N-Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α-Mannosidase II

Article abstract of DOI:10.1002/cmdc.201700607

Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α-mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co-inhibition of lysosomal α-mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N-substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α-mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (K_i=50–76 μm, as determined by enzyme assays) with a significant selectivity index of IC₅₀(LManII)/IC₅₀(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC₅₀=92–200 μm) and low cytotoxic activities in normal fibroblast cell lines (IC₅₀>200 μm). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2-mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α-mannosidase.

Full text of DOI:10.1002/cmdc.201700607

Accepted Article
Title: N-Benzyl substitution of polyhydroxypyrrolidines - the way to
selective inhibitors of Golgi α-mannosidase II
Authors: Sergej Šesták, Maroš Bella, Tomáš Klunda, Soňa Gurská,
Petr Džubák, Florian Wöls, Iain BH Wilson, Vladimir Sladek,
Marián Hajdúch, Monika Polakova, and Juraj Kóňa
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201700607
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700607
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N-Benzyl substitution of polyhydroxypyrrolidines - the way to
selective inhibitors of Golgi α-mannosidase II
Sergej Šesták, [a] Maroš Bella, [a] Tomáš Klunda, [a] Soňa Gurská, [b] Petr Džubák, [b] Florian Wöls, [c] Iain
B. H. Wilson, [c] Vladimir Sladek, [a] Marián Hajdúch, [b] Monika Poláková,*[a] Juraj Kóňa*[a]
[a]
Dr. Sergej Šesták, Dr. Maroš Bella, Tomáš Klunda, Dr. Vladimir Sladek, Dr. Monika Poláková, Dr. Juraj Kóňa
Institute of Chemistry, Center for Glycomics
Slovak Academy of Sciences
Dúbravská cesta 9, 845 38 Bratislava, Slovakia
E-mail: monika.polakova@savba.sk (M. Poláková), juraj.kona@savba.sk (J. Kóňa)
Dr. Soňa Gurská, Dr. Petr Džubák, Assoc. Prof. Marián Hajdúch
Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry
Palacky University and University Hospital in Olomouc
Puškinova 6, 775 20 Olomouc, Czech Republic
[b]
[c]
Florian Wöls, Dr. Iain B. H. Wilson
Department of Chemistry
University of Natural Resources and Life Sciences
A-1190 Vienna, Austria
Supporting information for this article is given via a link at the end of the document. It contains experimental procedures and analytical data for compounds
2 - 22, and calculated FMO-PIEDA interaction energies between the inhibitors and active-site amino acids of dGMII.
Abstract: Inhibition of biosynthesis of complex N-glycans in Golgi
apparatus influence progress of tumor growth and metastasis. Golgi
α-mannosidase II (GMII) has become a therapeutic target for a drug
with anticancer activities. One critical task for successful application
GMII drugs in medicine treatment is to reduce their unwanted co-
inhibition with lysosomal α-mannosidase (LMan) by which suffer all
known potent GMII inhibitors. A series of novel N-substituted
polyhydroxypyrrolidines was synthesized and tested with modeled
GH38 α-mannosidases from Drosophila melanogaster (GMIIb and
LManII). The most potent structures inhibited GMIIb (Ki = 50-76 µM
as determined by enzyme assays) with a significant selectivity index
of IC50(LManII)/IC50(GMIIb) > 100. They also showed the inhibitory
activity in vitro assays with MTS test towards cancer cell lines
(leukemia, IC50 = 92-200 µM) and low cytotoxic activity towards
normal fibroblast cell lines (IC50 > 200 µM). In addition, they did not
show any significant inhibitory activity towards GH47 Aspergillus
saitoi α1,2-mannosidase. An appropriate stereo configuration
of -CH2OH and benzyl functional groups on the pyrrolidine ring of the
inhibitor may lead to an inhibitor with the required selectivity to the
active site of a target α-mannosidase.
of short duration have demonstrated that swainsonine has a low
toxicity in humans and is well tolerated.[2g, 7] On the other hand,
swainsonine and all other known α-mannosidase inhibitors have
an additional serious side effect that potentially precludes their
use as therapeutics. These compounds also inhibit the broad-
specificity lysosomal α-mannosidase (LMan) (EC 3.2.1.24, GH
family 38)[8] resulting in the accumulation of mannose-containing
oligosaccharides in tissues, serum and urine.[2g, 4]
To apply swainsonine-mechanism-based compounds as
therapeutics, selective inhibitors of GMII with minimized side
effect to LMan are required. However, the potent inhibitors of
GMII exhibited negligible or no selectivity.[5a, 5f, 9] It was observed
that 3- and 5-substituted swainsonine derivatives exhibited a
weak selectivity towards GMII [IC50(LMan)/IC50(GMII) < 20] at
the nanomolar and micromolar levels, while 6- and 7-substituted
derivatives exhibited even poorer activity and selectivity at the
micromolar
polyhydroxypyrrolidine
selectivity.[9a, 9b]
and
millimolar
derivatives
levels.[5f, 10]
also showed
Some
weak
The active site of Drosophila melanogaster Golgi α-
mannosidase II (dGMII) is made up of three sugar-binding sites:
the catalytic, holding, and anchor subsites.[5a, 5b, 11] dGMII needs
Zn2+ ion co-factor for catalytic activity which resides at the
bottom of the catalytic subsite.[5b] Zn2+ ion is in octahedral
coordination with four amino acid residues (side chains of His90,
Asp92, Asp204, His471) and with an inhibitor (bidentate ligand).
All potent dGMII inhibitors have to bind properly to the bottom of
the active site by interaction with Zn2+ and neighboring amino
acid residues otherwise the potency of the inhibitor is dropped
dramatically.[9a, 9b, 9e, 9f, 9i-k, 10c, 12] The critical role of Zn2+ ion and
aspartic acid residues of the catalytic subsite for the interactions
with the dGMII inhibitors was recently confirmed by quantum
mechanics calculations.[13] From crystal structures of available
fruit fly dGMII[5b, 11a] and bovine bLMan[8a] it is evident that the
active site of both enzymes are structurally and chemically
almost identical in radius of 10 Å around Zn2+ ion co-factor. This
is one of reasons why structurally small potent GMII inhibitors
like swainsonine inhibit both enzymes effectively with no
Introduction
Swainsonine 1 [(1S,2R,8R,8aR)-trihydroxy-indolizidine] is an
alkaloid naturally occurring in a number of Australian and North
American plants.[1] It was found that it interferes with the
glycosylation pathway where it specifically inhibits glycoside
hydrolases from the GH family 38.[2] Swainsonine has attracted
attention as a potential anticancer agent as it inhibits tumor
growth and metastasis, augments natural killer and
macrophage-mediated tumor cell killing, and stimulates bone
3]
marrow cell proliferation.[2g,
The effect of swainsonine on
[2b, 2d, 2f, 4]
oligosaccharide processing was elucidated
as it binds
reversibly to the active site of Golgi α-mannosidase II (GMII) (EC
3.2.1.114)[5] and modulates N-glycoprotein synthesis resulting in
the accumulation of hybrid type of N-glycans.[2e, 6] Clinical trials
1
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Bn
N
significant selectivity observed. Thus, our strategy in the design
OH
OMs
O
O
OH
a
OH
b
OMs
of
a selective GMII inhibitor was based on a previous
RO
RO
RO
RO
c
proposal,[5a, 12c] and consists of two basic points: (i) design of the
core unit of the inhibitor (key interactions with catalytic subsite
which is identical in both dGMII and bLMan); and (ii) design of
O
O
O
O
O
O
O
: R = TBDMS
: R = Tr
: R = TBDMS
: R = Tr
: R = TBDMS
: R = Tr
: R = TBDMS
: R = Tr
8
9
2
3
4
5
6
7
structural linker (specific interactions with holding or anchor
8a, 11a]
subsites of dGMII which are missing in bLMan).[5a,
For
Bn
N
.
H
N
HCl
these purposes, polyhydroxypyrrolidines with N-substitution
were prepared. It is known that certain polyhydroxypyrrolidines
derived from natural 1,4-dideoxy-1,4-imino-D-mannitol (the
structure 24), an azafuranose analogue of mannose, are
inhibitors of both GMII and LMan at the micromolar or millimolar
level with a weak selectivity index to GMII observed for some N-
substituted derivatives [9a, 9b] (for more details about the design of
the structure of the inhibitors see the section Molecular
modeling). Our research group has recently published a series
of articles with mannose derivatives to find a structural feature of
the inhibitor with impact on selectivity.[9p-r] These studies
revealed that a selective GMII inhibitor should have a linker of
the benzyl type and modified 6-OH functional group. Structural
design was further developed and the present paper reports a
study with six novel N-arylalkyl substituted pyrrolidines from
which, to the best of our knowledge, some of them are the first
e
HO
d
HO
HO
OH
HO
OH
10
11
Scheme 1. Synthesis of the pyrrolidines 10 and 11. Reagents and conditions:
a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt,
overnight, 98% for 6, 95% for 7; c) BnNH2, 120 °C, 7-8 h, 87% for 8, 97% for
9; d) 6M HCl/MeOH 1:2 (v/v), rt, overnight, 62% from 8, 68% from 9; e) 1. H2,
10% Pd-C, MeOH, rt, overnight, 2. 10% HCl, 89%.
NaBH4 was performed in EtOH providing corresponding diols
4[20] and 5. Standard mesylation of diols 4 and 5 led smoothly to
dimesylated derivatives 6[20] and 7. The cyclization of the
dimesylate 6 with benzyl amine to access fully protected 1,4-
imino-L-lyxitol 8 was conducted previously in refluxing toluene
within 24 h.[20] Another substrate very similar to 7 was cyclized to
1,4-imino-L-lyxitol in neat benzyl amine under reflux for 18 h.[21]
However, optimization of reaction conditions showed that
optimal reaction time and temperature for the cyclization of
dimesylates 6 and 7 in neat benzyl amine were 7 h at 120 °C. In
addition, the work-up procedure was significantly simplified by
extraction of excess BnNH2 with cold 0.5M citric acid[22] instead
of its tedious removal by evaporation. Acid-sensitive protective
groups were stable under the condition used and the ring
closures were performed on a gram scale (~ 3.5 g, yield 87%).
Simultaneous removal of isopropylidene and trityl/silyl protective
groups from 9 under acidic conditions (6M HCl/MeOH) afforded
N-benzyl pyrrolidine 10 in moderate yield. In addition,
debenzylation of 10 under catalytic hydrogenation conditions
followed by treatment with diluted HCl provided the core
pyrrolidine 11[21] as its hydrochloride (Scheme 1).
GH38 inhibitors with
a
significant selectivity index of
IC50(LManII)/IC50(GMIIb) > 100 (for more details on the modeled
GH38 enzymes, LManII and GMIIb, see the section Enzyme
assays – experimental).
Results and Discussion
In the following subsections, the results from synthesis,
biological assays and molecular modeling are described and
discussed in connection with the selected Golgi and lysosomal
enzymes from the GH family 38 which are abbreviated as
follows: GMII (human Golgi α-mannosidase II),[9a, 9b] dGMII (fruit
fly Golgi α-mannosidase II, the homologue of mammalian Golgi
α-mannosidase II),[5b, 14] GMIIb (fruit fly Golgi α-mannosidase II
with some distinct differences in comparison to other
invertebrates and vertebrates),[15] LMan (human lysosomal α-
mannosidase),[8b, 9a, 9b] dLMan (fruit fly lysosomal α-mannosidase,
CG6206 gene product),[5f] LManII (fruit fly lysosomal α-
mannosidase, one of the acidic class II mannosidases),[15]
JBMan (Jack bean α-mannosidase, the plant homologue of the
acidic class II mannosidases)[16] and bLMan (bovine lysosomal
α-mannosidase).[8a] The fruit fly GMIIb, LManII and JBMan were
used for enzymatic assays in this study and our previous
studies;[9p-r] GMII, dGMII, LMan, dGMII and JBMan in other
previous studies,[5f, 9a-o] while dGMII and bLMan are the enzymes
with available crystal structures.[5a, 5b, 8a, 12c, 17]
R
N
R
N
a
b
7
TrO
HO
HO
OH
O
O
: R = CH
: R = CH
12
13
14
15
16
17
Ph
2CH2
Ph
2CH2
: R = p-methoxybenzyl
: R = pyridin-4-ylmethyl
: R = p-methoxybenzyl
: R = pyridin-4-ylmethyl
Scheme 2. Synthesis of target pyrrolidines 15-17. Reagents and conditions: a)
RNH2, 120 °C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6M HCl/MeOH
1:2 (v/v), rt, overnight, 52% for 15, 54% for 16, 62% for 17.
Following the optimized conditions for the cyclization reaction,
pyrrolidines 12-14 were prepared from dimesylate 7 and the
corresponding benzyl amine. Simultaneous hydrolysis of trityl
and acetonide protecting group furnished target pyrrolidines 15-
17 in moderate yields (Scheme 2).
Another approach based on the removal of N-benzyl group from
8 under standard conditions (H2, Pd/C in methanol) followed by
N-benzylation of the resulting pyrrolidine 18 with p-iodo- or p-
bromobenzyl bromide gave pyrrolidine 19 and 20 in 89% and
74% yield, respectively. Their treatment with 6M HCl in methanol
afforded target pyrrolidines 21 and 22 (Scheme 3).
Synthesis
Computer-assisted design[18] was used to predict
a core
structure and a selectivity structural linker of the dGMII inhibitor
(for more details see also the section Molecular modeling). The
synthesis of target compounds started from 2,3-O-
isopropylidene derivatives 2 and 3 which were prepared from D-
ribose according to the previously published procedure (Scheme
1).[19]
Highly efficient reductive ring opening of the lactols 2 and 3 with
2
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R
N
R
N
H
N
reduced selectivity was observed for N-phenethyl derivative 15.
It seems that a length of hydrocarbon chain between pyrrolidine
unit and phenyl function is a crucial factor for retaining the
selectivity of the GMIIb inhibitors. The similar inhibitory
properties were also observed in our previous study with sulfur-
containing α-D-mannopyranosides having the phenylalkyl
TBDMSO
HO
a
TBDMSO
b
c
8
HO
OH
O
O
O
O
: R = p-iodobenzyl
: R = p-bromobenzyl
: R = p-iodobenzyl
: R = p-bromobenzyl
21
22
19
20
18
functional group.[9p]
[5f]
As published previously
swainsonine 1, the nanomolar
Scheme 3. Synthesis of target pyrrolidines 21 and 22. Reagents and
conditions: a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 °C,
4h, 89% for 19, 74% for 20; c) 6M HCl/MeOH 1:2 (v/v), rt, overnight, 73% for
21, 66% for 22.
inhibitor of both GMII and LMan, gave an extremely poor
selectivity index of 2. A substitution at the position 5 in its
analogue 23 led to an increase in the selectivity index to a value
of 11. However, a swainsonine derivative with the benzyl
functional group itself at the position 5, which could mimic the
inhibitor 10 with the high selectivity index, is yet to be described.
For such a structure another increase of the selectivity index
could be expected to maintain its potency at the nanomolar level.
The core structure of the pyrrolidine inhibitors presented here is
closely related to the structure of natural 1,4-dideoxy-1,4-imino-
D-mannitol 24, namely its N-benzyl derivative 25 (Table 1).
When the potency and selectivity of 24 is compared to the
inhibitor 11, the deletion of −CH(OH)−CH2OH moiety in 24 and
the additional attachment of −CH2OH at the opposite site of the
ring in 11 led to a dramatic decrease in potency towards LManII
(from 13 µM to 7500 µM) and only to a slight decrease in
potency towards GMIIb (from 100 µM to 270 µM) resulting to the
required selectivity index. Similarly, when the potency and
selectivity of the N-benzyl derivatives 25 and the inhibitor 10 are
compared, the benzyl function further improved the potency and
selectivity (in case of 10) and only the selectivity (in case of 25).
These results suggest that a combination of an appropriate
stereo configuration of −CH2OH and benzyl functional groups on
a mannose mimicking core (polyhydroxylated pyrrolidine ring)
may lead to an inhibitor with the required selectivity for the active
site of GMIIb.
Biological assays
A series of seven polyhydroxylated pyrrolidines 10, 11, 15-17,
21 and 22 was evaluated towards the class II α-mannosidases
(GH family 38) GMIIb, LManII and JBMan and the class I α-
mannosidases (GH family 47) AspMan. All N-substituted
pyrrolidines showed inhibitory activity towards the target enzyme,
GMIIb, with IC50 values in the range from 52 µM to 770 µM
(Table 1).
However, unsubstituted pyrrolidine 11 exhibited only moderate
inhibitory activity (IC50 = 270 µM). Among the N-alkylated
pyrrolidines, phenethyl derivative 15 and pyridin-4-ylmethyl
derivative 17 were found to be the least potent inhibitors in
comparison with N-(4-substituted benzyl) analogues 10, 16, 21
and 22. The latter two N-(4-halobenzyl)pyrrolidines 21 and 22
were the most potent GMIIb inhibitors with IC50 values 52 and 55
µM, respectively. These data suggest that N-alkylation plays an
important role in the inhibitory activity of the N-substituted
polyhydroxylated pyrrolidines. For the compounds 10, 21 and 22
with the lowest IC50 values, their inhibition constants were also
determined (Table 1). It was observed that Ki values were
essentially the same as IC50 values, not differing more than by
10%.
A selective GMII inhibitor is required to exhibit none or
significantly reduced inhibitory activity towards LMan. All tested
polyhydroxylated pyrrolidines were found to be weak LManII
inhibitors with IC50 values at the millimolar level (IC50 in range of
1.2 mM to >8mM). N-Substituted derivatives were slightly
weaker inhibitors of LManII in comparison with the parent
pyrrolidine 11. It is noteworthy that N-phenethyl derivative 15
showed approximately 3-fold higher inhibition capacity than 11,
suggesting that two-carbon spacer between pyrrolidine and
benzene ring is a preferred arrangement for LManII inhibition.
Thus, it is not the suitable structural fragment of an effective
GMIIb inhibitor.
To verify the high selectivity index and weak inhibitory properties
of our pyrrolidine series towards LManII, the inhibition assay was
also measured with the α-mannosidase from Canavalia
ensiformis (Jack bean) (JBMan) (EC 3.2.1.24, GH family 38),
widely used as a model for acidic α-mannosides.[2b, 5f, 9e, 9q-r,10, 12a-
b]
This assay provided similar results as compared to LManII
(Table 1). All tested structures did not inhibit JBMan at the 2mM
concentration of the inhibitor except for 24. These results further
support the validity of the selectivity index of the N-benzyl
polyhydroxylated pyrrolidines. Since it is known[2b] that 24 and its
pyrrolidine analogues are also strong inhibitors of class I α-
mannosidases, further verification of the selectivity index of our
compounds (10, 11, 16, 21, 22) towards GMIIb was performed in
an enzymatic assay with Aspergillus saitoi α-1,2-mannosidase
(AspMan) (EC 3.2.1.113, GH family 47). All tested structures,
except for unsubstituted derivative 11 (IC50 = 1 mM), did not
inhibit AspMan at the 1-2 mM concentration of the inhibitor.
These results indicate that the most selective inhibitors (10 and
21) may maintain their specificity towards Golgi α-mannosidases
with no inhibitory effects towards acidic lysosomal and
endoplasmic reticulum α-mannosidases. Since the employed
model GMIIb from fruit fly showed some distinct biochemical
differences in comparison to mannosidase III or mannosidase IIx
from other invertebrates and vertebrates,[15]
Based on the IC50 values for LManII and GMIIb, a selectivity
index was calculated for each derivative as
a ratio of
IC50(LManII)/IC50(GMIIb). The most potent inhibitors of GMIIb 21
and 22 had the highest selectivity index 117 and 136,
respectively. N-Benzyl pyrrolidine 10 exhibited a slightly lower
index of 80. To the best of our knowledge, these structures are
the first known GH38 inhibitors with the significantly high
selectivity index since all known GH38 inhibitors had the
selectivity index <20 (for some example see 23, 25 and 26 in
Table 1) insufficient for clinical treatments. The selectivity index
of 16 was also satisfactory and higher than that of the weakest
GMIIb inhibitors 17 and 11. On the contrary, the significantly
3
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Table 1. Measured IC50 (and Ki) values [M] for class II (GMIIb, LManII and JBMan) and class I (AspMan) α-mannosidases (in M). For comparison representative
inhibitors with a known selectivity index to GMII (as appropriate to either GMII, dGMII or GMIIb) are also included.[6a, 6e, 6r]
Compound
GMIIb
LManII
IC50
JBMan
IC50
AspMan
IC50
Selectivity index
IC50(LManII)/IC50
(GMIIb)
R
IC50 [Ki]
N
(Inhibition in %)[a]
(Inhibition in %)[a]
(Inhibition in %)[b]
HO
HO
OH
11
(2.7 ± 0.75) x 10-4
[(2.2 ± 0.15) x 10-4]
(8.8 ± 0.06) x 10-5
[(7.6 ± 0.13) x 10-5)]
(1.9 ± 0.23) x 10-4
(n.d.)
7.5 x 10-3
7.0 x 10-3
1.2 x 10-3
0.7 x 10-3
(1.0 ± 0.2) x 10-3
28
80
(R = H)
(50)
n.i.
10
n.d.
(1)
(R = Bn)
15
2.7 x 10-3
n.m.
6
(R = EtPh)
16
(1.4 ± 0.17) x 10-4
n.d.
(9)
n.d.
(4)
n.i.
n.m.
n.d.
n.d.
117
136
2
(R= 4-MeO-Bn)
17
(n.d.)
(7.7 ± 0.40) x 10-4
[n.d.]
n.d.
n.d.
(R = 4-Py-Bn)
21
(2)
6.1 x 10-3
(9)
(5.2 ± 0.12) x 10-5
[(5.0 ± 0.12) x 10-5]
(5.5 ± 0.15) x 10-5
[(5.8 ± 0.06) x 10-5]
5.0 x 10-8 [c]
n.d.
n.i.
(R = 4-I-Bn)
22
(19)
(9)
7.5 x 10-3
(16)
n.d.
n.i.
(R = 4-Br-Bn)
(8)
11.0 x 10-8 [c]
2.0 x 10-7 [c]
>10.0 x 10-3 [h]
N
[3 x 10-9][d]
OH
H
HO
OH
1
2.9 x 10-8 [d]
3.1 x 10-7 [d]
2.5 x 10-7 [d]
n.m.
11
O
N
OH
H
HO
OH
23
1.0 x 10-4 [e]
1.3 x 10-5 [e]
n.m.
n.m.
n.m.
n.m.
<0
2
H
OH
OH
N
HO
OH
24
6.9 x 10-4 [e,f]
1.5 x 10-3 [e,f]
OH
OH
N
HO
OH
25
OH
O
2.0 x 10-3 [g]
n.i.g
2.0 x 10-3 [c]
n.i. [b,c]
>2
HO
HO
HO
O2S
26
n.m. - not measured. n.d. - not determined (the inhibition <50% at 8 mM). n.i. - no inhibition or inhibition <10% at 2 mM. [a] inhibition in the presence of 2 mM of
the inhibitor calculated towards control activity without inhibitor. [b] inhibition in the presence of 1 mM of the inhibitor calculated towards control activity without
inhibitor. [c] IC50 values measured for GMIIb, LManII, JBMan and AspMan by Poláková et al.[9q] [d] IC50 values measured for dGMII, dLMan and JBMan by Kuntz
[9a]
et al. [5f] [e] IC50 values measured for GMII and LMan by Winchester et al.
[f] calculated IC50 values by Bobovská et al.[18] based on measured in vitro assays
with human α-mannosidases. [9a] [g] IC50 values measured for GMIIb and LManII by Poláková et al.[9p] [h] Ki value measured for hERManI by Vallée et al..[23]
additional enzymatic assays with Co2+ independent forms of
Golgi mannosidase II, GMII, are required for any future
refinements of inhibitor design.
Compounds 1, 10, 15, 16, 21, 22 were tested in vitro for their
cytotoxic activity on an eight cancer cell lines: A549 (human
lung
leukemia),
adenocarcinoma),
CEM-DNR
CCRF-CEM
(T-lymphoblastic
(T-lymphoblastic
leukemia,
daunorubicin resistant overexpressing the LRP protein), K562
(acute myeloid leukemia), K562-TAX (acute myeloid leukemia,
overexpressing the P-glycoprotein), HCT116 (human
4
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Table 2. Cytotoxic activity of the selected pyrrolidines 10, 15, 16, 21, 22 and swainsonine 1 against eight tumor and two non-malignant cell lines (IC50 in µM).
Comp
A549
CCRF-CEM
CEM-DNR
K562
K562-TAX
HCT116
HCT116p53-/-
U2OS
BJ
MRC-5
10
> 200
101 ± 12
93 ± 11
194 ± 8
107 ± 13
> 200
> 200
> 200
> 200
> 200
15
16
21
22
> 200
> 200
> 200
> 200
95 ± 7
99 ± 11
94 ± 10
87 ± 11
122 ± 22
189 ± 12
192 ± 9
156 ± 10
> 200
117 ± 13
110 ± 11
103 ± 13
125 ± 13
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
105 ± 12
102 ± 11
94 ± 24
200 ± 0.3
169 ± 21
189 ± 18
1
> 200
113 ± 12
132 ± 20
> 200
137 ± 13
> 200
> 200
> 200
> 200
> 200
IC50 is the lowest concentration that kills 50 % of cells. The standard deviation in cytotoxicity assays is typically up to 15 % of the average value. Compounds with
IC50 > 200 μM are considered inactive.
colorectal cancer with wild-type p53), HCT116p53-/- (human
colorectal cancer with deleted p53), U2OS (Human Bone
Osteosarcoma Epithelial Cells) and on the two non-malignant
cell lines BJ (human fibroblast) and MRC-5 (human lung
fibroblasts). Cytotoxic activities are presented in Table 2.
In general, cytotoxic activities were observed in high
concentrations, and none of the tested compounds were
cytotoxic to the non-malignant cell lines BJ and MRC, and
solid tumor cell lines HCT116, HTC116p53-/- and A549. In
opposite, all compounds were cytotoxic to chemosensitive
CCRM-CEM acute lymphoblastic cell line as well as resistant
line CEM-DNR with IC50 in range of 87-132 µM. The
pyrrolidines 10, 16 a 21 showed slightly higher cytotoxicity for
CEM-DNR compared to the non-resistant CCRF-CEM. A
similar trend was also observed for the cell line K562 and its
resistant counterpart K562-TAX. In addition, pyrrolidine 22
and swainsonine 1 did not inhibit non-resistant K562 at all.
The tested N-substituted pyrrolidines showed slightly higher
cytotoxicity as swainsonine 1 (90 – 130 µM versus 110 – 140
µM) indicating that the pyrrolidine structures and the standard
1 may have similar pharmacokinetics properties.
proposed structures (compounds 10, 21 and 22) were further
refined by QSAR models with quantum mechanics interaction
energy descriptors developed in our laboratory.[18] Other
compounds (11 and 15-17) synthesized in this paper were
not proposed by molecular modeling. They were prepared to
compare their potency and selectivity with the proposed
structures 10, 21 and 22 as well as to understand an
interaction pattern for selective binding to GMIIb of the
proposed inhibitors.
To understand the observed selectivity towards GMIIb of the
polyhydroxypyrrolidines we focused on two factors which
would influence differential binding of inhibitors to GMIIb and
LManII (with GMII and LMan as surrogates with known 3D
structures); i) possible different ionization states of amino
group of the inhibitors and active-site amino acids; and ii)
quantum mechanics effects between the inhibitor and active-
site amino acids of the enzyme. For this purposes pKa
calculations in water and in both enzymes, docking, hybrid
QM/MM geometry optimizations and interaction energy FMO-
PIEDA calculations were performed.
pKa calculations. Polyhydroxypyrrolidines contain an amino
functional group incorporated in a ring moiety of the inhibitor.
The pKa values of such amino group may range from 5 to 9
depending on the position of the nitrogen atom in the ring as
well as on other structural factors (number and position of
hydroxyl groups in the ring, the conformation of the ring,
chemical external environment of the amino group, etc.).[24]
pH of GMII in Golgi apparatus is about 6 while pH of LMan is
more acidic (4.5-5.0) (for more details about pH of enzymatic
assays see the section 4.2. Enzyme assays). Thus, inhibitors
with a pKa value of 5-6 units could bind to GMII in neutral
form (as an amine) and to LMan in ionized form (as an
ammonium cation). This could induce different interaction
strength and binding mode as was proposed recently.[13] The
calculated pKa values of the synthesized inhibitors in aqueous
solution and in both enzymes are compiled in the Table 3.
The values in aqueous solution are close to a value of 7
being in the range from 6.3-7.9. This indicates that some
inhibitors may prefer protonated form and some neutral ones
in aqueous solution. For comparison, there are also pKa value
Molecular modeling
The main goals of molecular modeling were to design of a
selective GMII inhibitor, and to explain observed selective
binding to GMII of the synthesized polyhydroxypyrrolidines. In
proposals of new inhibitors computational design was
combined with available experimental data of known GH38
[9p, 9r]
inhibitors. We knew from our previous studies
that
appropriate attachment of the benzyl functional group and
deletion of 6-hydroxy group on the mannose core of the
inhibitor increased selectivity of the inhibitors towards GMIIb.
Because the mannoside inhibitors showed only a weak
inhibition at the millimolar level, a new core structure of the
selective
inhibitor
had
to
be
designed.
The
polyhydroxypyrrolidine ring was selected based on structural
analogy with α-mannosidase inhibitors swainsonine and 1,4-
dideoxy-1,4-imino-D-mannitol.
Positions
and
stereo
configurations of functional groups (hydroxy, hydroxylmethyl
and benzyl) were optimized by molecular docking. The
5
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10.1002/cmdc.201700607
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calculated[13] for swainsonine 1 and pyrrolidine 24, for which
also experimental pKa value are available.[24a] In the case of
dGMII, the inhibitors prefer protonated forms except for 11, 1
and 24. In contrast, for the 'acidic' bLMan, all inhibitors prefer
protonated forms with pKa values being in the range from 5.9
to 8.2. In case of the neutral form of the inhibitors the catalytic
acid Asp341 in dGMII (and Asp319 in bLMan) is in direct
contact with the inhibitor, the neutral (−COOH) form would
also be favoured, while for the protonated form of the
inhibitors, the ionized form (−COO-) of this Asp residue is
preferred. Interestingly, the adjacent Asp340 residue in dGMII
(Ser318 in bLMan) changed its pKa value depending on the
protonation states of Asp341 and the inhibitor. Although these
results do not explain a connection between measured IC50 of
the inhibitors and their protonation states, pKa calculations
clearly indicate that in the active sites, namely in dGMII, there
is a balance between protonation states of the inhibitor and
some active-site ionizable amino acids. This balance may
vary between inhibitors and be dependent on the binding
mode, thereby influencing the potency and observed
selectivity of the pyrrolidine inhibitors synthesized in this
study.
Table 3. Calculated pKa values for ligands and selected Asp residues in
water and in the enzymes.
R
pKa
N
HO
Water
pH=7
dGMII
pH=5.9
bLMan
pH=4.5
HO
OH
Compound
11 (R=H)
ligand ligand Asp341 Asp340 ligand Asp319
7.9
6.3
6.9
7.5
5.9
7.3
6.8
7.3
2.2
2.4
4.4
2.5
5.8
5.9
4.4
5.9
6.9
7.5
7.8
7.7
2.1
2.2
2.0
2.2
10 (R=Bn)
15 (R=EtPh)
16
(R=4-MeO-Bn)
17
6.4
7.5
2.3
5.9
7.5
1.8
(R=4-Py-Bn)
21 (R=4-I-Bn)
22 (R=4-Br-Bn)
1
6.9
7.3
7.4
2.4
2.5
5.6
5.9
5.9
4.3
7.3
7.3
8.2
2.3
2.3
3.0
7.3
7.8 [a]
(7.5)[b]
5.0 [a]
Figure 1. (a) A superposition of the bound swainsonine (PDB ID: 3BLB) [5f,
and computationally docked compound 21 into the active site of dGMII.
17]
Due to a steric effect of hydroxymethyl moiety of 21 its pyrrolidine ring (in
grey) is shift away from the catalytic nucleophile Asp204 compared with
the swainsonine ring (in green) (distances between oxygen of Asp204 and
24
6.6 [a]
(7.4)[b]
4.7 [a]
6.8
5.8
4.4
8.1
1.9
3.9
nitrogen of the inhibitors are in Å). (b) A superposition of the docked
17]
compound 21 in the active site of dGMII (PDB ID: 3BLB)[5f,
(21 with
Holoenzyme
carbons in grey) and bLMan (PDB ID: 1O7D)[8a] (21 with carbons in green)
indicates similar binding mode of both the pyrrolidine core and the benzyl
linker.
[a] calculated pKa value by Sladek et al.[13] [b] experimentally measured
pKa values.[2a, 9b]
Molecular docking. By molecular docking it was found that
polyhydroxypyrrolidines bind to the active site of both dGMII
and bLMan in a similar manner as swainsonine (Figure 1).
However, the pyrrolidine ring of our compounds is shifted
away from Asp204 and the Zn2+ ion as compared to the
indolizidine ring of the swainsonine. In Figure 1a there is in
superposition of the bound swainsonine (from the crystal
structure with ID PDB: 3BLB[5f, 17]) with the computationally
docked compound 21 in the active site of dGMII. Probably
due to a steric effect of hydroxymethyl moiety of 21, its
pyrrolidine ring may not bind so tightly to the bottom of the
active site. For example, the distances between the oxygen of
Asp204 and the ring nitrogen of the inhibitors are 2.75 Å (for
swainsonine) and 3.51 Å (for 21). The different position of the
6
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amino groups of both analyzed inhibitors may induce different
interactions with the active-site residues, namely with Asp204,
Asp341 and Zn2+, and influences binding affinities and pKa
values of the pyrrolidine inhibitors.
A superposition of the docked compound 21 in the active site
of GMII and bLMan clearly indicates the similar binding mode
of both pyrrolidine and benzyl moieties (Figure 1b).
that for swainsonine[13]. Similarly, Edisp interaction of 11 with
Phe206 is stronger than for swainsonine with some −15 kcal
mol−1. The remaining ligands show Edisp interaction with
Phe206 around −6 to −9 kcal mol−1. The largest Edisp
originates in the interaction with Trp95 with some −30 to −40
kcal mol−1.
FMO – PIEDA binding analysis. The molecular system
under study via the FMO method consisted of 24 fragments
including the ligand and the structural water which is always
present in the dGMII cavity[5f] for these ligands (for more
details see the Experimental section - Molecular modeling).
Each fragment corresponded to one of the following amino
acids residues: His90, Asp92−, Trp95, Asp204−, Phe206,
Arg228+, Tyr267, Ser268, Tyr269, Asp270−, Asp340−, Asp341,
Tyr407, Asp409−, Trp415, His470, His471, Asp472−, Tyr727,
Arg876+, Gly877, H2O, Zn2+, and the ligand. Since, as
discussed earlier, the main source of GH38 specificity is
expected to result from the different binding of the substituent
groups attached on the pyrrolidine core. It therefore appears
useful to point out residues of the binding site that interact
with different parts of the pharmacophores. Firstly, the core
structure which is essentially identical to the unsubstituted
ligand 11 interacts predominantly with the charge negative
residues Asp92, Asp204, Asp472, the charge neutral Tyr727
and Phe206 and the charge positive Zn2+ ion. Secondly, more
specifically, the interaction of the side-chain –CH2-OH, which
is present also in 11, is mediated through Asp341, Tyr269
and Asp340. Lastly, the residues in direct interaction with the
benzyl linker are Arg228, Arg876, H2O, Trp415 and Tyr267.
The strongest attractive forces (energies) stem from the
Coulomb electrostatic interactions with charged residues. The
Ees is around −90 to −150 kcal mol−1 between the ligand and
Asp92 and Asp204. Ees is below −150 to −220 kcal mol−1 for
the interaction with Asp472. This observation is in good
qualitative and quantitative agreement with the results for
swainsonine by Sladek et al.[13] It is noteworthy that the
interaction with Asp204 is considerably weakened if
substituents are introduced into the unsubstituted structure 11
(Table S1 of Supplementary data). Ees drops from about −156
kcal mol−1 in 11 to some −90 to −110 kcal mol−1 for the
substituted derivatives. Furthermore, the charge transfer +
polarisation term Ect+mix of all studied ligands is the largest for
11. Both may be easily rationalised if one considers the
difference in the Asp204···NH-ligand separation being ~1.6 Å
for 11 and ~2.5 Å for the remaining ligands. It is due to the
steric hindrance of the bulky substituent groups that prevent
the ligand from approaching Asp204 much closer.
Conclusions
Using available experimental data and computational
modeling the structures of the selective inhibitor of fruit fly
Golgi α-mannosidase IIb with minimal side-effects towards
fruit fly lysosomal α-mannosidase II were designed,
synthesized and tested in enzyme and in vitro cell line assays.
It was found that two functional groups, namely benzyl and
hydroxymethyl functions in appropriate stereo configurations,
at the core ring of the inhibitor can dictate potency and
different binding to the target and side-effect enzymes. It can
be concluded that N-benzylation of the core polyhydroxylated
pyrrolidine unit leads to increased IC50 (and Ki) as well as a
selectivity index towards GMIIb about 3-fold in comparison
with the non-substituted 11. Further increasing of the potency
and the selectivity index can be reached by appropriate
substitution at the position 4 on the benzene ring where 21
and 22 had the significant selectivity index >100. Compared
with known GMII, dGMII and GMIIb inhibitors, as natural
swainsonine (1) or 1,4-dideoxy-1,4-imino-D-mannitol (24), the
structures 10, 21 and 22 are the first known selective GMIIb
inhibitors with minimal side effect towards the lysosomal α-
mannosidase II. Since their potency is several orders of
magnitude lower than that of swainsonine, further structural
refinements will be required to obtain the selective and
sufficiently potent inhibitor. Also, additional enzymatic assays
with human GMII will be necessary to validate the high
selectivity index found for 10, 21 and 22 in assays with GMIIb.
The observed cytotoxic activity of the tested compounds in
72-hour MTS assay is rather low, demonstrating low toxicity
on non-tumor cell lines, on the other side is hard to predict its
real antitumor potential by the simple cytotoxic test. Such
activity should be more likely monitored by synergistic assay
with conventional antitumor drugs as doxorubicin, 5-FU or in
combination with X-ray irradiation and irinotecan as was
published previously for swainsonine and tunicamycin.[25]
Experiments with similar design might be a valuable tool in
the identification of the real therapeutic potential of GMIIb
inhibitors in future.
Based on the results from molecular modeling the potency
and the selectivity of the synthesized pyrrolidines might be
influenced by the binding position of the pyrrolidine ring (the
position of the ring nitrogen to Zn2+ and other active-site
amino acid residues) and its protonation state giving different
interactions with the key active-site residues (Zn2+, Asp341,
Asp204, Asp92 and Asp472 in case of dGMII) as was
demonstrated in our previous work.[13]
The main repulsive contribution is from the charge positive
Arg228 and Arg876 with Ees around 50 and 30 kcal mol−1,
respectively. Zn2+ is a special case - its total interaction with
the ligand is also repulsive. However, it is due to the
exchange energy term Eex rather than Ees. This can also be
interpreted in the same way as for swainsonine,[13] since the
interaction with the diol group on the ligand appears to be
essential for successful binding in the enzyme. Even though
Arg228 and Arg876 are in Coulomb repulsion with the ligand,
the benzyl ring exhibits non-negligible dispersion interactions
with both residues, which we believe to be important in the
stabilization within the enzyme cavity. The Edisp with Tyr269 is
in all cases around −5 kcal mol−1, which is somewhat more
Experimental Section
General chemistry.
7
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10.1002/cmdc.201700607
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FULL PAPER
TLC was performed on aluminium sheets precoated with silica gel 60
F254 (Merck). Flash column chromatography was carried out on silica
gel 60 (0.040-0.060 mm, Merck) with distilled solvents (hexanes, ethyl
acetate, chloroform, methanol). Dichloromethane was dried (CaH2)
and distilled before use. All reactions containing sensitive reagents
Discovery Ltd, UK) is a model of human cancers with p53 mutation
frequently associated with poor prognosis, A549 line is lung
adenocarcinoma. The daunorubicin resistant subline of CCRF-CEM
cells (CEM-DNR bulk) and paclitaxel-resistant subline K562-TAX
were selected in our laboratory by the cultivation of maternal cell lines
in increasing concentrations of daunorubicin or paclitaxel,
respectively. The CEM-DNR bulk cells overexpress MRP-1 and P-
glycoprotein protein, while K562-TAX cells overexpress P-
glycoprotein only. Both proteins belong to the family of ABC
transporters and are involved in the primary and/or acquired multidrug
resistance phenomenon.[27] MRC-5 and BJ cell lines were used as a
non-tumor control and represent human fibroblasts. The cells were
maintained in nunc/corning 80 cm2 plastic tissue culture flasks and
cultured in cell culture medium according to ATCC or Horizon
recommendations (DMEM/RPMI 1640 with 5 g/L glucose, 2 mM
glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal
calf serum, and NaHCO3).
Cytotoxic MTS assay. MTS assay was performed at Institute of
Molecular and Translational Medicine by robotic platform
(HighResBiosolutions). Cell suspensions were prepared and diluted
according to the particular cell type and the expected target cell
density (25000 – 35000 cells/mL based on cell growth characteristics).
Cells were added by automatic pipettor (30 μL) into 384 well microtiter
plates. All tested compounds were dissolved in 100% DMSO and
four-fold dilutions of the intended test concentration were added in
0.15 μL aliquots at time zero to the microtiter plate wells by the
echoacustic non-contact liquid handler Echo550 (Labcyte). The
experiments were performed in technical duplicates and three
biological replicates at least. The cells were incubated with the tested
compounds for 72 h at 37 °C, in a 5% CO2 atmosphere at 100%
humidity. At the end of the incubation period, the cells were assayed
by using the MTS test. Aliquots (5 μL) of the MTS stock solution were
pipetted into each well and incubated for additional 1–4 h. After this
incubation period, the optical density (OD) was measured at 490 nm
with an Envision reader (Perkin Elmer). Tumor cell survival (TCS) was
calculated by using the following equation: TCS = (ODdrug-exposed
well/mean ODcontrol wells) × 100%. The IC50 value, the drug concentration
that is lethal to 50% of the tumor cells, was calculated from the
appropriate dose-response curves in Dotmatics software.[27]
1
were carried out under an argon atmosphere. H NMR and 13C NMR
spectra were recorded at 25 ºC with Varian VNMRS 400 MHz, Bruker
AVANCE III HD 400 and Bruker AVANCE III HDX 600 spectrometers.
Chemical shifts are referenced to either TMS (δ 0.00, CDCl3 for 1H) or
HOD (δ 4.87, CD3OD for 1H), and to internal CDCl3 (δ 77.00) or
CD3OD (δ 49.00) for 13C. Optical rotations were measured on a Jasco
P2000 polarimeter at 20 °C. High resolution mass determination was
performed by ESI-MS on a Thermo Scientific Orbitrap Exactive
instrument operating in positive mode.
The lyxitols 10, 11, 15, 16, 17, 21 and 22 used in biological tests were
lyophilized before the use. p-Nitrophenyl α-D-mannopyranoside (pNP-
Manp) and jack bean α-mannosidase were purchased from Sigma;
swainsonine from Calbiochem; Aspergillus oryzae α1,2-mannosidase
from Prozyme.
Enzyme assays
Assays with Class II α-Mannosidases (GH family 38). The isolation
and purification of recombinant Drosophila melanogaster Golgi
(GMIIb) and lysosomal (LManII) α-mannosidases was carried out as
described by Nemčovičová et al.[15] The α-mannosidase from
Canavalia ensiformis (Jack bean) (JBMan) was purchased from
Sigma. Mannosidase activity of these enzyme preparations were
measured using p-nitrophenyl-α-D-mannopyranoside (pNP-Man;
Sigma; 100 mM stock in dimethylsulphoxide) as a substrate at 2 mM
final concentration in 50 mM acetate buffer at the relevant previously-
defined optimal pH) (Jack bean α-mannosidase, JBMan, at pH 5.0,
GMIIb at pH 6.0, and LManII at pH 5.2 ) and 0.5 μL of the enzyme
(0.05 µg of protein for JBMan), in a total volume of 50 μL for 1-2 h at
37 °C. GMIIb was assayed in the presence of 0.5 mM CoCl2.
The inhibitor was preincubated with the enzyme in the buffer for 5 min
at room temperature and the reaction was started by addition of the
substrate. The reactions were terminated with two volumes (0.1 mL)
of 0.5M sodium carbonate and the production of p-nitrophenol was
measured at 405 nm using a multimode reader Mithras LB943
(Berthold Technologies).
The average or representative result of three independent
experiments made in duplicate is presented. The IC50 value was
determined with 2 mM pNP-Man. The Ki values were determined from
Dixon plots of assays performed with pNP-Man at the indicated
concentration (0.5-4 mM).
Molecular modeling
Docking with Glide. The crystal structures of dGMII (PDB ID:
17]
[8a]
3BLB),[5f,
and bLMan (PDB ID: 1O7D)
were used as 3-D
enzyme models of human GMII and LMan for docking of synthesized
pyrrolidines with the GLIDE program
[28]
of the Schrödinger package.
Protonation states of amino acid residues of enzymes were calculated
for the pH = 6.0 ± 1 (dGMII) and 4.5 ± 1 (bLMan) by the Protein
Preparation Wizard of the Schrödinger package.[29] For docking with
dGMII all molecules of water at the active site of dGMII were deleted
Enzyme assays with Class I α-Mannosidase (GH family 47). As
pNP-Manp is not a substrate for GH family 47 enzymes, a MS-based
assay was developed using a 2-aminopyridine-labelled Man5GlcNAc2
N-glycan (Manα1,2Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,
4GlcNAc-PA; purified by RP-HPLC of the N-glycome of Trichomonas
vaginalis.[26] Approximately 5 pmol of the N-glycan was incubated with
0.3 µL (4 µU) Aspergillus saitoi α1,2-mannosidase in the absence or
presence of 0.25-2.0 mM inhibitor, 50 mM ammonium acetate buffer
(pH 5) in a total of 5 µL; all assays for 1 or 2 hours at 37 °C. The
reactions were heat inactivated for 5 minutes prior to MALDI-TOF MS
(Bruker Autoflex Speed); the peak heights of the m/z 989, 1151 and
1313 [M+H]+ ions (i.e., corresponding to the substrate and products
with one or two α1,2-mannose residues removed) were integrated to
estimate the conversion of substrate.
except one (WAT1820, numbering according to PDB ID: 3BLB)[17]
.
This water has been shown to be conserved in crystal structures of
dGMII either with intact substrates or inhibitors.[5f, 9d, 11a] In all docking
calculations the catalytic acid (Asp341 in dGMII and Asp319 in the
case of (bLMan) was modelled in the protonated form in accordance
with its catalytic role as a general acid. The receptor box for the
docking conformational search was centred at the Zn2+ ion co-factor
at the bottom of the active site with a size of 39×39×39 Å using partial
atomic charges for the receptor from the OPLS2005 force field except
for the Zn2+ and side chains of His90, Asp92, Asp204, Arg228, Tyr269,
Asp341 and His471 (analogous residues were selected for bLMan).
For these structural fragments the charges were calculated at the
In vitro MTS assays
Cell lines. All cells (if not indicated otherwise) were purchased from
the American Tissue Culture Collection (ATCC). The CCRF-CEM line
quantum mechanics level with the DFT (Density Functional Theory)
[30]
method (M06-2X)
using hybrid quantum mechanics/molecular
mechanics (QM/MM) model (M06-2X/LACVP**:OPLS2005) with the
[31]
is derived from
T
lymphoblastic leukemia, evincing high
QSite
program of the Schrödinger package. The grid maps were
chemosensitivity, K562 represent cells from an acute myeloid
leukemia patient sample with bcr-abl translocation, U2OS line is
derived from osteosarcoma, HCT116 is colorectal tumor cell line and
its p53 gene knock-down counterpart (HCT116p53-/-, Horizon
created with no Van der Waals radius and charge scaling for the
atoms of the receptor. Flexible docking in standard (SP) precision was
used. The partial charges of the ligands were calculated at the DFT
[32]
level (M06-2X/LACVP**) using the Jaguar program
of the
8
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10.1002/cmdc.201700607
ChemMedChem
FULL PAPER
Schrödinger package. The potential for nonpolar parts of the ligands
was softened by scaling the Van der Waals radii by a factor of 0.8 for
atoms of the ligands with partial atomic charges less than specified
cut-off of 0.15. The 5000 poses were kept per ligand for the initial
docking stage with scoring window of 100 kcal mol-1 for keeping initial
poses; the best 400 poses were kept per ligand for energy
minimization. The ligand poses with RMS deviations less than 0.5 Å
and maximum atomic displacement less than 1.3 Å were discarded as
duplicates. The post-docking minimization for 10 ligand poses with the
best docking score was performed and optimized structures were
saved for subsequent analyses using the MAESTRO [33] viewer of the
Schrödinger package.
The approach regarding the zinc atom in dGMII was described
earlier.[13]
Acknowledgements
This work was supported by the Slovak Research and
Development Agency (the project APVV-0484-12), Scientific
Grant Agency of the Ministry of Education of Slovak Republic
and Slovak Academy of Sciences (the project VEGA-
2/0064/15), the Slovak State Programme Project No.
2003SP200280203 and the Research
& Development
pKa calculations. The pKa values of ligands in water were calculated
at the quantum mechanics level, including empirical corrections from
the pKa prediction module[34] of the Jaguar program[32] of the
Schrödinger package. pKa values of ligands bound in the active site of
dGMII and bLMan were calculated at the empirical level considering
an enzyme pH optimum of 6.0 (dGMII) and 4.5 (bLMan) using the
PROPKA v.2 program.[35]
Operational Programme funded by the ERDF (Amplification
of the Centre of Excellence on Green Chemistry Methods and
Processes, CEGreenII, Contract No. 26240120025) and the
Austrian Science Fund (P29466). The MTS study was
supported by grants from the Czech Ministry of Education,
Youth and Sports (Grant numbers: LO1304, LM2015063) and
IGA-LF-2017-030 (Palacky University).
A
part of the
Quantum mechanics calculations. The geometries of complexes
calculations were performed in the Computing Centre of the
Slovak Academy of Sciences using the supercomputing
infrastructure acquired in project ITMS 26230120002 and
26210120002 (Slovak infrastructure for high-performance
computing) supported by the Research & Development
Operational Programme funded by the ERDF.
(GMII-inhibitor) were optimized at the hybrid QM/MM level using the
meta hybrid Minnesota functional with doubled amount of nonlocal
[30]
exchange (M06-2X)
with the Los Alamos national laboratory
[36]
effective core potential (LACVP**)
basis set employing the Qsite
[31b]
program
of the Schrödinger package. The QM part contained
ligands, Zn2+ and the following active-site amino acids (side chains of
His90, Asp92, Trp95, Asp204, Phe206, Arg228, Ser268, Tyr267,
Tyr269, Asp270, Asp340, Asp341, Asp409, Trp415, His471, Asp472,
Tyr727, Arg876, backbone of Gly877 and WAT1820). The MM part of
the system (the rest of the enzyme) was treated with the OPLS2005
force fields.[37] The starting geometries were taken from molecular
docking (see the section Docking with Glide).
The virtue of the Fragment Molecular Orbital – Pair Interaction Energy
Decomposition Analysis (FMO-PIEDA) technique is to enable
evaluation and analysis of pair interactions embedded within the
electrostatic potential of the surroundings.[38] This method was
successfully used to analyse the change of the binding motif of dGMII
ligands when they bind in different protonation states.[13] In this study,
we intented to explore its possible use in such way, that individual
energy terms could be used in simple QSAR models. That is, to
correlate any of the energy terms with experimentally assessed ΔG
values. The FMO – PIEDA allows the following energy decomposition
Keywords: pyrrolidines synthesis • swainsonine • Golgi
mannosidase II • cytotoxicity • molecular modeling
References:
[1]
[2]
S. M. Colegate, P. R. Dorling, C. R. Huxtable, Aust. J. Chem. 1979,
32, 2257-2264.
a) P. R. Dorling, C. R. Huxtable, S. M. Colegate, Biochem. J. 1980,
191, 649-651; b) K. W. Moremen, Biochim. Biophys. Acta, Gen.
Subj. 2002, 1573, 225-235; c) D. R. Tulsiani, T. M. Harris, O.
Touster, J. Biol. Chem. 1982, 257, 7936-7939; d) I. Cenci di Dello, P.
Dorling, B. Winchester, Biochem. J. 1983, 215, 693-696; e) D. R.
Tulsiani, O. Touster, Arch. Biochem. Biophys. 1983, 224, 594-600;
f) D. R. P. Tulsiani, H. P. Broquist, O. Touster, Arch. Biochem.
Biophys. 1985, 236, 427-434; g) P. E. Goss, M. A. Baker, J. P.
Carver, J. W. Dennis, Clin. Cancer Res. 1995, 1, 935-944.
a) K. Olden, P. Breton, K. Grzegorzewski, Y. Yasuda, B. L. Gause,
O. A. Oredipe, S. A. Newton, S. L. White, Pharmacol. Ther. 1991,
50, 285-290; b) J. W. Dennis, K. Koch, S. Yousefi, I. Vanderelst,
Cancer Res. 1990, 50, 1867-1872.
E
tot = Eels + Eexch + Ect-mix + Edisp
Here Eint or Etot is the total pair interaction energy, Eels is the
electrostatic energy, Eexch stands for the exchange energy, Ect+mix is
the charge-transfer + mixing energy (which is the not fully recovered
polarization energy) and Edisp is the dispersion energy. Eexch is always
repulsive as it represents the Pauli exchange term. The
electrostatic/Coulomb term can be either positive or negative while
the mixing and dispersion terms are negative, thus attractive and
stabilizing the interactions. In practice Edisp is often used to explain
ligand binding in enzymes.[39] We shall focus on Eint and Edisp. It
should be noted that these calculations at the current state of
implementation are not corrected for basis set superposition effect
(BSSE).[40] Some authors argue this not being a problem at close-to-
equilibrium separations of the interacting moieties.[41] General
remarks on the FMO accuracy may be found elsewhere.[38b, 42]
[3]
[4]
[5]
P. M. Novikoff, O. Touster, A. B. Novikoff, D. P. Tulsiani, J. Cell Biol.
1985, 101, 339-349.
a) D. R. Rose, Curr. Opin. Struct. Biol. 2012, 22, 558-562; b) J. M. H.
van den Elsen, D. A. Kuntz, D. R. Rose, EMBO J. 2001, 20, 3008-
3017; c) D. R. P. Tulsiani, D. J. Opheim, O. Touster, J. Biol. Chem.
1977, 252, 3227-3233; d) B. Dewald, O. Touster, J. Biol. Chem.
1973, 248, 7223-7233; e) K. W. Moremen, O. Touster, P. W.
Robbins, J. Biol. Chem. 1991, 266, 16876-16885; f) D. A. Kuntz, S.
Nakayama, K. Shea, H. Hori, Y. Uto, H. Nagasawa, D. R. Rose,
ChemBioChem 2010, 11, 673-680.
[43]
FMO-PIEDA analysis was carried out in the GAMESS package
[6]
[7]
D. R. P. Tulsiani, O. Touster, J. Biol. Chem. 1983, 258, 7578-7585.
a) P. E. Goss, C. L. Reid, D. Bailey, J. W. Dennis, Clin. Cancer Res.
1997, 3, 1077-1086; b) P. E. Goss, J. Baptiste, B. Fernandes, M.
Baker, J. W. Dennis, Cancer Res. 1994, 54, 1450-1457.
using the Def2-SVP basis sets.[44] For each residue (amino acid), its
own fragment was assigned and all were in the same layer (i.e. same
method applied). The single point calculations were done at the RI-
46]
MP2 level.[45] PIEDA background is documented elsewhere.[38a,
[8]
a) P. Heikinheimo, R. Helland, H. K. S. Leiros, I. Leiros, S. Karlsen,
G. Evjen, R. Ravelli, G. Schoehn, R. Ruigrok, O. K. Tollersrud, S.
McSweeney, E. Hough, J. Mol. Biol. 2003, 327, 631-644; b) Y. F.
The HOP – Hybrid Operator Projection technique was adopter in the
generation of fragments for the covalently bounded amino acids.[46b]
Metal atoms in enzymes represent a challenge for QM description.[47]
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700607
ChemMedChem
FULL PAPER
Liao, A. Lal, K. W. Moremen, J. Biol. Chem. 1996, 271, 28348-
Supercomputer Center, and University of Wisconsin-Madison;
http://www.rcsb.org. Accessed June 04, 2012; PDB ID: 3BLB.
[18] A. Bobovská, I. Tvaroška, J. Kóňa, J. Mol. Graph. Model. 2016, 66,
47-57.
28358.
[9]
a) B. Winchester, S. Aldaher, N. C. Carpenter, I. C. Dibello, S. S.
Choi, A. J. Fairbanks, G. W. J. Fleet, Biochem. J. 1993, 290, 743-
749; b) I. C. Dibello, G. Fleet, S. K. Namgoong, K. Tadano, B.
Winchester, Biochem. J. 1989, 259, 855-861; c) M. Bols, O. Lopez,
F. Ortega-Caballero, in Comprehensive Glycoscience, Vol. 1, 1st ed.
(Ed.: J. P. Kamerling), Elsevier, Oxford, 2007, pp. 815-867; d) D. A.
Kuntz, W. Zhong, J. Guo, D. R. Rose, G. J. Boons, ChemBioChem
2009, 10, 268-277; e) H. Fiaux, D. A. Kuntz, D. Hoffman, R. C.
Janzer, S. Gerber-Lemaire, D. R. Rose, L. Juillerat-Jeanneret,
Bioorg. Med. Chem. 2008, 16, 7337-7346; f) A. Siriwardena, H.
Strachan, S. El-Daher, G. Way, B. Winchester, J. Glushka, K.
Moremen, G. J. Boons, ChemBioChem 2005, 6, 845-848; g) B. Li, S.
P. Kawatkar, S. George, H. Strachan, R. J. Woods, A. Siriwardena,
K. W. Moremen, G. J. Boons, ChemBioChem 2004, 5, 1220-1227;
h) S. George, B. Li, H. Strachan, R. Ayres, R. Collins, A.
Siriwardena, K. W. Moremen, G. J. Boons, Glycobiology 2002, 12,
668-668; i) P. Englebienne, H. Fiaux, D. A. Kuntz, C. R. Corbeil, S.
Gerber-Lemaire, D. R. Rose, N. Moitessier, Proteins 2007, 69, 160-
176; j) H. Fiaux, F. Popowycz, S. Favre, C. Schutz, P. Vogel, S.
Gerber-Lemaire, L. Juillerat-Jeanneret, J. Med. Chem. 2005, 48,
4237-4246; k) L. M. Kavlekar, D. A. Kuntz, X. Wen, B. D. Johnston,
B. Svensson, D. R. Rose, B. M. Pinto, Tetrahedron-Asymmetry
2005, 16, 1035-1046; l) D. A. Kuntz, A. Ghavami, B. D. Johnston, B.
M. Pinto, D. R. Rose, Tetrahedron-Asymmetry 2005, 16, 25-32; m)
X. Wen, Y. Yuan, D. A. Kuntz, D. R. Rose, B. M. Pinto, Biochemistry
2005, 44, 6729-6737; n) W. Chen, D. A. Kuntz, T. Hamlet, L. Sim, D.
R. Rose, B. M. Pinto, Bioorg. Med. Chem. 2006, 14, 8332-8340; o)
S. P. Kawatkar, D. A. Kuntz, R. J. Woods, D. R. Rose, G. J. Boons,
J. Am. Chem. Soc. 2006, 128, 8310-8319; p) M. Poláková, S.
Šesták, E. Lattová, L. Petruš, J. Mucha, I. Tvaroška, J. Kóňa, Eur. J.
Med. Chem. 2011, 46, 944-952; q) M. Poláková, R. Stanton, I. B. H.
Wilson, I. Holková, S. Šesták, E. Machová, Z. Jandová, J. Kóňa,
Carbohydr. Res. 2015, 406, 34-40; r) M. Poláková, R. Horák, S.
Šesták, I. Holková, Carbohydr. Res. 2016, 428, 62-71.
[19] a) K. Vamshikrishna, P. Srihari, Tetrahedron 2012, 68, 1540-1546;
b) J. H. Cho, D. L. Bernard, R. W. Sidwell, E. R. Kern, C. K. Chu, J.
Med. Chem. 2006, 49, 1140-1148; c) R. Furst, C. Lentsch, U.
Rinner, Eur. J. Org. Chem. 2013, 2293-2297.
[20] D. K. Kim, G. H. Kim, Y. W. Kim, J. Chem. Soc., Perkin Trans. 1
1996, 803-808.
[21] T. B. Mercer, S. F. Jenkinson, B. Bartholomew, R. J. Nash, S.
Miyauchi, A. Kato, G. W. J. Fleet, Tetrahedron-Asymmetry 2009, 20,
2368-2373.
[22] M. Corsi, M. Bonanni, G. Catelani, F. D'Andrea, T. Gragnani, R.
Bianchini, Int. J. Org. Chem. 2013, 3, 41-48.
[23] F. Vallee, P. Yip, F. Lipari, B. Sleno, P. Romero, K. Karaveg, K.
Moremen, A. Herscovics, P. L. Howell, Glycobiology 2000, 10,
1141-1141.
[24] a) G. Legler, in Inimosugars as Glycosidase Inhibitors: Nojirimycin
and beyond (Ed.: A. E. Stütz), Wiley-VCH Verlag GmbH, Weinheim,
1999, pp. 31-67; b) O. Lopez, F. L. Qing, C. M. Pedersen, M. Bols,
Bioorg. Med. Chem. 2013, 21, 4755-4761.
[25] J. C. M. De-Freitas-Junior, L. G. Bastos, C. A. Freire-Neto, B. Du
Rocher, E. S. F. W. Abdelhay, J. A. Morgado-Diaz, J. Cell. Biochem.
2012, 113, 2957-296.
[26] K. Paschinger, A. Hykollari, E. Razzazi-Fazeli, P. Greenwell, D.
Leitsch, J. Walochnik, I. B. H. Wilson, Glycobiology 2012, 22, 300-
313.
[27] a) V. Nosková, P. Džubák, G. Kuzmina, A. Ludková, D. Stehlik, R.
Trojanec, A. Janošťáková, G. Kořínková, V. Mihal, M. Hajduch,
Neoplasma 2002, 49, 418-425; b) P. Perlíková, G. Rylová, P. Nauš,
T. Elbert, E. Tloušťová, A. Bourderioux, L. P. Slavětínská, K. Motyka,
D. Doležal, P. Znojek, A. Nová, M. Harvanová, P. Džubák, M. Šiller,
J. Hlaváč, M. Hajdúch, M. Hocek, Mol. Cancer Ther. 2016, 15, 922-
937.
[28] a) R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J.
Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, M. Shelley, J. K.
Perry, D. E. Shaw, P. Francis, P. S. Shenkin, J. Med. Chem. 2004,
47, 1739-1749; b) Glide, version 7.0, Schrödinger, LLC: New York,
NY, 2016.
[10] a) W. H. Pearson, L. Y. Guo, Tetrahedron Lett. 2001, 42, 8267-
8271; b) E. J. Hembre, W. H. Pearson, Tetrahedron 1997, 53,
11021-11032; c) R. Shah, J. Carver, J. Marino-Albernas, I.
Tvaroška, F. D. Tropper, J. Dennis, in United States Patent
Application Publication, US 2003/0236229 A1, GlycoDesign Inc.,
Toronto (CA), 2003; d) T. Fujita, H. Nagasawa, Y. Uto, T.
Hashimoto, Y. Asakawa, H. Hori, Org. Lett. 2004, 6, 827-830.
[11] a) N. Shah, D. A. Kuntzt, D. R. Rose, Proc. Natl. Acad. Sci. U. S. A.
2008, 105, 9570-9575; b) W. Zhong, D. A. Kuntz, B. Ernber, H.
Singh, K. W. Moremen, D. R. Rose, G. J. Boons, J. Am. Chem. Soc.
2008, 130, 8975-8983.
[29] Schrödinger Suite 2016 Protein Preparation Wizard; Epik v. 3.5;
Impact v. 7.0, Schrödinger; Maestro v.10.5, LLC: New York, NY,
2016.
[30] Y. Zhao, D. G. Truhlar, Theor. Chem. Res. 2008, 120, 215-241.
[31] a) R. B. Murphy, D. M. Philipp, R. A. Friesner, J. Comput. Chem.
2000, 21, 1442-1457; b) Qsite; Schrödinger, LLC: New York, NY,
2016.
[12] a) Y. Bleriot, T. Dintinger, A. Genregrandpierre, M. Padrines, C.
Tellier, Bioorg. Med. Chem. Lett. 1995, 5, 2655-2660; b) M. P. Heck,
S. P. Vincent, B. W. Murray, F. Bellamy, C. H. Wong, C. Mioskowski,
J. Am. Chem. Soc. 2004, 126, 1971-1979; c) D. A. Kuntz, C. A.
Tarling, S. G. Withers, D. R. Rose, Biochemistry 2008, 47, 10058-
10068; d) N. S. Kumar, D. A. Kuntz, X. Wen, B. M. Pinto, D. R.
Rose, Proteins 2008, 71, 1484-1496.
[32] Jaguar, version 9.1, Schrödinger, LLC: New York, NY, 2016.
[33] Maestro, version 10.5; Schrödinger, LLC: New York, NY, 2016.
[34] J. J. Klicic, R. A. Friesner, S. Y. Liu, W. C. Guida, J. Phys. Chem. A
2002, 106, 1327-1335.
[35] a) H. Li, A. D. Robertson, J. H. Jensen, Proteins 2005, 61, 704-721;
b) D. C. Bas, D. M. Rogers, J. H. Jensen, Proteins 2008, 73, 765-
783.
[13] V. Sladek, J. Kóňa, H. Tokiwa, Phys. Chem. Chem. Phys. 2017, 19,
12527-12537.
[36] a) W. R. Wadt, P. J. Hay, J. Chem. Phys. 1985, 82, 284-298; b) P. J.
Hay, W. R. Wadt, J. Chem. Phys. 1985, 82, 270-283.
[37] G. A. Kaminski, R. A. Friesner, J. Tirado-Rives, W. L. Jorgensen, J.
Phys. Chem. B 2001, 105, 6474-6487.
[14] C. Rabouille, D. A. Kuntz, A. Lockyer, R. Watson, T. Signorelli, D. R.
Rose, M. van den Heuvel, D. B. Roberts, J. Cell Sci. 1999, 112,
3319-3330.
[38] a) D. G. Fedorov, T. Nagata, K. Kitaura, Phys. Chem. Chem. Phys.
2012, 14, 7562-7577; b) S. Tanaka, Y. Mochizuki, Y. Komeiji, Y.
Okiyama, K. Fukuzawa, Phys. Chem. Chem. Phys. 2014, 16,
10310-10344; c) D. G. Fedorov, K. Kitaura, The Fragnemt
Molecular Orbital Method - Practical Applications to Large Molecular
Systems, CRC Press, Taylor and Francis Group, 2009; d) M. S.
Gordon, D. G. Fedorov, S. R. Pruitt, L. V. Slipchenko, Chem. Rev.
2012, 112, 632-672.
[15] I. Nemčovičová, S. Šesták, D. Rendić, M. Plšková, J. Mucha, I. B. H.
Wilson, Glycoconjugate J. 2013, 30, 899-909.
[16] a) B. S. G. Kumar, G. Pohlentz, M. Schulte, M. Mormann, N. S.
Kumar, Glycobiology 2014, 24, 252-261; b) S. Howard, C. Braun, J.
McCarter, K. W. Moremen, Y. F. Liao, S. G. Withers, Biochem.
Biophys. Res. Commun. 1997, 238, 896-898.
[17] D. A. Kuntz, D. R. Rose, Golgi Mannosidase II in complex with
swainsonine at 1.3 Angstrom. The Research Collaboratory for
Structural Bioinformatics (RCSB): RCSB-Rutgers, RCSB-San Diego
[39] a) S. Arulmozhiraja, N. Matsuo, E. Ishitsubo, S. Okazaki, H.
Shimano, H. Tokiwa, PLoS One 2016, 11; b) N. Sriwilaijaroen, S.
Magesh, A. Imamura, H. Ando, H. Ishida, M. Sakai, E. Ishitsubo, T.
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700607
ChemMedChem
FULL PAPER
Hori, S. Moriya, T. Ishikawa, K. Kuwata, T. Odagiri, M. Tashiro, H.
Hiramatsu, K. Tsukamoto, T. Miyagi, H. Tokiwa, M. Kiso, Y. Suzuki,
J. Med. Chem. 2016, 59, 4563-4577.
[40] S. F. Boys, F. Bernardi, Mol. Phys. 1970, 19, 553-566.
[41] X. W. Sheng, L. Mentel, O. V. Gritsenko, E. J. Baerends, J. Comput.
Chem. 2011, 32, 2896-2901.
[42] K. Fukuzawa, C. Watanabe, I. Kurisaki, N. Taguchi, Y. Mochizuki, T.
Nakano, S. Tanaka, Y. Komeiji, Comput. Theor. Chem. 2014, 1034,
7-16.
[43] a) M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S.
Gordon, J. H. Jensen, S. Koseki, N. Matsunaga, K. A. Nguyen, S. J.
Su, T. L. Windus, M. Dupuis, J. A. Montgomery, J. Comput. Chem.
1993, 14, 1347-1363; b) M. S. Gordon, M. W. Schmidt, Theory and
Applications of Computational Chemistry, Elsevier, 2005.
[44] a) F. Weigend, Phys. Chem. Chem. Phys. 2006, 8, 1057-1065; b) F.
Weigend, R. Ahlrichs, Phys. Chem. Chem. Phys. 2005, 7, 3297-
3305.
[45] X. G. Ren, P. Rinke, V. Blum, J. Wieferink, A. Tkatchenko, A.
Sanfilippo, K. Reuter, M. Scheffler, New J. Phys. 2012, 14, 053020.
[46] a) M. C. Green, D. G. Fedorov, K. Kitaura, J. S. Francisco, L. V.
Slipchenko, J. Chem. Phys. 2013, 138; b) D. G. Fedorov, K. Kitaura,
J. Comput. Chem. 2007, 28, 222-237.
[47] V. Sladek, I. Tvaroška, J. Phys. Chem. B 2017, 121, 6148-6162.
11
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Entry for the Table of Contents
Selective GMIIb inhibitors: The synthesized N-benzyl
substituted polyhydroxypyrrolidines represent the first known
selective inhibitors of Golgi α-mannosidase IIb. The
attachment of two functional groups, namely benzyl and
hydroxymethyl function in appropriate stereo configuration, at
the core ring of the inhibitor can dictate potency and different
binding to the target and side-effect enzymes.
12
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