High consistency of structure-based design and X-ray crystallography: Design, synthesis, kinetic evaluation and crystallographic binding mode determination of biphenyl-n-acyl-β-d-glucopyranosylamines as glycogen phosphorylase inhibitors

Article abstract of DOI:10.3390/molecules24071322

Structure-based design and synthesis of two biphenyl-N-acyl-β-D-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crysta

Full text of DOI:10.3390/molecules24071322

molecules
Article
High Consistency of Structure-Based Design
and X-Ray Crystallography: Design, Synthesis,
Kinetic Evaluation and Crystallographic
Binding Mode Determination of
Biphenyl-N-acyl-β-D-Glucopyranosylamines
as Glycogen Phosphorylase Inhibitors
Thomas Fischer 1,†, Symeon M. Koulas 2,†, Anastasia S. Tsagkarakou 2,† , Efthimios Kyriakis 2,
George A. Stravodimos 2, Vassiliki T. Skamnaki 2, Panagiota G.V. Liggri 2,3
,
Spyros E. Zographos 3, Rainer Riedl 1,
*
and Demetres D. Leonidas 2,
*
1
Institute of Chemistry and Biotechnology, Center of Organic and Medicinal Chemistry, Zurich University of
Applied Sciences, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland; thomas.fischer@zhaw.ch
Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500 Larissa, Greece;
sym.koulas@gmail.com (S.M.K.); anastasiatsagk@hotmail.com (A.S.T.); kyriakis.ef@gmail.com (E.K.);
stravodimos@windowslive.com (G.A.S.); vskamnaki@bio.uth.gr (V.T.S.);
b_liggri@windowslive.com (P.G.V.L.)
2
3
Institute of Biology, Pharmaceutical Chemistry and Biotechnology, National Hellenic Research Foundation,
48 Vassileos Constantinou Avenue, 11635 Athens, Greece; sez@eie.gr
*
Correspondence: rainer.riedl@zhaw.ch (R.R.); ddleonidas@bio.uth.gr (D.D.L.); Tel.: +41-58-934-5618 (R.R.);
+30-2410-565278 (D.D.L.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 March 2019; Accepted: 1 April 2019; Published: 3 April 2019
Abstract: Structure-based design and synthesis of two biphenyl-N-acyl-β-D-glucopyranosylamine
derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa,
a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the
importance of structural water molecules and that the inhibitory efficacy correlates with the degree
of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in
silico-derived models of the binding mode generated during the design process corresponded very
well with the crystallographic data.
Keywords: structure-based design; glycogen phosphorylase inhibitor; glycogen metabolism; type 2
diabetes; X-ray crystallography; N-acyl-β-D-glucopyranosylamine
1. Introduction
Glycogen phosphorylase (GP) is a validated pharmaceutical target for the discovery of new
antidiabetic drugs [
in the liver and muscle to produce glucose-1-phosphate. The human hepatic enzyme (hlGPa) is
responsible to maintain blood glucose homeostasis [ ]. GP senses plasma levels of glucose to ensure
1,2]. GP is an allosteric enzyme that catalyses the first step of glycogen degradation
3
that glycogenolysis is halted when glucose is abundant in plasma. This is achieved through allosteric
binding of glucose to GP and stabilization of a conformation that enables the inactivation of the
enzyme [
levels have to be maintained. Thus, the development of potent GP inhibitors was based on glucose
derivatives by modifying glucose to generate inhibitors with better binding affinity [ ]. Early structural
4]. However, glucose is not a potent inhibitor of GP (Ki = 3.2 mM) [5] since blood glucose
6
Molecules 2019, 24, 1322; doi:10.3390/molecules24071322
www.mdpi.com/journal/molecules

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studies proposed the modification of the
β-1 position and the addition of suitable hydrophobic groups
to exploit interactions with a hydrophobic pocket [
adjacent to the active site formed mostly by hydrophobic residues (termed as the
A –CONH– linker between glucopyranose and the hydrophobic groups has proven to be particularly
efficient since NH forms a hydrogen bond to the side chain of His377 [ ]. One of the first attempts to
designing potent GP inhibitors was to add a methyl or a phenyl group ( and , Figure 1) that led to a
100- and 39.5-fold decrease of the Ki value, respectively compared to that of glucose [7].
7,8] in the direction of the
β-anomeric substituent,
β
-channel) [ ].
9
6
1
2
OH
OH
H
H
O
O
HO
HO
HO
HO
N
N
OH
OH
O
O
1
2
Figure 1. Previously described glucopyranosyl containing GP inhibitors 1 and 2.
Here, we present the structure-based design and organic synthesis of two new GP glucopyranosyl
inhibitors as well as the kinetic and X-ray crystallography assessment of the new compounds.
2. Results
2.1. Molecular Modelling
We designed the two molecules 3 and 4, as depicted in Figure 2, as potential inhibitors of GP.
OH
OH
H
O
HO
HO
N
H
O
HO
HO
N
OH
O
OH
F
O
3
4
Figure 2. Structure-based designed GP inhibitors containing a para fluorinated phenyl group in the
meta position (3), and with a phenyl residue in para position (4).
For the structure-based design of novel GP inhibitors, we examined the co-crystal structure of
2
bound to GP (PDB entry 3G2N) [
network of hydrogen bonds with the amino acid residues His377, Asn484, Glu672 and Gly675 whereas
the phenyl group points into a spacious cavity ( -channel) that provides sufficient room for further
9]. As is visible in Figure 3a, the glucopyranose moiety forms a
β
ligand growing. We hypothesized that an aromatic ring attached in para position of the phenyl group
can interact with His341 by aromatic interactions for inhibitor enhancement. The 4-fluorophenyl unit
in the meta position was intended to populate the β-channel and engage in constructive interactions
with the amino acids Asn282 and His341 in close proximity. The strategic placement of a single fluorine
atom is reported to have also positive effects on the pharmacokinetic profile of drug molecules, and by
the formation of hydrogen bonds it can contribute to elevated inhibitor potency [10–14]. We performed
docking experiments applying pharmacophore queries for the conservation of the hydrogen bonds
formed by the glucopyranose residue and to maintain optimal ligand orientation.

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Figure 3.
scored pose;
and ( ) compound
carbons in green, docked ligands with carbons in yellow.
(
a
−
) Co-crystal structure PDB 3G2N; (
9.1160); ( ) compound docked to PDB 3G2N (second highest scored pose;
docked to PDB 3G2N (highest scored pose; 9.4502), co-crystallized ligand with
b
) compound
3
docked to PDB 3G2N (highest
c
3
−
8.9981);
d
4
−
Both postulated inhibitors could be docked into the cavity with the glucopyranose head group
being anchored to fulfil the pharmacophore query. Compound displayed two almost equally ranked
3
docking poses that differ significantly in orientation by populating two opposite sites within the
cavity. In the highest ranked pose (Figure 3b) the fluorine atom points towards the amino acids
Asp339 and Ala383 and might form a water mediated interaction with one of the solvent molecules
present in close proximity. In the second highest ranked docking pose, represented in Figure 3c,
the fluorine penetrates the cavity towards Tyr280. In this pose, the fluorine containing phenyl ring
overlaps with a water molecule that is present in the crystal structure PDB 3G2N which has to
be replaced by the incoming ligand (solvent was set to inactive in the force field for the docking
experiment). For compound
4 only one highly ranked orientation was obtained in the molecular
modeling (Figure 3d) with the ligand being oriented in a straight line. We decided to synthesize
the designed compounds to examine their inhibitory potential as well as their actual orientation in
biological assays and crystallographic experiments.
2.2. Chemistry
Driven by our encouraging docking results we synthesized the designed molecules
according to the synthetic route summarized in Figure 4. The amino functionalized protected
glucopyranoside derivative could be obtained through hydrogenation of the commercially available
azide . The formation of an amide bond between the amine and either 4’-fluorobiphenyl-3-carboxylic
and . Elimination of the protecting
3 and
4
6
5
6
acid or biphenyl-4-carbonylchloride afforded the intermediates
7
8
acyl groups yielded the designed GP inhibitors 3 and 4.

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O
O
O
O
O
O
a)
O
O
O
O
O
O
O
N3
NH2
O
O
O
O
O
5
6
b) c)
O
OH
O
O
d)
H
R
O
H
R
O
O
HO
HO
N
O
N
O
O
OH
O
O
O
3: R = 3-(4'-fluorophenyl)
4: R = 4-phenyl
7: b) R = 3-(4'-fluorophenyl)
8: c) R = 4-phenyl
Figure 4. Synthesis of the designed compounds
RT, 12 h, 97%; (
) 4’-fluorobiphenyl-3-carboxylic acid, COMU, DIPEA, DMF, 0 ◦C
) biphenyl-4-carbonylchloride, DIPEA, THF, RT, 2 h, 75%; ( ) NaOMe, methanol, RT, 2 h,
73%.
3
and
4
. (
a
) Pd/C 10%, hydrogen, ethyl acetate,
RT, 12 h, 26%;
: 53%, 4:
b
→
(
c
d
3
2.3. Biological Evaluation
The synthesized compounds were examined in in vitro assays to determine their inhibitory
potential against GP. Inhibition constant (Ki) values were measured using hlGPa (the pharmacologically
relevant target) and the rabbit muscle enzymes (rmGPa and rmGPb) for comparison reasons since
previously reported Ki values for other inhibitors were determined for the rabbit muscle enzymes.
Production of rmGPa, rmGPb, and hlGPa, as well as enzyme kinetics experiments were performed
according to previously described protocols [15]. As depicted in Table 1, both compounds 3 and 4 could
be identified as GP inhibitors. They displayed competitive inhibition with respect to the substrate
Glc-1-P (kinetics plots are presented in the Supplementary Materials, Figure S1). Within the series
of tested molecules,
4 proved to be the most potent inhibitor with a Ki of 9.7 µM against rmGPb and
19.4 M against the pharmacologically relevant hlGPa. Compared with the co-crystallized ligand
µ
2
(PDB 3G2N), which was used for the design of the novel compounds, inhibitor
potency by almost one order of magnitude.
4 displayed elevated

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Table 1. Inhibitory data for glycogen phosphorylases.
Ki [µM]
rmGPa
Inhibitor
Structure
OH
hlGPa
rmGPb
H
N
O
HO
HO
1
1
35 ± 1 [16] 32 ± 1 [16]
n.r.
OH
O
OH
H
N
1
1
O
2
81 ± 7 [7]
n.r.
n.r.
HO
HO
OH
O
O6'
C5
OH
C4
C3
C6'
C4'
C6
O5'
O4'
C5' O
H
C14
C9
C ' N
HO
HO
1
C2
C13
C12
3 2
C7
N1
45.6 ± 1.8
19.4 ± 0.2
31.4 ± 2.2
12.8 ± 0.6
56.1 ± 4.4
C2' OH
C3'
C8
O3'
O
O2
O2'
C10
F
F1
C11
C13
C14
C9
C12
C11
O6'
C5
OH
C4
C3
C6'
C4'
O5'
O4'
C6
C7
4 2
C5' O
C10
H
9.7 ± 0.7
C1' N
HO
C2
HO
O3'
N1
C8
C3' C2'OH
O
O2
O2'
1
Not reported; 2 crystallographic numbering.
2.4. X-Ray Crystallography
Motivated by the positive results in the biological assays, we performed structural studies of the
two novel inhibitors and . The studies were performed using rmGPb crystals since they are easier to
grow and the active site is fully conserved in terms of amino acid sequence and structural architecture
compared to hlGPa [17 18]. Previous studies revealed that structural data deriving from rmGPb can be
3
4
,
directly correlated to hlGPa [15,19,20].
Both inhibitors were found bound to the active site with the glucopyranose ring anchoring to the
region known to bind glucose analogues in previous rmGPb-inhibitor complexes [2,21]. However, the
two compounds bind significantly different at the enzyme active site and the biphenyl moieties point
to different directions (Figure 5) as a result of their meta and para connection, respectively. As expected,
the biphenyl fragment ◦itself is not planar, and one benzene ring is twisted in relation to the other and
◦
the dihedral angle is 30 and 42 in
3 and 4, respectively. Upon binding to rmGPb 3 and 4 engage in 16
and 14 hydrogen bond interactions with protein residues in the active site when applying a distance
cut off of 3.3 Å using the program CONTACT as implemented in CCP4 [22] (Table S2), respectively.
The –CONH- linker in the
His377 and to the side chain of Asn284, both of which are not present in the
complex, atom O2 of the linker forms a hydrogen bond to the main chain amide of Leu136 (Figure 5;
Table S2), a hydrogen bond previously observed for other acyl-glucopyranose derivative complexes.
3
complex forms two hydrogen bonds, the N1 to the carbonyl oxygen of
4
complex. Instead, in the
4

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Figure 5. The binding mode of
4 (left) and 3 (right) at the active site of rmGPb. The inhibitor is shown
in thick sticks, hydrogen bonds as dashed lines, and water molecules as black spheres.
The fluorine of
3 forms a halogen [13] bond to the side chain of Asn282 (Figure 5) and this
interaction seems to govern the orientation of the biphenyl moiety within the active site of rmGPb.
Superposition of the two complex structures onto the native unliganded rmGPb structure reveals that
the binding of
(termed 280s loop) [
rmGPb- and rmGPb-
residues with the greatest difference. The new conformation of the 280s loop brings the side chain of
Asn282 to a halogen bonding distance [13] to F1 of (Figure 6). This conformation is further stabilized
3
triggers a significant conformational change of a loop composed by residues 282–289
]. The root mean square distance for all atoms of residues 282–285 between the
complex structures is 0.9 Å, with Asn282, Asp283, and Asn284, being the
4
3
4
3
through the formation of a hydrogen bond between the main chain carbonyl oxygen of Asp283 and
the main chain amide of Phe285.
Figure 6. Stereo diagram of the superposition of the rmGPb-
3
complex (brown) onto the rmGPb-
4
complex (green) showing the different conformations of the 280s loop.
3. Discussion
Structural comparison of the rmGPb-
4
complex with the rmGPb-
2
complex [
9
] (Figure 7) reveals
that although the glucopyranose atoms are at almost◦identical positions at the active site C10 shifts by
~0.4 Å to best orient the biphenyl moiety (there is a 47 inclination between the phenyl rings of 2 and 4)
and hence the hydrogen bond of N1 to the main chain carbonyl oxygen of His377 is lost (distance in the

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rmGPb-4 complex, 4.2 Å). However, this loss is compensated by the formation of a hydrogen bond of
O2 to the main chain amide of Leu136. Therefore, it seems that the 15 additional van der Waals contacts
(applying a distance cut off of 4.0 Å using the program CONTACT as implemented in CCP4 [22]) of
the terminal phenyl group with the protein residues at the active site are the reason for the 8.3 times
lower Ki value of
and the rmGPb-
4
1
with respect to that of
2. Similarly, structural comparison of the rmGPb-4 complex
complex [16] (Figure 7) also reveals a C10 shift by ~0.6 Å with a resulting loss of
the hydrogen bond of N1 to the main chain carbonyl oxygen of His377 which is compensated by the
formation of a hydrogen bond between O2 and Leu136. The van der Waals interactions of the biphenyl
moiety once again seem to be the reason for the 3.3 times difference between the Ki values of 4 and 1.
Figure 7. Superposition of the rmGPb-3 complex (brown) onto the rmGPb-2 (left, green)) and the
rmGPb-1 (right, grey) complexes.
The biphenyl moiety in both inhibitors,
3
and
4, is involved in 23 van der Waals interactions
within a distance cut off of 4.0 Å with protein residues at the
β-pocket, in each protein ligand complex.
Therefore, it seems that despite their different location within the active site, non-polar interactions
support the binding of the biphenyl moiety. Furthermore, despite the fact that the two terminal phenyl
◦
groups of
3
and
4
are inclined by ~15 to each other, both are involved in cation-
π
stacking interactions
to the imidazole ring of His341 (Figure 5). The hydrogen bonds of N1 in the
4 complex to Asn284
and His377 and the halogen bond of F1 to Asn282 are partly counterbalanced in the
hydrogen bond of O2 to Leu136. Thus, the structural basis of the variation in the inhibitory potency
between and seems to lie within the energy cost associated with the significant conformational
3 complex by the
3
4
change of the 280s loop triggered by the binding of 3 but not by the binding of 4.
The observed crystallographic data correspond very well with our design strategy and our
molecular docking experiments. The second highest ranked docking pose of
co-crystallized ligand, pointing its para fluorinated phenyl ring towards the
For compound the congruence is even more striking as the docked pose and the observed orientation
in the co-crystal structure are identical. Even the torsion between the two phenyl rings is modelled
correctly (Figure 8b). The orientation of with the para fluorinated aromatic ring in the -channel
offers a further potential explanation for its diminished inhibitory potency compared to . Compared
with the co-crystal structure PDB 3G2N, an ordered water molecule bound in this area by the formation
of two hydrogen bonds with protein residues is displaced by . As this area of the pocket is rather
3
is oriented like the
β-channel (Figure 8a).
4
3
β
4
3
polar, it is plausible that the displacement of a water molecule by a hydrophobic system declines the
constructive interactions and therefore worsens the inhibitors potency.

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Figure 8.
in yellow, and the crystal structure of
ranked docking pose of in PDB 3G2N with carbon atoms in yellow, and the crystal structure of
GP with carbon atoms in green.
(
a
) Overlay of the second highest ranked docking pose of
3
in PDB 3G2N with carbon atoms
) overlay of the highest
in
3
in GP with carbon atoms in green, (
b
4
4
Given the unexpected significant difference in the potency of the two inhibitors, which can
be explained by the X-ray crystallography results, the present study highlights the importance of
obtaining structural data in every step of the inhibitor optimization process.
4. Materials and Methods
4.1. General
All NMR spectra were recorded on a Bruker (Fällanden, Switzerland) AVANCE III HD 500 One
Bay spectrometer with a magnetic field of 11.75 T. For 1H NMR spectra a frequency of 500 MHz resulted.
Chemical shifts are reported in ppm from tetramethylsilane as internal standard. Data are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintet,
br. = broad, m = multiplet), coupling constants (Hz), integration. For the 13C NMR spectra a frequency
of 125 MHz resulted. Chemical shifts are reported in ppm from tetramethylsilane as internal standard,
whereas fluorine coupling was observed, it was reported with multiplicity (d = doublet), coupling
constants (Hz), and number of carbon atoms. The multiplicities of the signals were determined by
DEPT measurements. Low-resolution mass spectrometry was performed on a MSQ Plus device
(Thermo Scientific, Basel, Switzerland). NMR spectra of all synthesized compounds as well as LC-MS
and HPLC spectra of the test compounds, graphs of the kinetics studies and crystallographic data can
be found in the Supplementary Materials.
4.2. Chemistry
All reagents and solvents were purchased from Sigma Aldrich (Buchs, Switzerland), TCI
(Zwijndrecht, Belgium) or Fluorochem (Hadfield, UK) and used as received. Solvents were stored over
4 Å molecular sieves.
2-(acetoxymethyl)-6-aminotetrahydro-2H-pyran-3,4,5-triyl triacetate (6; ZHAWOC6075): 1-Azido-1-deoxy-
b-D-glucopyranoside tetraacetate ( ) (0.55 g, 1.78 mmol) was dissolved in ethyl acetate (20 mL).
5
Palladium 10% on activated charcoal (0.11 g, 0.1 mmol) was added and a hydrogen atmosphere was
applied at 1 bar. After stirring at ambient temperature for 12 h the mixture was filtered over celite
and concentrated in vacuum. The title compound
6
(0.49 g, 97% yield) was used in further synthesis
= 5.23 (t, J = 9.6 Hz, 1H), 4.82
1
without purification: H-NMR (500 MHz, [D6]DMSO, 25 ◦C, TMS):
δ
(t, J = 10.0 Hz, 1H), 4.62 (dd, J = 9.6 Hz, 9.0 Hz, 1H), 4.29 (d, J = 7.6 Hz, 1H), 4.09 (dd, J = 12.3 Hz, 5.3
Hz, 1H), 3.97 (dd, J = 12.3 Hz, 2.4 Hz, 1H), 3.88 (ddd, J = 10.0 Hz, 5.3 Hz, 2.4 Hz, 1H), 2.54 (br. s, 2H),
2.19 (s, 3H), 1.97 (s, 3H), 1.97 (s, 3H), 1.93 (s, 3H) ppm. 13C-NMR (125 MHz, [D6]DMSO, 25 ◦C, TMS):
δ = 170.54, 170.01, 196.83, 169.79, 84.58, 73.39, 72.69, 71.61, 69.22, 62.82, 21.06, 21.03, 20.87, 20.80 ppm.
MS (m/z): 284 [M + H]+.

Molecules 2019, 24, 1322
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2-(acetoxymethyl)-6-(4’-fluorobiphenyl-3-ylcarboxamido)tetrahydro-2H-pyran-3,4,5-triyl triacetate
(7;
ZHAWOC6074): The amine
6
(100 mg, 0.353 mmol) and 4’-fluorobiphenyl-3-ca◦rboxylic acid (77 mg,
0.353 mmol) were dissolved in dimethylformamide (6 mL) and cooled to 0 C. COMU (151 mg,
0.353 mmol) was added followed by diisopropylethylamine (0.12 mL) and the mixture was stirred
at 0 ◦C. The reaction was stirred overnight and allowed to reach ambient temperature. Ethyl
acetate (25 mL) was added and the organic phase was washed with 1N HCl (2
×
10 mL), NaHCO3
10% (2 × 10 mL) and brine (2
×
10 mL). The organic layer was dried over sodium sulphate and
concentrated in vacuum. Purification by chromatography on silica gel (Gradient 0-100% ethyl acetate
1
in cyclohexane) afforded the title compound
7
in 26% yield: H-NMR (500 MHz, CDCl3, 25 ◦C, TMS):
δ
= 7.99 (t, J = 1.7 Hz, 1H), 7.73–7.70 (m, 1H), 7.69-7.66 (m, 1H), 7.60–7.56 (m, 2H), 7.51 (t, J = 7.7 Hz,
1H), 7.18–7.13 (m, 2H), 5.47 (t, J = 9.2 Hz, 1H), 5.41 (t, J = 9.5 Hz, 1H), 5.13 (dd, J = 10.0 Hz, 9.5 Hz,
1H), 5.08 (t, J = 9.6 Hz, 1H), 4.35 (dd, J = 12.6 Hz, 4.4 Hz, 1H), 4.12 (dd, J = 12.6 Hz, 2.3 Hz, 1H), 3.93
(ddd, J = 10.0 Hz, 4.4 Hz, 2.3 Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.05 (s, 3H), 2.05 (s, 3H) ppm. 13C-NMR
(125 MHz, CDCl3, 25 ◦C, TMS):
δ = 171.61, 170.63, 169.88, 169.61, 167.04, 162.79 (d, J = 247.4 Hz, 1C),
140.89, 136.01 (d, J = 3.3 Hz, 1C), 133.42, 130.84, 129.29, 128.76 (d, J = 8.2, 2C), 126.12, 125.62, 115.88
(d, J = 21.5 Hz, 2C), 78.99, 73.66, 72.58, 70.90, 68.22, 61.64, 20.76, 20.74, 20.62, 20.61 ppm. MS (m/z):
546 [M + H]+.
2-(acetoxymethyl)-6-biphenyl-4-ylcarboxamidotetrahydro-2H-pyran-3,4,5-triyl triacetate (
8; ZHAWOC6076):
Under an argon atmosphere (50 mg, 0.176 mmol) was dissolved in tetrahydrofuran (3 mL).
6
Biphenyl-4-carbonylchloride (38 mg, 0.176 mmol) and diisopropylethylamine (0.05 mL) were added
and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuum
and the crude material was purified by chromatography on silica gel (Gradient: 0–100% methanol in
◦
8
in 75% yield: 1H-NMR (500 MHz, CDCl3, 25 C, TMS):
dichloromethane) to obtain the title compound
= 7.84–7.83 (m, 2H), 7.70–7.66 (m, 2H), 7.64–7.60 (m, 2H), 7.50–7.45 (m,2H), 7.43–7.38 (m, 1H), 7.15
δ
(d, J = 9.1 Hz, 1H), 5.49 (t, J = 9.3 Hz, 1H), 5.49 (t, J = 9.5 Hz, 1H), 5.14 (t, J = 10.0, 1H), 5.10 (t, J = 9.5 Hz,
1H), 4.37 (dd, J = 12.7 Hz, 4.4 Hz, 1H), 4.13 (dd, J = 12.7 Hz, 2.2 Hz, 1H), 3.◦94 (ddd, J = 10.0 Hz, 4.4 Hz,
2.2 Hz, 1H), 2.10 (s, 3H), 2.07 (s, 9H) ppm. 13C-NMR (125 MHz, CDCl3, 25 C, TMS): δ = 171.57, 170.63,
169.88, 169.61, 166.85, 145.19, 139.73, 131.35, 128.96, 128.19, 127.80, 127.38, 127.21, 78.97, 73.63, 72.63,
70.86, 68.25, 61.66, 20.75, 20.74, 20.62, 20.61 ppm. MS (m/z): 528 [M + H]+.
4’-fluoro-N-(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)biphenyl-3-carboxamide
(3;
ZHAWOC6072): The acyl protected compound (50 mg, 0.10 mmol) was dissolved in methanol
7
(15 mL) and sodium methanolate (54 mg, 1.00 mmol in 1 mL methanol) was added and the mixture
kept stirring at ambient temperature for 2h. After neutralization with Amberlyst 15 H+ form and
further stirring for 5 min. the mixture was filtrated and the solvent was removed in vacuum.
Purification by chromatography on reversed phase silica gel (Gradient 0–100% methanol in water)
1
afforded the title compound
(500 MHz, [D6]DMSO, 25 C, TMS):
3
as a white solid with purity > 99.8% (0.02 g, 53% yield): H-NMR
◦
δ = 8.99 (d, J = 9.0 Hz, 1H), 8.19 (t, J = 1.6 Hz, 1H), 7.89 (dt,
J = 7.8 Hz, 1.1 Hz, 1H), 7.85–7.79 (m, 3H), 7.58–7.54 (m, 1H), 7.37–7.30 (m, 2H), 5.05–4.89 (m, 4H),
4.52 (t, J = 5.6Hz, 1H), 3.72–3.65 (m, 1H), 3.48–3.41 (m, 1H), 3.37–3.31 (m, 1H), 3.25 (td, J = 8.8 Hz,
4.2 Hz, 1H), 3.20 (ddd, J =◦9.4 Hz, 5.6 Hz, 2.0 Hz, 1H), 3.11 (td, J = 9.3 Hz, 4.8 Hz, 1H) ppm. 13C-NMR
(125 MHz, [D6]DMSO, 25 C, TMS):
δ = 166.88, 162.53 (d, J = 244.8 Hz, 1C), 139.48, 136.45 (d, J = 3.2 Hz,
1C), 135.27, 130.04, 129.47, 129.39 (d, J = 8.2 Hz, 2C), 127.33, 126.06, 116.24, (d, J = 21.4 Hz, 2C), 80.79,
79.26, 77.99, 72.67, 70.52, 61.47 ppm. MS (m/z): 378 [M + H]+.
N-(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)biphenyl-4-carboxamide (
4; ZHAWOC6077):
The acyl protected compound (70 mg, 0.133 mmol) was dissolved in methanol (15 mL) and sodium
8
methanolate (72 mg, 1.33 mmol in 1 mL methanol) was added and the mixture kept stirring at ambient
temperature for 2 h. After neutralization with Amberlyst 15 H+ form and further stirring for 5 min.
the mixture was filtrated and the solvent was removed in vacuum. Purification by chromatography on
reversed phase silica gel (gradient 0–100% methanol in water) afforded the title compound 4 as a white

Molecules 2019, 24, 1322
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1
◦
solid with purity >99.8% (0.04 g, 73% yield): H-NMR (500 MHz, [D6]DMSO, 25 C, TMS): δ = 8.89 (d,
J = 8.9 Hz, 1H), 8.04–8.00 (m, 2H), 7.81–7.77 (m, 2H), 7.76–7.72 (m, 2H), 7.52–7.47 (m, 2H), 7.44–7.39
(m, 1H), 5.04–4.89 (m, 4H), 4.52 (t, J = 5.7 Hz, 1H), 3.69 (m, 1H), 3.45 (dt, J = 11.5 Hz, 5.6 Hz, 1H), 3.36
(td, J = 9.0 Hz, 4.7 Hz, 1H), 3.25 (td, J = 8.8 Hz, 4.4 Hz, 1H), 3.19 (ddd, J =◦9.7 Hz, 5.6 Hz, 2.1 Hz, 1H),
3.11 (td, J = 9.2 Hz, 5.0 Hz, 1H) ppm. 13C-NMR (125 MHz, [D6]DMSO, 25 C, TMS): δ = 166.74, 143.40,
139.58, 133.41, 129.51, 128.79, 128.57, 127.36, 126.86, 80.81, 79.26, 78.08, 72.56, 70.55, 61.48 ppm. MS
(m/z): 360 [M + H]+.
4.3. In Silico Studies
Molecular modelling experiments were performed using the Molecular Operating Environment
MOE 2015.10 from Chemical Computing Group. Co-crystal structures of GP are available from
the Protein Data Bank. For the actual work pdb entry: 3G2N was selected for the computational
studies. In MOE the pocket was prepared for the dockings via the Protonate 3D method applying
the default values for temperature 300 K, pH 7 and salt 0.1. The ligands to be docked to the protein
were imported from SD files to receive a MOE compatible molecular database. As the SD files did not
contain 3D coordinates, they were generated directly using MOE rebuild3D with an RMSD gradient
of 0.1. For docking experiments the Amber10:EHT force field was used [23,24]. The pharmacophore
placement was applied with a rigid receptor. The docked poses were subsequently analysed with
respect to their scores and interactions with the target enzyme.
4.4. Kinetics
Rabbit muscle GPb (rmGPb) was purified from rabbit skeletal muscles following the protocol
developed by Fischer and Krebs [25] with a slight modification (L-cysteine was replaced with
2-mercaptoethanol). Human liver GPb (hlGPb) was produced as described previously [15]. rmGPa
and hlGPa were prepared by phosphorylation of rmGPb and hlGPb, respectively, performed using a
truncated form of the
γ (catalytic) subunit of rabbit skeletal muscle phosphorylase kinase produced as
described previously [26].
Kinetic studies were performed at 30 ◦C in the direction of glycogen synthesis by measuring the
inorganic phosphate released in the reaction using the method by Saheki et al. [27] 3
µg/mL rmGPb,
rmGPa, or 1 g/mL hlGPa were assayed in a 30 mM imidazole/HCl buffer (pH 6.8) containing
µ
60 mM KCl, 0.6 mM EDTA, and 0.6 mM dithiothreitol using constant concentrations of glycogen (0.2%
w/v), AMP (1 mM; only for the rmGPb experiments), and various concentrations of Glc-1-P (2, 3, 4,
6, and 10 mM for rmGP and 1, 2, 3, 4, and 6 mM for hlGPa) and inhibitors. Briefly, absorption at
850 nm of each sample is transformed to µmoles of phosphates by using a standard curve. Initial
velocities were calculated from the pseudo-first order rate constants (k) using the first-order rate
equation ([A]=[A]0 · ekt) where [A]0 and [A] are the initial and the sample’s concentration of substrate
at various times, and t is the corresponding time (min). The apparent KM values (KM(app.)) are then
calculated by plotting pseudo-first order rate constants (k) vs. [Glc-1-P] using the Michaelis–Menten
equation. The inhibition constant (Ki) values were then calculated from the intercept to horizontal axis
of the plot of KM(app.) vs. [inhibitor] using non-linear regression program GRAFIT [28] and an explicit
value for the standard deviation of each point.
4.5. X-Ray Crystallography
Tetragonal (space group P43212) T state rmGPb crystals were grown by the batch method. Briefly,
an rmGPb (100 mg/mL) solution in a 50 mM
50 mM
β
-glycerol phosphate buffer pH 6.8, supplemented with
◦
β
-mercaptoethanol and 1 mM EDTA was dialyzed overnight at 4 C against a solution
of a 10 mM BES (N,N-bis-(2-hydroxyethyl)-2-aminoethane sulfonic acid/NaOH) buffer pH 6.7,
supplemented with 0.1 mM EDTA, 0.02% (w/v) sodium azide and active charcoal (rmGPb—charcoal
ratio 1:1.2) to remove any nucleotides bound to the enzyme. The enzyme solution was then diluted to
25–30 mg/mL with the dialysis buffer and the addition of spermine and DTT to final concentration

Molecules 2019, 24, 1322
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of 1 and 3 mM, respectively. Microseeds, prepared from previously grown rmGPb crystals, were
also added in the crystallization solution. The final crystallization solution was placed in small
tubes (diameter 3 mm; length 3 cm) and left at 16 ◦C. rmGPb crystals appeared after 3–4 days. X-ray
crystallographic binding studies were performed by diffusion of either
3 or 4 (1 mM; 24 h), solution in
the crystallization media supplemented with 10% (v/v) DMSO in preformed rmGPb crystals at room
temperature prior to data collection. X-ray diffraction data were collected using a Cu X-ray microfocus
source (Oxford Diffraction SuperNova) equipped with a 4-kappa goniometer and the ATLAS CCD
(135 mm) detector at room temperature. Crystal orientation, integration of reflections, inter-frame
scaling, partial reflection summation, and data reduction was performed by the program CrysalisPro
(Agilent Technologies UK Ltd.) [29]. Scaling and merging of intensities were performed by Aimless [30
]
and the optimum resolution was selected based on the CC1/2 criterion [30]. Crystallographic refinement
of the complexes was performed by maximum-likelihood methods using REFMAC [31]. The starting
model employed for the refinement of the complexes was the structure of the native T state rmGPb
complex determined at 1.9 Å resolution (Leonidas et al., unpublished results). Ligand molecule
coordinates and topologies were constructed using AceDRG [32] within Coot [33] and they were
fitted to the electron density maps after adjustment of their torsion angles. A summary of the data
processing and refinement statistics for the inhibitor complex structures is given in Table S1 in the
Supplementary Materials. The validity of the refinement procedure was checked using the PDB_REDO
server [34]. As there were more than five reflections per atom available, both an isotropic and an
anisotropic B-factor model were considered, and the isotropic B-factor model was selected based on
the Hamilton R ratio test. A TLS model for grouped atom movement with one TLS group was used.
The stereochemistry of the protein residues was validated by MolProbity [35]. Figures were prepared
with CCP4 Molecular Graphics [36]. The coordinates of the new structures have been deposited with
the RCSB Protein Data Bank (http://www.rcsb.org/pdb) with codes presented in Table S1.
5. Conclusions
Two novel inhibitors of GP were structure-based designed in silico, synthesized and evaluated
towards their inhibitory properties from a kinetic and crystallographic point of view. Both inhibitors
displayed higher potency towards rmGPb compared to the co-crystallized template ligand that was
employed as the starting point for our targeted design. With a Ki of 9.7
enzyme the most potent inhibitor was almost one order of magnitude more potent than the template
compound. The pathologically relevant human target enzyme hlGPa was inhibited with a Ki of 19.4 M.
µM for the rabbit muscle
µ
Crystallographic studies of the two inhibitors revealed that they bind as expected to the enzyme with
the glucopyranose moiety anchored by the formation of multiple hydrogen bonds. Nevertheless,
deviations were observed for the enzyme structure in the 280 s loop upon binding the two inhibitors.
The resulting differences in constructive interactions to the inhibitor serve as the basis to explain the
difference in potency of the two inhibitors. Comparison of the computationally derived ligand poses
with the effectively determined coordinates obtained by X-ray crystallography demonstrated very
high similarity, confirming the validity of the in silico drug design strategy.
Supplementary Materials: Supplementary materials related to this article, including complete analytical data of
the synthesized compounds, kinetics plots and crystallographic data, are available online.
Author Contributions: Conceptualization: R.R. and D.D.L.; formal analysis: T.F., S.M.K., A.S.T., E.K., G.A.S.,
V.T.S., P.G.V.L., and S.E.Z.; funding acquisition: R.R. and D.D.L.; investigation: T.F., S.M.K., A.S.T., E.K., G.A.S.,
V.T.S., P.G.V.L., and S.E.Z.; project administration: R.R. and D.D.L.; supervision: R.R. and D.D.L.; validation: T.F.,
S.M.K., A.S.T., E.K., G.A.S., V.T.S., P.G.V.L., S.E.Z., R.R., and D.D.L.; visualization, T.F., E.K., V.T.S., P.G.V.L., and
S.E.Z.; writing—original draft: T.F., E.K., V.T.S., R.R., and D.D.L.; writing—review and editing: T.F., E.K., V.T.S.,
R.R., and D.D.L.
Funding: This work was supported in part by the Postgraduate Program “Application of Molecular
Biology-Molecular Genetics-Molecular Markers”, Department of Biochemistry and Biotechnology, University of
Thessaly. E.K. would like to acknowledge support by Greece and the European Union (European Social Fund-
ESF) through the Operational Program «Human Resources Development, Education and Lifelong Learning» in the
context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432),

Molecules 2019, 24, 1322
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implemented by the State Scholarships Foundation (IK
Υ
). T.F. and R.R. are grateful to the Zurich University
of Applied Sciences (ZHAW) for financial support. The APC was funded by the Zurich University of Applied
Sciences (ZHAW) and by discount vouchers for publication in MDPI journals from R.R. and D.D.L.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3 and 4 are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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