to design new structures aiming at
selectivity for hOGA inhibition.
10.2.2.1 PUGNAc
In the search for OGA inhibitors, Vasella, and coworkers, in 1990, were inspired by
aldonolactones, known as potent glycosidase inhibitors, and particularly by 2acetamido-2-deoxyglucono-1,5-lactone, reported to inhibit OGA, although with a μM
. As both glucono-1,5-lactone oxime and its phenylcarbamoyl derivative were better
K
i
inhibitors of β-glucosidase than the corresponding lactone, Vasella synthesized for the
first time 2-acetamido-2-deoxy--glucono-1,5-lactone O-(phenylcarbamoyl)oxime
(PUGNAc) (Scheme 10.5)[45] to explore its ability to inhibit OGA. Starting from
oxime 1 oxidized with MnO
, they prepared the cyclized benzylidene protected (Z)-2
2
in 50% yield, with starting material recovered in 35% yield. Debenzylidenation with
Na/NH
and acetylation was followed by partial deacetylation with CH3NH2 remain-
3
ing the sugar hydroxy groups acetylated (compound 3). Reaction with phenyl isocyanate gave carbamate 5, which was deprotected with ammonia in methanol to afford
PUGNAc in 16.8% overall yield starting from oxime 1 (Scheme10.1).
Vasella and coworkers have then tested PUGNAc for the inhibition of OGA from
animal, fungal, and plant origin. They found out that PUGNAc was very effective,
particularly for the inhibition of the fungal enzyme, exhibiting K
= 40 nM[47]. This
i
potent OGA inhibitor was also the best of any glycosidase inhibitors described at
that time. However, the first synthesis has severe problems with the scale-up,
301
Scheme 10.1 The first synthesis of PUGNAc by Beer etal.[45, 46]. Reagents/solvent and
yield: (a) Activated MnO
CH
OH, 71%, (e) PhNCO, Et3N, THF, 94%; (f) saturated NH3, MeOH, 61%.
3
, MeOH, 50%; (b) Na, NH3, MeOH, 86%; (c) Ac2O, Py, 96%; (d) CH3NH2,
2
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302
Scheme 10.2 Improved approach to prepare PUGNAc by Vasella and Mohan[48].
Reactions/solvent and yield: (a) (NH
(c) DBU, NCS/DCM, 59%; (d) PhNCO, Et
, THF/MeOH, 74%; (b) NH2OH·HCl, py/MeOH, 75%;
4)2CO3
N, DCM, 70%; (e) Aq. NH3, 73%.
3
particularly the oxidation step with MnO2, which encouraged Vasella group to
improve the synthetic approach for PUGNAc (Scheme 10.2)[48]. Starting from
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α--glucopyranose 6, the anomeric deacetylation was carried out by ammonium carbonate to give 7 in 74% yield from 100
starting material. The open chain oxime 8 was obtained by treatment of 7 with
hydroxylammonium chloride and pyridine in MeOH under reflux. Reaction with
N-chlorosuccinimide in DBU afforded compound 9, which reacted with PhNCO in
THF to yield phenyl carbamate 10. De-O-acetylation with saturated aqueous NH
afforded PUGNAc in 16.7% overall yield.
As shown by Vocadlo and coworkers[39], PUGNAc is a potent inhibitor of hOGA
= 46 nM) but unfortunately, it is also a potent inhibitor of β-hexosaminidase
(K
i
= 36 nM). The dysfunction of this enzyme results in the accumulation of ganglio-
(K
i
sides and other glycoconjugates in the lysosome, causing Tay Sachs and Sandhoff
neurodegenerative diseases, as the enzyme is localized in the lysosome. This lack of
selectivity encouraged the modification of PUGNAc structure by varying the 2-N-acyl
chain and Stubbs etal.[49] reported the synthesis of PUGNAc and analogs starting
from glucosamine hydrochloride to prepare the 2-N-Boc protected precursor 11, submitted to the reaction conditions previously reported by Mohan and Vasella[48] to
prepare 12 (Scheme10.3)[49]. Treatment with phenyl isocyanate yields the carbamate 13, whose Boc deprotection is achieved with trifluoroacetic acid (TFA). Further
reaction with acyl chloride in pyridine and acetyl deprotection to release the sugar
hydroxy groups with NH
and its desired analogs. Unfortunately, they showed K
/MeOH afforded PUGNAc in 5.7% overall yield from 11
3
for hOGA and for human
i
hexosaminidase in the μM range and their selectivity was very poor[49].
The crucial step in both the synthetic approaches followed by Vasella group[48]
and Vocadlo group[49] is the N-chlorosuccinimide-mediated oxidative ring closure
of the oxime precursor in relatively low yields, as a secondary product with a fivemembered ring resulting from acetyl migration is formed. Moreover, the separation
3
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Scheme 10.3 Synthesis of PUGNAc and N-acylated derivatives as reported by Stubbs
etal.[49] in 2006. Reagents/solvent and yield: (a) i. NH
DCM, 52% over the two steps; (b) PhNCO, Et
(48%); (e) NH
, MeOH, PUGNAc (32%), 16a (32%), 16b (26%), 16c (23%), 16d (26%), 16e
3
N, THF, 71%; (c) CF3COOH, DCM; (d) RCOCl, py, 5
3
OH·HCl, py, MeOH; ii. DBU, NCS,
2
(29%) 16f (23%) yield over two steps.
of both products was found to be quite difficult[48, 49]. Aiming to overcome this
problem, Goddard–Borger and Stubbs[50] developed a new approach, starting from
the peracetylated 2-azido precursor 17 (Scheme10.4), obtained from glucosamine in
two steps and good overall yield[48, 50]. This azido sugar reacted with benzylamine
to deacetylate the anomeric position. Synthesis of the open chain oxime succeeded in
very high yield (97%) and ring closure with DBU and N-chlorosuccinimide gave 19 in
93% yield with the desired (Z)-stereochemistry. The reductive acylation of the azido
303
Scheme 10.4 PUGNAc synthesis developed by Goddard-Borger and Stubbs[50]. Reagents/
solvent and yield: (a) NH
THP, 91%; (d) 1. PMe
OH·HCl, py, MeOH, 97%; (b) DBU, NCS, DCM, 93%; (c) PhNCO, Et3N,
2
, AcOH, 2-(PhSe)2, CH2Cl2, toluene, water, 89%; 2. NH3/MeOH, 61%.
3
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304
group was carried out by reaction of 19 with acetic acid, trimethylphosphine, and a
catalytic amount of 2,2′-diphenyldiselenide to afford PUGNAc in 89% yield. This
was, indeed, the most fruitful and high-yielding methodology to synthesize PUGNAc,
whose access in reasonable quantities has been required for research, due to its broad
spectrum of inhibitory activity acting on enzymes interfering in a wide number of
biological processes. Interestingly, it was used for the synthesis of PUGNAc analog
with galacto configuration, termed Gal-PUGNAc[50], which is a potent and selective
inhibitor of lysosomal exo-N-acetyl--glucosaminidases[51].
Pursuing their research to control PUGNAc selectivity, Stubbs and coworkers[44], in 2016, synthesized new PUGNAc analogs, where the carbamoyl moiety is
varied in size, hydrophobicity, and shape. For the purpose, they envisioned three
series of compounds. In one of them, the PUGNAc phenyl group is substituted in
the para-position with methyl or methoxy groups, or with a bromine atom, or
replaced by a benzyl group. Another series has a cyclic or acyclic aliphatic structure
replacing the phenyl group and the third one has NHPh of the carbamate replaced
by an amino acid derivative. To access PUGNAc and the designed 63 compounds,
the authors developed a new methodology, in which a colorogenic intermediate carbonate 21 is formed in situ by reaction of 9 with 4-nitrophenyl chloroformate, and
then transformed into carbamates in good overall yield by reaction with anilines or
amines, in a one-pot reaction (Scheme 10.5). The inhibition of OGA and of
Scheme 10.5 Synthesis of PUGNAc and representative analogs with modified carbamate
moiety as developed by Stubbs and coworkers[44]. Reagents/solvent: (a) 1. 4-nitrophenyl
chloroformate, DIPEA, THF; 2. Amine or aniline-based compound, DIPEA; (b) NH
, MeOH.
3
10.2 O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA) in Neurodegeneration
Table10.1 Inhibition constant of compounds 23a–23i over OGA and HEXB.
K
(μM)
i
305
Inhibitor
PUGNAc 0.036[34] 0.046[34]
23a 0.021 ± 0.005 0.028±0.007
23b 0.111 ± 0.019 0.178 ± 0.042
23c 0.047 ± 0.002 0.056 ± 0.016
23d 14 ± 3.5 230 ± 78
23e 1.3 ± 0.17 78 ± 26
23f 45 ± 16 420 ± 140
23g 17 ± 4 220 ± 75
23h 161 ± 8 188 ± 72
23i 0.77 ± 0.031 11 ± 4.8
Source: Adapted from Hattie etal.[44].
HEXB OGA
β-hexosaminidase B found for this small library of compounds and the Ki values of
the most representative compounds (Table10.1) showed that the aryl derivatives
have, in general, a similar potency as PUGNAc, although the electron-donating
methoxy group decreased potency as compared to PUGNAc. All the aliphatic derivatives were less potent than PUGNAc for both OGA and HEXB, which is the product of the lysosomal hexosaminidase gene HEXB. Nonetheless, the results obtained
demonstrate that the envisioned structural changes were efficient in tuning selectivity to HEXB over OGA, leading to the discovery of potent and selective inhibitors
of HEXB.
In conclusion, all efforts to achieve selectivity of PUGNAc for OGA failed, either
by changing the carbamate moiety, or the sugar N-acylation. Also, the Gal-PUGNAc
analog, in which sugar position 4has the opposite configuration of that exhibited by
PUGNAc, is a selective inhibitor of the human lysosomal β-hexosaminidases,
changing GM2ganglioside levels in cultured cells, but is not able to affect O-GlcNAc
levels [51]. Nonetheless, these selective HEXB inhibitors may become powerful
small molecules for defining the role played by GM2in biological processes at the
molecular level in diseases with abnormal accumulation of GM2, and for creating
models to follow up disease stages.
10.2.2.2 GlcNAcstatins
In 2006, van Aalten and coworkers[52] presented the first rationally designed glucoimidazole GlcNAcstatin, inspired by the structure of nagstatin (Figure 10.6), a
natural product and potent inhibitor of hexosaminidase[55]. GlcNAcstatin has a
molecular architecture similar to that of nagstatin, bearing an isopropylamido
group on position 8 and a phenethyl group at position 2. The authors expected that
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