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G aphical abs ac
pp xxx–xxxN-(4-Subs i u ed-benzoyl)-N0-(b-
DD
-glucopy anosyl)u eas as inhibi o s o glycogen phospho ylase: Syn hesis
and e alua ion by kine ic, c ys allog aphic, and molecula modelling me hods
Ve onika Nagy, Nó a Fel öldi, Bálin Kónya, Jean-Pie e P aly, Tibo Docsa, Pál Ge gely, E angelia D. Ch ysina
*
,
Cos as Ti aidis, Magda N. Kosmopoulou, Ky a-Melinda Alexacou, Ma ia Kons an akaki, Deme es D. Leonidas,
Spy os E. Zog aphos, Nikos G. Oikonomakos, S anisla Kozmon, Igo T a oška, László Somsák
*
OH
N
OH
HO
HO
HO H
N
O
O
R
R = Me, Ph, Cl, OH, OMe, NO2, NH2, COOH, COOMe
Bes inhibi o : R = Me
K
i = 2.3 μM
BMC 9770 No. o Pages 1, Model 5G
23 Janua y 2012
1
N-(4-Subs i u ed-benzoyl)-N0-(b-D-glucopy anosyl)u eas as inhibi o s
o glycogen phospho ylase: Syn hesis and e alua ion by kine ic,
c ys allog aphic, and molecula modelling me hods
q
Ve onika Nagy
a,b
,Nó a Fel öldi
a
,Bálin Kónya
a
,Jean-Pie e P aly
b
,Tibo Docsa
c
,Pál Ge gely
d
,
E angelia D. Ch ysina
e,
⇑
,Cos as Ti aidis
e
,Magda N. Kosmopoulou
e
,Ky a-Melinda Alexacou
e
,
Ma ia Kons an akaki
e
,Deme es D. Leonidas
e,
,Spy os E. Zog aphos
e
,Nikos G. Oikonomakos
e,z
,
S anisla Kozmon
,Igo T a oška
,László Somsák
a,
⇑
a
Depa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, H-4010 Deb ecen, Hunga y
10
b
Uni e si é Claude-Be na d Lyon 1, ICBMS, UMR UCBL-CNRS-INSA-CPE 5246, CPE-Lyon, 43 Boule a d du 11 No emb e 1918, F-69622 Villeu banne, F ance
c
Cell Biology and Signaling Resea ch G oup o The Hunga ian Academy o Sciences, Uni e si y o Deb ecen, Egye em é 1, H-4032 Deb ecen, Hunga y
d
Depa men o Medical Chemis y, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Egye em é 1, H-4032 Deb ecen, Hunga y
e
Ins i u e o O ganic and Pha maceu ical Chemis y, The Na ional Hellenic Resea ch Founda ion, 48, Vassileos Cons an inou A enue, GR-116 35 A hens, G eece
Ins i u e o Chemis y, Slo ak Academy o Sciences, Dub a ska ces a 9, SK-845 38 B a isla a, Slo akia
a icle in o
A icle his o y:
20 Recei ed 5 Oc obe 2011
Re ised 28 Decembe 2011
Accep ed 29 Decembe 2011
A ailable online xxxx
Keywo ds:
N-Acyl-N
0
-b-
D
-glucopy anosyl u eas
Glycogen phospho ylase
Inhibi o
X- ay c ys allog aphy
Molecula docking
30
abs ac
N-(4-Subs i u ed-benzoyl)-N
0
-b-D-glucopy anosyl u eas (subs i uen s: Me, Ph, Cl, OH, OMe, NO
2
,NH
2
,
COOH, and COOMe) we e syn hesised by ZnCl
2
ca alysed acyla ion o O-pe ace yla ed b-D-glucopy anosyl
u ea as well as in eac ions o O-pe ace yla ed o O-unp o ec ed glucopy anosylamines and acyl-isocya-
na es. O-dep o ec ions we e ca ied ou by base o acid ca alysed anses e ifica ions whe e necessa y.
Kine ic s udies e ealed ha mos o hese compounds we e low mic omola inhibi o s o abbi muscle
glycogen phospho ylase b(RMGPb). The bes inhibi o was he 4-me hylbenzoyl compound (K
i
= 2.3
l
M).
C ys allog aphic analyses o complexes o se e al o he compounds wi h RMGPbshowed ha he ana-
logues exploi ed, oge he wi h wa e molecules, he a ailable space a he b-pocke subsi e and induced
a mo e ex ended shi o he 280s loop compa ed o RMGPbin complex wi h he unsubs i u ed benzoyl
u ea. The esul s sugges he key ole o he wa e molecules in ligand binding and s uc u e-based ligand
design. Molecula docking s udy o selec ed inhibi o s was done o show he abili y o he binding a fini y
p edic ion. The binding a fini y o he highes sco ed docked poses was calcula ed and co ela ed wi h
expe imen ally measu ed K
i
alues. Resul s show ha co ela ion is high wi h he R-squa ed (R
2
) coe fi-
cien o e 0.9.
Ó2012 Else ie L d. All igh s ese ed.
1. In oduc ion
50
Type 2 diabe es melli us is cu en ly es ima ed o a ec mo e
han 5% o he adul popula ion in Wes e n socie ies, and i s inci-
dence is expec ed o inc ease conside ably in he u u e, in pa ic-
ula owing o he d ama ic inc ease in obesi y. The global incidence
o ype 2 diabe es is p ojec ed o a flic mo e han 300 million
people wo ldwide o e he nex 20 yea s, and many o hose a -
ec ed will be young adul s.
1
The disease is cha ac e ised by hype glycaemia associa ed wi h
de ec i e insulin p oduc ion and hepa ic and pe iphe al insulin
esis ance. Due o long e m complica ions, i is a majo cause o
60
blindness and enal disease, and is known o significan ly inc ease
he isk o ca dio ascula diso de s.
2
Cu en p e en i e and he -
apeu ic s a egies do no achie e adequa e con ol o blood glucose
o p e en ch onic mo bidi y, and a e comple ely ine ec i e in
40% o all diagnosed cases.
3
As a consequence o his, he e is a
p essu e on he esea ch communi y (bo h academic and indus-
ial) o de elop no el heal hca e in e en ions o add ess his sub-
s an ial biomedical challenge.
4,5
Hepa ic glucose ou pu is ele a ed in ype 2 diabe ic pa ien s
and cu en e idence indica es ha glycogenolysis ( elease o
70
monome ic glucose om he glycogen polyme s o age o m) is
0968-0896/$ - see on ma e Ó2012 Else ie L d. All igh s ese ed.
doi:10.1016/j.bmc.2011.12.059
q
PDB ID Codes: 2QNB, 2QLM, 2QLN, 2QN3, 2QN7, 2QN8, 2QN9.
⇑
Co esponding au ho s. Tel.: +30 2107273851; ax: +30 2107273831 (E.D.C.);
el.: +36 52512900/22453; ax: +36 52453836 (L.S.).
E-mail add esses: [email p o ec ed],[email p o ec ed] (E.D. Ch ysina), somsak@
ig is.unideb.hu (L. Somsák).

P esen add ess: Depa men o Biochemis y and Bio echnology, Uni e si y o
Thessaly, 26 Plou onos S ., 41221 La issa, G eece.
z
D . Nikos G. Oikonomakos sadly passed away on he 31s o Augus 2008 while
his manusc ip was in p epa a ion. This pape is dedica ed o his memo y.
Q3
Q1
Q2
Bioo ganic & Medicinal Chemis y xxx (2012) xxx–xxx
Con en s lis s a ailable a SciVe se ScienceDi ec
Bioo ganic & Medicinal Chemis y
jou nal homepage: www.else ie .com/loca e/bmc
BMC 9770 No. o Pages 17, Model 5G
25 Janua y 2012
Please ci e his a icle in p ess as: Nagy, V.; e al. Bioo g. Med. Chem. (2012), doi:10.1016/j.bmc.2011.12.059
an impo an con ibu o o he abno mally high p oduc ion o
glucose by he li e .
2,6,7
Glycogen phospho ylase (GP) is he en-
zyme esponsible o glycogen b eakdown o p oduce glucose
and ela ed me aboli es o ene gy supply.
4,8,9
Due o i s key ole
in modula ion o glycogen me abolism, pha macological inhibi ion
o GP has been ega ded as an e ec i e he apeu ic app oach o
ea ing ype 2 diabe es.
10,11
Besides he li e issue GP isoenzymes
a e also loca ed in muscle and b ain issue wi h inc eased o e all
homology and high le els o iden i y a he ca aly ic si e. Hence,
80
se ies o s udies ha e been unde way, employing bo h he li e
and he muscle isoenzyme o he design o new an idiabe ic
agen s. Se e al classes o compounds ha e been epo ed and
ex ensi ely su eyed
3,10,12,13
o he inhibi ion o and in complex
wi h li e and abbi muscle GP (RMGP).
Dis inc binding si es iden ified in GP
14
include he ca aly ic si e,
he pu ine inhibi o y si e (also known as I-si e), he allos e ic
si e, he glycogen s o age si e, a no el allos e ic inhibi o si e and
he newly disco e ed benzimidazole-binding si e. The ques o
compounds wi h highe po ency han glucose o inhibi GP ac i i y
90
led o he design o a ious glucose-based analogues. Fo his pu -
pose he p ope ies o he ca aly ic si e o GP in he Ts a e (inac i e)
con o ma ion we e in es iga ed. Specifically, he c ys al s uc u e
o abbi muscle enzyme e ealed ha he ca aly ic si e is a long
channel secluded om he bulk sol en . The en ance o his chan-
nel is cons ained by a flexible loop (280s) comp ising esidues
282–286. Du ing allos e ic ansi ion he 280s loop changes con o -
ma ion and ac s as a ollga e allowing access o he subs a e o he
co e o he molecule. Dissec ion o he ca aly ic si e in he p esence
o a collec ion o b-subs i uen s o
D
-glucose un eiled wo sub-si es
100
o he ca aly ic channel, p e iously filled wi h wa e molecules in
he na i e enzyme. These si es a e lined by esidues o mixed io-
nic/hyd ophobic cha ac e .
10
Glucose analogues, binding p ima ily
o he ca aly ic si e, ep esen he mos popula ed amily o GP
inhibi o s
3,15
comp ising among o he s a ious de i a i es o N-
acyl-b-
D
-glucopy anosylamines, glucopy anosylidene-spi o-he -
e ocycles, N- and C-b-
D
-glucopy anosyl he e ocycles as well as b-
D
-glucopy anosyl hiosemica bazone de i a i es.
A selec ion o N-acyl-b-
D
-glucopy anosylamine ype com-
pounds collec ed in Cha 1 indica e ha inc easing hyd ophobici y
110
o he acyl moie y makes he inhibi ion s onge (among 1 o 8,4is
he bes inhibi o ) while he
a
-
D
-anome 9is ine ec i e. Phospho -
yla ed b-
D
-glucopy anosylamine 10 has a e y weak e ec . Spi o-
cycliza ion wi h he p ope ‘anome ic’ configu a ion (11 and 12
s 13) makes e y good inhibi o s. A achmen o a u he amide
moie y o he fi s one (14,15) wi h a hyd ophobic pa esul s
in inhibi o 15 whose e ficiency is compa able o ha o he
spi o-hydan oins 11 and 12. C ys allog aphic analyses o com-
plexes o he abo e compounds wi h abbi muscle glycogen phos-
pho ylase b(RMGPb) demons a ed ha in mos cases a H-b idge
120
exis s be ween he b-
D
-glucopy anosylamine NH and main-chain
ca bonyl o His377 nex o he ac i e si e ( o illus a ion and
e e ences see Cha 1). This hyd ogen bond in 1–4,6,7, and 10–
12 makes an impo an con ibu ion o he binding. Consequen ly,
a
-
D
-configu a ed de i a i es 9and 13 a e much weake inhibi o s.
This hyd ogen b idge is absen in he complexes o compounds 5,
8, and 14 which again show poo binding. In he case o benzoyl
u ea 15 his H-b idge has no been obse ed in he c ys al
16
bu
he binding is s onge han in any o he N-acyl-b-
D
-glucopy ano-
sylamine de i a i e. This poin s o he ole o in e ac ions o he
130
inhibi o molecule in he b-channel o he enzyme.
The aim o he p esen s udy is o in es iga e in e ac ions o N-
benzoyl-N
0
-b-
D
-glucopy anosyl u ea de i a i es in he b-channel.
To his end 15 was modified by placing neu al apola and pola ,
as well as acidic and basic subs i uen s in posi ion 4 o he phenyl
ing. Expe imen ally de e mined and compu ed inhibi ion con-
s an s we e compa ed o e alua e he p edic i epowe o molecula
docking me hods applied o he ca aly ic si e o GP, while c ys allo-
g aphic s udies o he enzyme-inhibi o complexes allowed o
assess binding peculia i ies o he molecules.
140
2. Resul s & discussion
2.1. Syn hesis
Al hough he e a e many examples o N-subs i u ed-N
0
-glycosyl
u ea de i a i es in he li e a u e, a he ou se o his wo k we
could find only wo s uc u es o N-acyl-N
0
-glycosyl u eas: pe -
O-ace yla ed N-ace yl- and N-benzoyl-N
0
-(b-
D
-glucopy ano-
syl)u ea
18
(pe -O-ace yl p o ec ed 14 and 15, espec i ely). These
de i a i es we e made by exhaus i e ace yla ion o b-
D
-glucopy -
anosyl u ea by Ac
2
O/ZnCl
2
, and N-benzoyla ion o 2,3,4,6- e a-O-
ace yl-b-
D
-glucopy anosyl u ea by BzCl/Py, espec i ely. Fo he
150
syn hesis o he planned new de i a i es disconnec ions Aand B
(Scheme 1) we e en isaged equi ing in es iga ion o eac ions be-
ween b-
D
-glucopy anosylamines and acyl-isocyana es (A) and
acyla ions o b-
D
-glucopy anosyl u ea (B).
In e media es o he p epa a ion o he a ge compounds
(Scheme 2) we e ob ained om 2,3,4,6- e a-O-ace yl-b-
D
-gluco-
py anosyl azide (17). Pe -O-ace yla ed b-
D
-glucopy anosylamine
18 was p epa ed by Raney-nickel educ ion o 17. The p o ec ed
b-
D
-glucopy anosyl u ea 16 was syn hesized by a sligh modifica-
ion o a published p o ocol. N-Acyla ion o 16 wi h acid chlo ides
160
ca alysed by ZnCl
2
in CHCl
3
ga e compounds 20–24 in accep able
yields in mos cases. Fo de ails o hese ans o ma ions and e e -
encing, please, consul Supplemen a y da a.
The eac ion o glucosylamine 18 wi h in si u p epa ed acyl-iso-
cyana es
19
was p obed nex . In hese eac ions he sugges ed sol-
en CH
3
CN had o be d ied wi h ex eme ca e because e en
aces o mois u e p e en ed he o ma ion o he acyl-isocyana e.
The yields o hese ans o ma ions we e also good excep ha o
23. I can be concluded ha o he p epa a ion o he pe -O-ace -
yla ed a ge compounds 19–24 N-acyla ion o u ea 16 appea s
170
supe io as compa ed o he o he ou e aking in o accoun he
numbe and simplici y o he necessa y manipula ions.
Remo al o he p o ec ing g oups was pe o med by he
Zemplén me hod o ge 15,27, and 28. These eac ions had o be
closely con olled by TLC in o de o a oid loss o he N-acyl g oup
a e longe eac ion imes (c . Scheme 2). This could no be
achie ed du ing Zemplén dep o ec ion o 20,21, and 24, he e o e,
he espec i e 25,26, and 29 we e ob ained by mild acid ca alyzed
anses e ifica ion. These p oblems associa ed wi h he dep o ec-
ion we e ci cum en ed by elabo a ing a p ocedu e o con e
180
unp o ec ed glucopy anosylammonium ca bama e
20
(35) and
acyl-isocyana es o N-acyl-N
0
-b-
D
-glucopy anosyl u eas, and o
he p epa a ion o 31–33 hese condi ions we e used. Ca aly ic
educ ion o 29 u nished amino de i a i e 30, while ha o 33
ga e ca boxylic acid 34.
S uc u e elucida ion o he new compounds was s aigh o -
wa d by NMR me hods and needs no de ailed commen s (see Sup-
plemen a y da a o da a). Ca bonyl esonances o he acyl u ea
moie ies appea ed be ween 149–157 ppm (–NHCONH–) and
162–171 ppm (–NHCOA ).
190
2.2. Enzyme kine ics and X- ay c ys allog aphic esul s
The inhibi o y e ec o en new N-b-
D
-glucopy anosyl u ea ana-
logues 25–34 (Table 1) on he enzyme ac i i y was e alua ed by ki-
ne ic assays pe o med in he di ec ion o glycogen syn hesis a pH
6.8 and 30 °C. The esul s showed ha all compounds a e compe -
i i e inhibi o s o he enzyme ac i i y wi h K
i
alues in he low
l
M
ange (Table 1). Among he subs i uen s in oduced in he pa a-
posi ion o he phenyl ing only he –CH
3
(compound 25) appea ed
2V. Nagy e al. / Bioo g. Med. Chem. xxx (2012) xxx–xxx
BMC 9770 No. o Pages 17, Model 5G
25 Janua y 2012
Please ci e his a icle in p ess as: Nagy, V.; e al. Bioo g. Med. Chem. (2012), doi:10.1016/j.bmc.2011.12.059
o b ing abou sligh ly imp o ed a fini y (K
i
alue o 2.3
l
M) com-
pa ed o he lead compound 15.
200
Wi h he aim o in e p e he esul s ob ained om he kine ic
expe imen s and p o ide a ionaliza ions o he binding a fini ies,
s uc u al s udies o RMGPbin complex wi h six new compounds
25–30 we e pe o med a high esolu ion (Table 1). All de i a i es
o 15 bound a he ca aly ic si e, as i was clea ly indica ed by he
2F
o
F
c
and F
o
F
c
elec on densi y maps (Fig. 1a– ). Addi ional
elec on densi y was obse ed a he new allos e ic si e o com-
pound 29 (Fig. 1g), sugges ing weak binding. All six inhibi o s in-
duced ex ended con o ma ional changes mainly in he 280s loop
upon binding, simila , no iden ical hough, o hose obse ed in
210
he RMGPb:15 complex.
16
Mo e de ailed analysis o he complex
s uc u es was pe o med by mapping he key hyd ogen bond
and an de Waals in e ac ions o med wi h esidues lining he
ca aly ic si e (Tables S2b and S2c). The in e ac ions o med by
he pe iphe al hyd oxyl g oups o he glucopy anose moie y a
he ca aly ic si e a e main ained in almos all complexes. The e-
o e, he analyses ocused on he al e a ions in he ne wo k o con-
ac s ha occu when a new neu al, apola and pola o sligh ly
acidic and basic unc ional g oup was in oduced o he pa en
molecule (a posi ion 4 o he phenyl ing).
220
2.2.1. Compound 15
The c ys al s uc u e o RMGPb–15 complex (Bzu ea) was p e i-
ously de e mined o 1.8 Å esolu ion
16
; he s uc u al esul s
showed ha upon binding o 15 a he ca aly ic si e, he e is a sig-
nifican ea angemen o he 280s loop. In specific, Asn284 is
sandwiched be ween he side chains o esidues Phe285 and
Ty 613, esul ing in he dis up ion o he inhibi o si e loca ed a
he en ance o he ca aly ic channel. The d ama ic shi obse ed
in he 280s loop (shi s 1.3 o 3.7 Å o C
a
a oms compa ed o
N–H···O=C
H-b idge
Ki [μM]
+ 1 R = CH3 32
+ 2 R = CF3 75
+ 3 R = C6H5 81
+
4 R = C10H7
(2-naph hyl) 10
– 5 R = NH2 140
+ 6 R = CH2N3 49
+
7 R = CH2CH2CO2H
20
OH
N
OH
HO
HO
HO
R
O
– 8 R = COOCH3 210
ON
OH
HO
HO
O
R
O
N
H
N
HN
O
H
H
His-377
H-b idges be ween His-377 and
N-acyl-β-D-glucopy anosylamine ype inhibi o s
in he ca aly ic si e o GP
O
HO
HO
HO
HO
HN
O
CF3
– 9 non inhibi o y
OH
N
OH
HO
HO
HO
P
OCH3
O
OCH3
+ 10 5900
N–H···O=C
H-b idge
Ki [μM]
+ 11 X = O 3.1
O
HO
HO
HO
HO
N
H
H
N
X
O+ 12 X = S 5.1
OH
N
OH
HO
HO
HO
NH
O
CH3
O
– 14 370
O
HO
HO
HO
HO
HN NH
O
O
–
29
13 320
105
OH
N
OH
HO
HO
HO H
N
O O
– 15 4.6
Cha 1. Selec ed N-acyl-
D
-glucopy anosylamine ype inhibi o s o GP.
15,17
OH
N
OR
RO
RO
RO H
N
O
A
O
ONH2
OR
CN
O
A
O
OH
N
OR
NH2
O
A
O
X
++
AB
AB
Scheme 1. Re osyn he ic disconnec ions o N-acyl-N
0
-(b-
D
-glucopy anosyl) u ea.
V. Nagy e al. /Bioo g. Med. Chem. xxx (2012) xxx–xxx 3
BMC 9770 No. o Pages 17, Model 5G
25 Janua y 2012
Please ci e his a icle in p ess as: Nagy, V.; e al. Bioo g. Med. Chem. (2012), doi:10.1016/j.bmc.2011.12.059

he na i e s uc u e) was a ibu ed o he bulky benzoyl moie y;
230
howe e , hese shi s we e no in he di ec ion o T o Rallos e ic
con e sion. The con o ma ional changes esul ed in inc eased
con ac s be ween he inhibi o and he p o ein, p o iding a a io-
naliza ion o he K
i
wi h a alue o 4.6
l
M. Howe e , he ‘cha ac-
e is ic’ hyd ogen bond be ween amide ni ogen N1 and he
backbone O o His377 was dis up ed in RMGPb–15 complex.
16
This
hyd ogen bond was p esen in mos RMGPbs uc u es de e mined
in complex wi h b-
D
-glucopy anosylamine analogues illus a ed in
Cha 1.
2.2.2. Compound 25
240
In oduc ion o a me hyl (–CH
3
) g oup in he pa a-posi ion o he
phenyl ing esul ed in an imp o ed inhibi o wi h a K
i
alue o
2.3
l
M compa ed o he lead compound 15. S uc u al s udies o
RMGPb–25 complex showed ha 25 bound a he ca aly ic si e
and o med a o al o 18 hyd ogen bonds and 94 an de Waals
in e ac ions (sligh ly educed compa ed o 15, 20 and 103, espec-
i ely) (Tables S2b and S2c and Fig. 2a and b). The a o emen ioned
‘cha ac e is ic’ hyd ogen bond be ween N1 and he main chain O
o His377 was dis up ed in acco dance wi h he esul s obse ed
o Bzu ea. Thesubs i u ed phenyl ing poin eda asimila di ec ion
250
o ha o 15 wi h anegligible incline(a omsshi ed by0.4 o0.7 Å)
owa ds Ala383 (Fig. 3a). The sol en s uc u e was compa able in
bo h complex s uc u es excep o wo wa e molecules, Wa 161
O and Wa 235 O (numbe ing om RMGPb:15 complex s uc u e)
ha we e displaced o a oid clashes wi h C15 o he me hyl g oup.
A ew mino shi s we e also eco ded in he sol en lying in he
icini y o he hyd ophobic –CH
3
g oup, ha is Wa 84 O shi s by
0.7 Å, Wa 215 O by 0.6 Å and Wa 209 O by 0.5 Å (Wa 93 O,
Wa 251 O, Wa 183 O numbe ing om RMGPb–15 complex, espec-
i ely). The ea angemen o he sol en s uc u e induces mo e
260
p o ound changes in he 280s loop compa ed o hose obse ed in
he Bzu ea complex. In pa icula , he side chain o Asn282 became
less s able mainly due o he displacemen o Wa 235 O in he p es-
ence o he –CH
3
g oup and changed con o ma ion by o a ion o i s
dihed als (
1
,
2
)by(120°,33°), espec i ely. As a esul he side
chain o Asn284 is subjec ed o a mino shi in all a oms by 0.5 Å.
Simila mo e (by 0.5 Å in all a oms) was eco ded o Phe285 since
he hyd ogen bond o med be ween he backbone oxygen and
Asn282 ND2 was dis up ed. Analogous changes we e induced o
he side-chain a oms o Phe286 (a oms shi ed by 0.5 o 0.7 Å and
0w
o a ed by 10°). Howe e , he mos p o ound modifica ions,
possibly in e ela ed o he new posi ion o Asn282, we e obse ed
in Glu287 ha adop ed a di e en con o ma ion ( o a ion o dihe-
d al angles (
1
,
2
,
3
)by(55°,18°,170°), espec i ely) com-
pa ed o RMGPb:15 complex s uc u e. The pep ide bond be ween
Glu287 and Gly288 also flipped o es o e s e eochemis y.
ON
3
OAc
AcO
AcO
Ac
O
OH
N
OAc
AcO
A
cO
AcO OH
N
OH
HO
HO
HO H
N
O O
16
19-24 15, 25-34
b
d
H
N
O
ONH
2
OAc
AcO
AcO
Ac
O
a
17
c
R
OH
N
OAc
AcO
AcO
Ac
O
18
NH
2
O
O
R
ONH
2
.HO
2
CNH
2
OH
HO
HO
HO
e
o
o
g
i
35
Condi ions Yield (%) R R Reac ion ime Yield (%)
c
50 H 19
e
15 H 2 h 90
c
34 CH320
25 CH3 12 d 54
c
43 C6H521
26 C
6H5 6 d 86
d
67 Cl 22
e
27 Cl 1 h 86
c
d
72
20 OAc 23
e
28 OH 0.5 h 87
c
d
60
65 NO224
g
29 NO2 5 d 99
29
h
30 NH2 1 h 99
35
i
31 OCH3 2 d 50
35
i
32 COOMe 5 d 39
35
i
33 COOBn 4 d 43
33
j
34 COOH 3 h 73
Scheme 2. Reagen s and condi ions: (a) PPh
3
, E OAc, NH
3
,CO
2
, , 88%; (b) H
2
, Raney-Ni, E OAc, , 71%; (c) 4-R-C
6
H
4
–COCl, ZnCl
2
, CHCl
3
, eflux; (d) 4-R-C
6
H
4
–CONCO, CH
3
CN,
A , ; (e) NaOMe, MeOH, ; ( ) AcCl, MeOH, ; (g) KHSO
4
, MeOH, ; (h) H
2
, Raney-Ni, MeOH, ; (i) 4-R-C
6
H
4
–CONH
2
con e ed o 4-R-C
6
H
4
–CONCO (by (COCl)
2
in
ClCH
2
CH
2
Cl a eflux emp., hen eac ion wi h 35 in Py, ; (j) H
2
, Pd/C, MeOH, eflux.
4V. Nagy e al. / Bioo g. Med. Chem. xxx (2012) xxx–xxx
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Al hough binding o 25 seemed o p o oke mo e p o ound changes
in he 280s loop han hose o 15, and some pe u ba ion in he
sol en s uc u e, in oduc ion o –CH
3
appea ed o be o e all ene -
ge ically a ou able since i ou weighed hose changes and esul ed
280
in a be e inhibi o han 15.
2.2.3. Compound 26
Replacemen o he me hyl g oup by ano he hyd ophobic bu
much bulkie subs i uen (–C
6
H
5
) in he pa a-posi ion o he
phenyl ing was also in es iga ed. The new compound exhibi ed
simila a fini y o 25 (K
i
alue o 3.4
l
M) and bound a he ca aly ic
si e wi h he phenyl ing been subjec ed o mino incline (a oms
shi ed by 0.3 o 0.5 Å) compa ed o he co esponding one in
15 complex (Fig. 3b) owa ds Asn133 and Glu88 (unlike RMGPb:25
ha mo es sligh ly owa ds Ala383). The new a oma ic ing in o-
290
duced was o a ed by 38° o op imize i s in e ac ions wi h esi-
dues in he icini y Asn282, Phe286, A g292, and His341. The
(–C
6
H
5
) g oup occupied he subsi e o he b-pocke a he ca aly ic
channel
21
displacing h ee wa e molecules Wa 84 O, Wa 161 O
and Wa 215 O upon binding o a oid clashes wi h C17 a om o
he second phenyl ing. These wo wa e s we e hyd ogen bonded
o Wa 235 O, also absen in he new complex s uc u e ha
was in u n in e ac ing wi h Asn282 ND2. Dis up ion o his
wa e -media ed ne wo k des abilized he side chain o Asn282
ha adop ed a di e en con o ma ion by o a ion o i s dihed als
300
(
1
,
2
)by(130°,35°) simila o ha in RMGPb:25. The
al e a ion o Asn282 side chain esul ed in shi s in he ange o
0.3 o 0.7 Å in bo h Asn284 and Phe285 side chain a oms and
mo e p onounced modifica ions in Glu287. The pep ide bond
o med be ween Glu287 and Gly288 flipped as in RMGPb:26 com-
plex and he side chain o he o me also changed ( o a ion o
dihed al angle
3
by 61°). In oduc ion o such a bulky subs i u-
en a ec s also he side chain o A g292 he a oms o which a e
sligh ly shi ed away om he ligand (by 0.5 Å). O e all, 26 upon
binding main ains he numbe o hyd ogen bond in e ac ions
310
o med compa ed wi h he me hyl compound 25; howe e , he
inc eased numbe o an de Waals in e ac ions (113) did no seem
o p omo e ligand binding (Fig. 2c, Tables S2b and S2c). The p e e -
ence hough o he
b-pocke subsi e o mo e hyd ophobic and he space a ailabili y
o e en bulkie subs i uen s became e iden .
2.2.4. Compound 27
To u he in es iga e he ype o in e ac ions o med a he
ca aly ic si e a chlo ine a om (–Cl) was placed in posi ion 4 o
he phenyl ing. Kine ic esul s indica ed ha a fini y o he new
320
analogue was equi alen o ha o he pa en molecule (K
i
alue
o 4.4
l
M). Analysis o he RMGPb:27 c ys al s uc u e showed ha
i bound a he ca aly ic si e in a simila ashion o he p e ious
analogues (Fig. 3c) making a o al o 19 hyd ogen bond and 91
Van de Waals in e ac ions (Fig. 2d, Tables S2b and S2c). The phe-
nyl ing was
Table 1
Kine ic da a ob ained wi h abbi muscle GPband c ys allog aphic numbe ing o he compounds
O
HO
HO
OH
OH
O5
O2
O6
O3
O4 C5
C3
C2
C6
C4
H
N
O
H
N
O
R
C8
O8O7
N2
C7
N1
Compound R K
i
(
l
M) Compound R K
i
(
l
M)
15
C10
C11
C12
C13
C14
C9
4.6 ± 0.90
16
30
NH
2
C13
C12
C11
C10
C14
C9
N3
6.0 ± 0.60
25
CH3
C11
C12
C13
C14
C10
C9
C15
2.3 ± 0.03
a
31
OCH3
3.2 ± 0.16
26
C17
C18
C19
C20
C16
C15
C11
C10
C13
C14
C9
C12
3.7 ± 0.06 32
COOCH
3
4.0 ± 0.16
27
Cl
C11
C12
C13
C14
C9
C10
4.4 ± 0.09
a
33
COOCH
2
Ph
150 ± 12
a
28
OH
C13
C12
C11
C10
C9
C14
O12
6.3 ± 0.30 34
COOH
85 ± 6.5
a
29
N
C11
C10
C13
C14
C9
O
O
O9
O10
C12 N3
3.3 ± 0.30
a
K
i
alues o hese compounds we e calcula ed o compa ison pu poses by he Cheng–P uso equa ion: K
i
=IC
50
/(1 + [S]/K
m
).
V. Nagy e al. /Bioo g. Med. Chem. xxx (2012) xxx–xxx 5
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almos coplana wi h he one in 15, and only sligh shi (by 0.3 o
0.5 Å) o he ca bon a oms owa ds Ala383 was obse ed compa ed
o 15 when bound o RMGPb(Fig. 3c). Two wa e molecules a e
displaced upon binding o 27 a he ca aly ic si e; Wa 161 O o
Figu e 1. Schema ic ep esen a ion o he 2F
o
F
c
elec on densi y maps con ou ed a 1.0
le el o he efined Bzu ea analogues bound a he ca aly ic si e o RMGPb(a, b, c,
d, e, o compounds 25,26,27,28,29,30) and he new allos e ic si e (g, compound 29).
6V. Nagy e al. / Bioo g. Med. Chem. xxx (2012) xxx–xxx
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330
a oid clashes wi h he chlo ine a om and i s neighbo ing Wa 235
O ha was no igh ly bound a he ca aly ic si e any mo e also
due o he con o ma ional change o Asn282 side chain (dihed al
angles
1
,
2
o a e by 120°,40°, espec i ely) ha esul ed in
he dis up ion o he second hyd ogen bond in e ac ion i o med
wi h ND2 a om o he same esidue. Addi ional al e a ions ob-
se ed in he sol en s uc u e in ol ed shi s o Wa 215 O
(Wa 257 O numbe ing om GPb:25 complex), Wa 84 O (Wa 87
O), Wa 236 O (Wa 186 O), Wa 148 O (Wa 270 O) by 0.8, 0.7,
0.3, 0.9 Å, espec i ely. O e all, esidues o he 280s loop ollow
340
he same ea angemen obse ed o in he RMGPb:15 c ys al
s uc u e excep o Asn282. Upon ligand binding, esidues
Asn284, Phe285, Phe286 a e subjec ed o mino dis u bance (in
he ange o 0.3 o 0.7 Å). Mo e ex ensi e changes a e eco ded
o Glu287 he pep ide bond o which wi h Gly288 flipped in his
complex s uc u e as well. Despi e he inc eased pe u ba ion o
Figu e 2. S e eo ep esen a ion o he molecula in e ac ions o compounds 15 (a), 25 (b) 26 (c), 27 (d), 28 (e), 29 ( ), 30 (g) when bound a he ca aly ic si e o RMGPband
p o ein a oms in he icini y. Wa e molecules a e labeled as w. Emphasis is gi en in he new subs i uen in oduced in he pa a-posi ion o he phenyl ing o 15.
V. Nagy e al. /Bioo g. Med. Chem. xxx (2012) xxx–xxx 7
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we ha e no a ional o such a la ge e o . Howe e , we canno ex-
590
clude a possibili y o a di e en binding mode o hese inhibi o s,
caused by he so called 280s loop (280–289 amino acid esidues)
flexibili y. Also i is known ha lipophilic and s acking in e ac ions
o he me hylphenyl g oup a e usually poo ly desc ibed by sco ing
unc ions. We also no e ha ou case is no unique, e. g. swainso-
nine ha is mos ac i e inhibi o o mannosidase II was p edic ed
o be one o he weakes inhibi o s wi h se e al sco ing unc ions,
hough i s binding mode was p edic ed co ec ly. This shows ha
he e is a oom o imp o emen o sco ing unc ions.
3. Conclusions
600
N-(4-Subs i u ed-benzoyl)-N
0
-b-
D
-glucopy anosyl u ea de i a-
i es we e p epa ed by ZnCl
2
ca alysed acyla ion o O-pe ace yla ed
b-
D
-glucopy anosyl u ea o eac ing O-pe ace yla ed b-
D
-glucopy -
anosylamine wi h acyl-isocyana es and subsequen dep o ec ion.
Some compounds we e p epa ed, o a oid p o ec ing g oup manip-
ula ions, in eac ions o b-
D
-glucopy anosylammonium ca bama e
wi h acyl-isocyana es. Subs i uen s o neu al pola and apola as
well as acidic and basic cha ac e we e in oduced in he 4-posi ion
o he phenyl ing wi h he aim o exploi he a ailable space a he
subsi e o he b-pocke o he ac i e si e o glycogen phospho ylase
610
aking in o accoun he sol en s uc u e. Mos o he new com-
pounds we e low mic omola inhibi o s o abbi muscle glycogen
phospho ylase b, howe e , no significan imp o emen o he
inhibi o y e ficiency as compa ed o ha o N-benzoyl-N
0
-b-
D
-glu-
copy anosyl u ea could be obse ed. These findings migh indica e
he lack o a specific and c ucial in e ac ion om his posi ion wi h-
in he ca aly ic si e.
C ys allog aphic analyses o he 3D s uc u es o he new com-
pounds wi h RMGPbshowed clea ly ha all analogues induced a
mo e ex ended shi o he backbone a oms o he 280s loop com-
620
pa ed o RMGPb:15. This shi was acili a ed by he flipping o he
pep ide bond o med be ween Glu287 and Gly288 o c ea e mo e
space a he ca aly ic si e o he enzyme. The mos p o ound di e -
ences we e obse ed in he side chain o Asn282 ha adop ed a
modified con o ma ion upon ligand binding in all complexes
excep o –OH and –NH
2
g oups. O e all, he s uc u al esul s
we e in acco dance wi h hose ob ained by he kine ic e alua ion
o he compounds. The bes inhibi o iden ified was compound
25 (4-CH
3
g oup) ha o med inc eased an de Waals in e ac ions
wi h he esidues lining he ca aly ic si e. The second bes inhibi-
630
o , ou o hose s udied by X- ay c ys allog aphy, was compound
29 (4-NO
2
g oup) ha s abilized he closed con o ma ion o he
280s loop and as well as esidues a he a end o he b-pocke
such A g292 h ough an ex ended ne wo k o wa e -media ed
in e ac ions. Compound 29 o med he la ges numbe o hyd ogen
bonds a he ca aly ic si e compa ed o he es o he compounds.
The esul s ob ained sugges he key ole o he sol en s uc u e in
s uc u e-based ligand design. This could be exploi ed o he
design o mo e po en inhibi o s o enzyme ac i i y coupling he
knowledge de i ed om each complex s uc u e wi h geome ic
640
algo i hms.
25
The compounds 15,25–32 we e docked in o he Bzu ea and
2-Nap-u ea c ys al s uc u e o he glycogen phospho ylase,
espec i ely. The binding a fini y o he highes anked docked
poses was p edic ed by he
LIAISON
p og am (LiaSco e) and co e-
la ed wi h expe imen ally measu ed K
i
alues o he s udied inhib-
i o s. The quali y o he co ela ion was assessed using he R-
squa ed (R
2
) coe ficien . The alue o he R
2
anges om 0.1080
up o 0.9180. Gene ally, a be e co ela ion was ound o he
s uc u es docked in o he 2-Nap-u ea s uc u e. The bes co ela-
650
ion was achie ed o he co ela ion whe e wo ou line s, 25 and
32, we e excluded om he linea fi , wi h linea co ela ion coe -
ficien s A and B (A + B⁄x) ha ing alues o 23.89 and 7.12. In e -
es ingly, he bes measu ed inhibi o appea s as an ou line . This
si ua ion migh be caused by he di e en binding mode p edic ed
by he docking and absence o he wa e molecules p esen in he
ac i e si e as ha e been shown in p esen ed c ys al s uc u e. De-
spi e all his, p esen ed linea fi can be used o p edic he binding
a fini y o compounds ha ing he same sca old s uc u e by he
LIAISON
p og am.
660
4. Expe imen al
4.1. Gene al syn he ic me hods
Mel ing poin s we e measu ed in open capilla y ubes o on a
Kofle ho -s age and a e unco ec ed. Op ical o a ions we e de e -
mined wi h a Pe kin-Elme 241 pola ime e a oom empe a u e.
NMR spec a we e eco ded wi h B uke WP 200 SY (200/50 MHz
o
1
H/
13
C), B uke AM 360 (360/90 MHz o
1
H/
13
C) o B uke
AM 400 (400/100 MHz o
1
H/
13
C) spec ome e s. Chemical shi s
a e e e enced o Me
4
Si (
1
H), o o he esidual sol en signals
(
13
C). TLC was pe o med on DC-Alu olle Kieselgel 60 F
254
(Me ck)
670
(eluen E OAc–hexane 1:2, unless s a ed o he wise), and he spo s
we e isualized unde UV ligh and by gen le hea ing. Fo column
ch oma og aphy Kieselgel 60 (Me ck, pa icle size 0.063–
0.200 mm) was used. O ganic solu ions we e d ied o e anhyd ous
MgSO
4
and concen a ed unde diminished p essu e a 40–50 °C
(ba h empe a u e). Ace oni ile was dis illed om P
4
O
10
and
s o ed o e molecula sie es (3 Å). D y me hanol was dis illed
om magnesium me hyla e. O he sol en s o comme cial analy -
ical g ade quali y we e used wi hou u he pu ifica ion.
4.2. Gene al me hods o he p epa a ion o N-acyl-N
0
-(2,3,4,6-
680
e a-O-ace yl-b-
D
-glucopy anosyl)u eas
4.2.1. Me hod A
To a solu ion o an acyl chlo ide (7.7 mmol) in 20 ml o d y chlo-
o o m anhyd ous zinc chlo ide (80 mg, 0.59 mmol) and 2,3,4,6- e -
a-O-ace yl-b-
D
-glucopy anosyl u ea (16, 1 g, 2.56 mmol) we e
added wi h s i ing. The eac ion mix u e was efluxed un il TLC
showed he comple e ans o ma ion o 16. Then he eac ion mix-
u e was pou ed in o ice-wa e and was ex ac ed wi h chlo o o m
(2). The o ganic phases we e collec ed and washed wi h sa d aq
Na
2
CO
3
solu ion and wa e . A e d ying he sol en was e apo a ed
690
and he esidue was pu ified by column ch oma og aphy (eluen :
E OAc–hexane = 1:2).
4.2.2. Me hod B
To a suspension o NaOCN (1.21 g, 18.7 mmol) in d y ace oni-
ile an acyl chlo ide (14.4 mmol) in ace oni ile (30 ml) and SnCl
4
(84
l
l, 0.72 mmol) we e added unde a gon wi h s i ing. The mix-
u e was s i ed unde eflux o 8 h and a e cooling o 2,3,4,6-
e a-O-ace yl-b-
D
-glucopy anosyl amine (18, 1 g, 2.88 mmol) was
added unde a gon. A e s i ing o 30 min some d ops o wa e
we e added and he mix u e was fil e ed. The sol en was e apo-
700
a ed and he esidue pu ified by column ch oma og aphy (eluen :
E OAc–hexane = 1:1).
4.3. Gene al me hods o he p epa a ion o N-acyl-N
0
-
(b-
D
-glucopy anosyl)u eas
4.3.1. Me hod C
A solu ion o an N-acyl-N
0
-(2,3,4,6- e a-O-ace yl-b-
D
-glucopy -
anosyl)u ea in d y me hanol was ea ed wi h a ca aly ic amoun
14 V. Nagy e al. / Bioo g. Med. Chem. xxx (2012) xxx–xxx
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o a me hanolic solu ion o NaOMe a . A e TLC had shown
disappea ance o he s a ing ma e ial he eac ion mix u e was
neu alized wi h a ca ion exchange esin (Ambe lys 15, H
+
o m).
710
A e fil a ion he sol en was emo ed and he esidue was pu i-
fied by c ys allisa ion.
4.3.2. Me hod D
A solu ion o an N-acyl-N
0
-(2,3,4,6- e a-O-ace yl-b-
D
-glucopy -
anosyl)u ea in d y me hanol was ea ed wi h a ca aly ic amoun
o ace yl chlo ide a . A e TLC had shown disappea ance o
he s a ing ma e ial he eac ion mix u e was neu alized wi h
solid NaHCO
3
. A e fil a ion he sol en was emo ed and he es-
idue was pu ified by column ch oma og aphy (eluen : CHCl
3
–
MeOH =9:1).
720
4.3.3. Me hod E
Oxalylchlo ide (1.1 equi ) was added o a suspension o a ca -
boxamide (0.15 mmol) in anhyd ous 1,2-dichlo oe hane (2 mL)
and he mix u e was hea ed a eflux emp. o 1 d. The ola iles
we e dis illed o unde diminished p essu e and oluene
(2 5 mL) was e apo a ed om he esidue o emo e he es
o oxalylchlo ide. The acyl-isocyana e ob ained in his way was
mixed wi h a solu ion o b-
D
-glucopy anosylammonium-ca ba-
ma e
20
(1 equi ) in anhyd ous py idine (100 mg/14 mL) and he
mix u e was s i ed un il TLC (CHCl
3
–MeOH =7:1) showed no
730
mo e change ( ans o ma ions we e incomple e). Py idine was dis-
illed o unde diminished p essu e and e apo a ion o oluene
(2 30 mL) emo ed aces o py idine. The c ude was pu ified
by column ch oma og aphy (CHCl
3
–MeOH =7:1).
Pa icula syn heses and compound cha ac e iza ion da a a e
p esen ed in he Supplemen a y da a.
4.4. Enzyme p epa a ion
RMGPbwas isola ed om abbi skele al muscle and pu ified as
desc ibed p e iously.
26
Kine ic s udies we e pe o med in he
di ec ion o glycogen syn hesis in he p esence o a ious concen-
740
a ions o inhibi o s as indica ed in Table 1. Enzyme ac i i y was
measu ed a pH 6.8 by he elease o ino ganic phospha e as de-
sc ibed p e iously by Oikonomakos e al.
27
(compounds 27,29
and 30) and Saheki e al.
28
(compounds 21,23 and 25).
4.5. C ys al complex o ma ion and X- ay c ys allog aphic da a
collec ion and p ocessing
Na i e T-s a e GPbc ys als we e g own in he e agonal la ice,
space g oup P4
3
2
1
2
29
and p io o da a collec ion we e soaked in a
bu e ed solu ion (10 mM Bes, pH 6.7) wi h 6.2 mM o 21 ( o 2 h),
0.76 mM o 23 (in 20% DMSO o 84 h), 1mMo 25 (in 2% DMSO o
750
10 h), 10 mM o 29 ( o 1 h). Co-c ys als o RMGPbcomplexed wi h
27 and 30 we e ob ained in a medium consis ing o 20 mg/ml en-
zyme, 1 mM spe mine, 3 mM DTT, 10 mM BES, 0.1 mM EDTA,
0.02% sodium azide, pH 6.7 (16 °C) wi h ei he 5 mM o 27 o
7mMo 30. Di ac ion da a o all complexes we e collec ed om
single c ys als a oom empe a u e, using synch o on adia ion
sou ce a EMBL-Hambu g ou s a ion, Ge many, beamlines X31
(k= 0.8123 Å), X11 (k= 0.813 o 0.8468 Å), BW7A (k= 0.9076 Å)
and BW7B (k= 0.8441 Å), and SRS-Da esbu y Labo a o y, beamline
PX9.6 (k= 0.92 Å). In eg a ion o he eflec ions and da a educ ion
760
was pe o med using he p og ams DENZO and SCALEPACK om
HKL-package.
30
4.6. C ys al s uc u e de e mina ion
The s uc u e o RMGPbin complex wi h 15, p e iously de e -
mined a 1.8 Å esolu ion
16
wi h XPLOR (pdb code 1K06) was
efined using REFMAC.
31
The esul s showed ha esidues lining
he 280s loop exhibi ed di e ences in hei side chain o ien a ions
wi h he mos p o ound ones localized in esidues Asp283 (
u
,
w
,
1
,
2
change by 14°,51°, 166°,20°, espec i ely) and Glu287 (
u
,
w
,
3
) change by 22°,35°, 156°, espec i ely). The new model
770
deposi ed wi h he p o ein da a bank (pdb code 2QNB), as eplace-
men o 1K06, was used as a s a ing model o he s uc u e de e -
mina ion o all six complexes. C ys allog aphic efinemen was
ca ied ou using a s anda d p o ocol as implemen ed by REF-
MAC.
31
2F
o
F
c
and F
o
F
c
elec on densi y maps calcula ed we e
isualized using he p og am o molecula g aphics ‘O’.
32
Ligand
models we e fi ed o he elec on densi y maps a e adjus men
o hei o sion angles. Al e na e cycles o manual ebuilding wi h
‘O’ and efinemen wi h REFMAC imp o ed he quali y o he mod-
els. The da a collec ion and efinemen s a is ics along wi h he
780
model quali y a e summa ized in Supplemen a y da a Table S2a.
The s e eochemis y o he p o ein esidues was alida ed by
PROCHECK.
33
The analyses o he complex s uc u es comp ised
mapping o he hyd ogen bond and an de Waals in e ac ions o
he analogues wi h he esidues lining he ca aly ic si e using
CONTACT
33
applying a dis ance cu o 3.3 and 4.0 Å be ween he
elec onega i e a oms, espec i ely. The p og am calcula es he
angle OHN(whe e he hyd ogen posi ion is unambiguous)
and he angle sou ceoxygen-bonded ca bon a om. Sui able al-
ues a e 120°and 90°. The ne wo k o in e ac ions is desc ibed in
790
Tables S2b and S2c. S uc u al compa isons we e pe o med wi h
he p og am ‘O’
32
by supe posi ion o he a omic coo dina es o
he new complexes wi h hose o he RMGPb:Bzu ea s uc u e.
The oo mean squa e de ia ion in C
a
posi ions we e de e mined
o esidues (24–249), (261–281), (289–313), (326–549) and
(558–830) using LSQKAB.
33
All figu es we e p epa ed wi h he p og ams MolSc ip
34
and
BobSc ip
35
and ende ed wi h Ras e 3D.
36
The coo dina es o he new s uc u es ha e been deposi ed wi h
he RCSB P o ein Da a Bank (h p://www. csb.o g/pdb) wi h codes:
800
2QNB-15, 2QLM-25, 2QLN-26, 2QN3-27, 2QN7-28, 2QN8-29 and
2QN9-30.
4.7. P epa a ion o p o ein and ligand s uc u es o docking
The ini ial s uc u es o he glycogen phospho ylase in a com-
plex wi h py idoxal-5
0
-phospha e and N-benzoyl-N
0
-b-
D
-glucopy -
anosyl u ea 15 and N-(2-naph hoyl)-N
0
-b-
D
-glucopy anosyl u ea
(2-Nap-u ea) we e ob ained om he PDB da abase unde he
codes Bzu ea and 2-Nap-u ea, espec i ely, and p epa ed using
Sch odinge ’s Maes o and P o ein P epa a ion Wiza d
37
as ol-
lows. Wa e molecules we e emo ed, hyd ogen a oms we e added
810
and p o ona ion s a es we e assigned based on a esidue pK
a
’s a
hei no mal pH (7.0). A om ypes and pa ial cha ges we e as-
signed acco ding o he OPLS_2001 o ce field, also known as
OPLS-AA.
38
Docking g ids we e p epa ed o each o he p epa ed p o ein
s uc u es, Bzu ea and 2-Nap-u ea, using he same p ocedu e.
The cen e s o he cubic g id boxes we e placed on he cen oid
o he bound ligands and he box sizes we e se o 14 Å in all h ee
dimensions.
Nine inhibi o s wi h a ailable expe imen al K
i
da a we e se-
820
lec ed o his s udy. The s uc u es o all inhibi o s we e ob ained
h ough he ene gy minimiza ion using Jagua 7.0 p og am in-
cluded in he Sch odinge so wa e
37
a he DFT B3LYP
39,40
le el
wi h he 6-31+G⁄basis se
41–46
p io o he docking. The calcula ed
ESP cha ges we e used as inpu pa ial cha ges o ligand a oms in
he docking calcula ions.
Docking was ca ied ou using he Glide 4.5 package o Sch o-
dinge sui e 2007.
37
Recen e iew o docking p og ams showed
47
ha Glide belongs amongs he mos accu a e docking p og ams.
V. Nagy e al. /Bioo g. Med. Chem. xxx (2012) xxx–xxx 15
BMC 9770 No. o Pages 17, Model 5G
25 Janua y 2012
Please ci e his a icle in p ess as: Nagy, V.; e al. Bioo g. Med. Chem. (2012), doi:10.1016/j.bmc.2011.12.059
We ha e used flexible ligand docking wi h he s anda d p ecision
830
(SP) algo i hm.
48
The pa ame e s o an de Waals adii we e
scaled by 0.80 o ligand a oms wi h pa ial a omic cha ge less hen
0.15. Ligand poses we e clus e ed wi h RMSDs less han 0.5 Å and
wi hin maximum a omic displacemen less han 1.3 Å. A e he
docking p ocedu e 20 poses o each docked ligand wi h he bes
GlideSco e
49
we e sa ed and used o he analysis.
Binding a fini ies we e ecalcula ed o all sa ed docking poses
employing he p og am Liaison included in he Sch odinge so -
wa e.
37
The s uc u es o he complexes using he OPLS2005 o ce
field
50
we e op imized and he LiaSco e’s (‘GlideSco e in Liaison’)
840
analyzed o he elaxed complexes. Enzyme ligand complexes
we e op imized wi h a es ained mobili y o ecep o esidues a
dis ance la ge han 12 Å and wi h ozen esidues a dis ance la -
ge han 16 Å om ligand, espec i ely. The same DFT calcula ed
pa ial a omic cha ges we e used o he ligand as o he docking.
Implici sol en con inuum model ha e been used in compu a ion
o model sol en e ec s. The calcula ed LiaSco e o he bes
docked pose we e co ela ed wi h he expe imen ally measu ed
lnK
i
alues.
Acknowledgmen s
850
This wo k was suppo ed by he Hunga ian Scien ific Resea ch
Fund (OTKA T37210, T46081, CK77712) as well as by he TÁMOP
4.2.1/B-09/1/KONV-2010007 p ojec co-financed by he Eu opean
Union and he Eu opean Social Fund. Financial aid o he collabo a-
ion be ween A hens and Deb ecen (GR-4/03) as well as Lyon and
Deb ecen (F-11/05) was p o ided by GSRT (G eece) as well as
EGIDE (F ance), espec i ely, and he Agency o Resea ch Fund
Managemen and Resea ch Exploi a ion (KPI, Hunga y), and also
by CNRS (F ance) and he Hunga ian Academy o Sciences (PICS
4576). VN hanks he F ench Embassy in Budapes o suppo ing
860
he co- u o ed PhD Thesis p epa ed in Lyon and Deb ecen. Addi-
ional suppo o his wo k was p o ided by G eek GSRT h ough
ENTER-EP6/2001, PENED-204/2001, he Join Resea ch and Tech-
nology P ojec s be ween G eece and Hunga y (2005–2007), Ma ie
Cu ie Hos Fellowship o he T ans e o Knowledge (ToK) con ac
no MTKD-CT-2006-042776 unde FP6; he FP7 Capaci ies coo di-
na ion and suppo ac ions REGPOT-2008-1-No 230146 ‘EURO-
STRUCT’ and REGPOT-2009-1-No 245866 ‘ARCADE’; The esea ch
leading o hese esul s has ecei ed unding om he Eu opean
Communi y’s Six h (RII3/CT/2004/5060008, IHPP HPRI-CT-1999-
870
00012) and Se en h F amewo k P og amme (FP7/2007-2013) un-
de g an ag eemen No. 226716 awa ded o bo h SRS-Da esbu y
Labo a o y and EMBL-Hambu g ou s a ion. We would also like o
hank Agence Na ionale de la Reche che o unding ANR-08-
BLAN-0305 ‘GPdia’ p ojec coo dina ed by JPP o suppo ing ou
collabo a i e esea ch ne wo k and acknowledge he assis ance
o he s a a EMBL beamlines X31, X13, BW7A, BW7B, a he
DORIS s o age ing in Hambu g and a SRS-Da esbu y beamline
PX9.6 o p o iding excellen acili ies o X- ay da a collec ion.
Supplemen a y da a
880
Supplemen a y da a (de ails o syn heses and c ys allog aphic
analyses) associa ed wi h his a icle can be ound, in he online
e sion, a doi:10.1016/j.bmc.2011.12.059.
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16 V. Nagy e al. / Bioo g. Med. Chem. xxx (2012) xxx–xxx
BMC 9770 No. o Pages 17, Model 5G
25 Janua y 2012
Please ci e his a icle in p ess as: Nagy, V.; e al. Bioo g. Med. Chem. (2012), doi:10.1016/j.bmc.2011.12.059