3D-QSAR,homology modeling,and molecular docking studies on
spiropiperidines analogues as agonists of nociceptin/orphanin FQ receptor
Ming Liu,Lin He,Xiaopeng Hu,Peiqing Liu,Hai-Bin Luo ⇑
School of Pharmaceutical Sciences,Sun Yat-Sen University,Guangzhou 510006,PR China
a r t i c l e i n f o Article history:
社会主义劳动者Received 3May 2010
Revid 22September 2010Accepted 24September 2010
Available online 29September 2010Keywords:NOP Agonist 3D-QSAR CoMFA
Homology modeling Molecular docking
a b s t r a c t
The nociceptin/orphanin FQ receptor (NOP)has been implicated in a wide range of biological functions,including pain,anxiety,depression and drug abu.Especially,its agonists have a great potential to be developed into anxiolytics.However,the crystal structure of NOP is still not available.In the prent work,both structure-bad and ligand-bad modeling methods have been ud to achieve a comprehen-sive understanding on 67N-substituted spiropiperidine analogues as NOP agonists.The comparative molecular-field analysis method was performed to formulate a reasonable 3D-QSAR model (cross-vali-dated coefficient q 2=0.819and conventional r 2=0.950),who robustness and predictability were fur-ther verified by leave-eight-out,Y-randomization,and external test-t validations.The excellent performance of CoMFA to the affinity differences among the compounds was attributed to the contri-butions of electrostatic/hydrogen-bonding and steric/hydrophobic interactions,which was supported by the Surflex-Dock and CDOCKER molecular-
docking simulations bad on the 3D model of NOP built by the homology modeling method.The CoMFA contour maps and the molecular docking simulations were integrated to propo a binding mode for the spiropiperidine analogues at the binding site of NOP.
Ó2010Elvier Ltd.All rights rerved.
The nociceptin/orphanin FQ receptor (NOP),also known as ORL1,OP4,or LC132,is a deorphanized member of the G-protein coupled receptor (GPCR)superfamily.This receptor is distributed in the brain and periphery.1It shares considerable structural and localization features with other opioid receptors,2but it is classi-fied as a non-opioid member of the opioid receptor family by the International Union of Basic and Clinical Pharmacology (IUPHAR).Its endogenous ligand,nociceptin (or orphanin FQ),which activates NOP,is a 17-amino acid neuropeptide isolated from the brain in 1995.3,4The neuropeptide acts as an inhibitive agent on synaptic transmission in the CNS,and reduces responsiveness to stress.The nociceptin-NOP system has been implicated in a wide range of biological functions,including pain,mood disorders,drug abu,cardiovascular control,and immunity.5–7NOP is receiving consid-erable attention as a potential target for the treatment of anxiety and depression.
A number of drugs are ud for the treatment of anxiety and depression.But they have some drawbacks due to their poor and/or variable efficacy,long run in to peak behavioral effect,and a wide range of side effects leading to tolerability and compli-ance problems.8Considerable evidences indicate that nociceptin and veral non-peptide NOP agonists rve as anxiolytics with fewer side effects.9–17
Several different class of NOP agonists have been reported.Among them,spiropiperidine analogues exhibit relatively higher NOP binding affinities,18,19however,there is not a QSAR (quantita-tive structure–activity relationship)or pharmacophore model re-ported in their work.In the prent study,comparative molecular-field analysis (CoMFA)was performed to formulate 3D-QSAR models of 67spiropiperidine analogues 19as NOP ago-nists.Subquently,a homology model was build bad on the crystal structure of Beta2-Adrenergic G Protein-Coupled receptor (PDB code:2RH1)20as a template.Finally,molecular docking sim-ulations were performed to obtain a complete picture of the struc-tural characteristics of the most active agonist P67within the putative active site of this protein.Results of the study not only support the u of spiropiperidine analogues as a potential thera-peutic agent by targeting NOP,but also aid the rational design of novel and more effective NOP agonists as desired.
Sixty-ven N-substituted spiropiperidine analogues with hu-man NOP binding affinities (K i )determined from competition bind-ing assays were collected from the literature.19The 67molecules were divided into a training t of 57compounds and a test t of 10compounds,as shown in Table 1.The test t covers the range of biological activities and indicates a moderate diversity in their chemical structures.The experimental p K i values (Àlog K i )were ud for the 3D-QSAR analysis.
The three-dimensional structures were constructed using SYBYL programming package (version 7.3.5).21The MMFF94force field and MMFF94partial atomic charges were applied to the
0960-894X/$-e front matter Ó2010Elvier Ltd.All rights rerved.doi:10.1016/j.bmcl.2010.09.116
Corresponding author.Tel.:+862039943031.
E-mail address:luohb77@mail.sysu.edu (H.-B.Luo).
Table1
Structures,experimental,19and CoMFA-predicted activities of spiropiperidines analogues as nociceptin/orphanin FQ receptor(NOP)agonists
N N
N
O
税制R1
R2A B
Cl
Cl
C
Cl
Cl
D
Compound R1R2Àlog(K i/nM)
Experimental Predicted Res
P09A H-7.6387.654À0.016
P10a A Me7.5097.1830.326
P11A Et7.0767.112À0.036
P12A Pr7.4697.2420.227
P13A Bu7.2447.344À0.100
P14A i-Pr7.1807.242À0.062
P15A c-PrCH27.081 6.9670.114
P16A c-BuCH27.2767.2090.067
P17a A c-HexylCH27.0517.154À0.103
P18A Propargyl 6.631 6.800À0.169
P19A Allyl 6.693 6.750À0.057
P02B H–8.1678.0670.100
P20B Bu–7.3127.587À0.275
贺寿祝词P21B i-Amyl7.2527.476À0.224
P22B CH3OC(O)CH2–7.7107.7050.005
P23B HO(CH2)27.7338.052À0.319
P24B MeO(CH2)27.5857.826À0.241
P25a B NH2(CH2)2–7.3217.621À0.300
P26B CH3NH(CH2)2–8.3938.2740.119
P27B EtNH(CH2)2–8.6788.5310.147
P28B i-PrNH(CH2)2–8.5938.3760.217秦门
P29B c-PentylNH(CH2)2–8.0638.395À0.332
P30B c-HexylNH(CH2)2–8.3018.369À0.068
P31B(CH3)2N(CH2)2–8.4568.1380.318
P32B c-PrNH(CH2)2–8.4328.4200.012
P33B(i-Pr)2N(CH2)2–7.9177.8020.115
P34a B BuNH(CH2)2–8.6688.6620.006
P35B i-BuNH(CH2)2–8.5618.698À0.137
P36B BuNH(CH2)2–8.4208.601À0.181
P37B N(CH2)28.6488.3240.324
P38B N(CH2)38.4958.2880.207
P39B N(CH2)28.0977.9000.197
P40C H–8.6388.729À0.091
P41C CH3NH(CH2)2–9.0978.8450.252
桃花诺歌词P42C EtNH(CH2)2–9.1559.167À0.012
P43a C i-PrCH2NH(CH2)2–9.1559.0410.114
P44C c-PrCH2NH(CH2)2–9.3019.470À0.169
P45C c-BuNH(CH2)2–9.3019.0480.262
P46C PrNH(CH2)2–9.2229.333À0.111
P47C i-BuNH(CH2)2–9.3019.371À0.070
P48C BuNH(CH2)2–9.3989.2820.116
P49C Et2N(CH2)2–9.0008.9920.008
P50C N(CH2)28.6388.939À0.301
P03D H–8.8868.5380.348
P51a D Pr–8.2688.1320.136
P52D CH3C(O)CH2–8.3478.541À0.194
P53D HO(CH2)2–8.7708.6020.098
P54D CH3NH(CH2)2–8.6788.702À0.024
P55D EtNH(CH2)2–8.7969.029À0.233
P56a D i-PrNH(CH2)2–8.8548.970À0.116
P57D c-PentylNH(CH2)2–9.0468.9650.081
P58D c-HexylNH(CH2)2–9.0469.0160.030
P59D PrNH(CH2)2–9.0009.204À0.204
P60D CH2@CHCH2NH(CH2)2–9.0469.090À0.044
P61D c-BuNH(CH2)2–8.8248.910À0.086
P62D c-PrCH2NH(CH2)2–9.0979.123À0.026
P63D i-BuNH(CH2)2–9.3019.2370.064
P64a D(i Pr)2-N–(CH2)2–8.1748.634À0.460
(continued on next page)
M.Liu et al./Bioorg.Med.Chem.Lett.20(2010)7004–70107005
compounds.The nitrogen atoms clo to the R1group for tho li-gands are positively charged.And the compounds were minimized with a non-bond cut-off of8Åusing Powell conjugate-gradient algorithm.The convergence criterion was t to0.05kcal/mol. The most active compound P67rved as an alignment template for superposition.
Following the standard CoMFA procedure,each compound was mapped onto a3D lattice with grid points2.0Åapart.An sp3car-bon atom with+1charge was employed to probe the steric(Len-nard-Jones)and electrostatic(Coulombic)field energies.The cut-off interaction energies were t to30kcal/
mol.Columnfiltering, for any column(lattice points)of computed molecular-field ener-gies with a variance less than2.0kcal/mol,was applied to reduce computation time without negatively affecting the quality of the CoMFA models.
The robustness of the partial-least-squares(PLS)analysis embedded in CoMFA was addresd by internal cross-validation using the leave-one-out(LOO)procedure.21–23As an acceptable practice in3D-QSAR studies,a cross-validated regression coeffi-cient q2being higher than0.5can be considered as a statistical proof with high predictability,22,23while the conventional regres-sion coefficient r2should be more than0.9.
婴儿吃手是什么原因
Four GPCRs,including human A2A Adenosine Receptor(3EML), Beta1-Adrenergic G Protein-Coupled Receptor(Protein code: 2VT4),Beta2-Adrenergic G Protein-Coupled Receptor(2RH1),and Rhodopsin(3DQB),were evaluated.Considering the quence identity as well as some key residues(Supplementary data Appen-dix1),the Beta2-Adrenergic G Protein-Coupled Receptor was lected as a template for the homology modeling.The quences of the template and human ORL1-receptor(SwissProt P41146) were aligned using the quence alignment protocol within Accel-rys Discovery Studio2.5.24The structure information predicted by the TRANSMEM method is considered in the alignment.NOP indicates a quence identity of24.6%to the template in
i
Experimental Predicted Res
P65D N(CH2)28.8548.888À0.034
P66a D BuNH(CH2)2–9.3019.1710.130 P67D i-AmylNH(CH2)2–9.3989.2510.147 P68D c-HexylCH2NH(CH2)2–9.1559.212À0.057 P69D BnNH(CH2)2–8.4448.3820.062
P70
Cl
BuNH–7.7997.7210.078
P71a
Cl
BuNH–8.7458.941À0.196
P72
Cl
BuNH–9.2229.1510.071
P73
Cl
Cl
BuNH–7.9147.8870.027
a Test t.Res=p K
i Experimental
Àp K i Predicted.
Table2
Statistical parameters of the CoMFA models
Training t
q20.819
q2LEO(average of30runs)0.795
r20.950
SEE0.179
F160.1
Optimal components6
Field fraction(%)
S57.0
E43.0
Test t
q20.855
r20.912
k0.900
S=steric and E=electrostatic.r,q,SEE,and F are the
conventional Pearson regression coefficient,cross-val-
idated regression coefficient,standard error of esti-
mate,and statistical F value,respectively.LEO means
the leave-eight-out partial least-squares analysis.k is
the slope of the linear regression for the test t.
7006M.Liu et al./Bioorg.Med.Chem.Lett.20(2010)7004–7010
Figure1.Plot of predicted versus experimental p K i values of the3D-QSAR CoMFA
model.
Figure2.CoMFA contour maps:the steric/electrostatic maps for four compounds.In a and b,green(labeled as‘SF1’and‘SF2’)and yellow(‘SD1’and‘SD2’) the sterically higher NOP binding affinity)and disfavored areas,respectively,while blue(‘EP1’,‘EP2’,‘EP3’,and‘EP4’)and red(‘EN1’,and‘EN2’) regions where electropositive substituents are favorable and unfavorable in c,d,e,and f,respectively.Plea refer to the web-version for the alternative color (nitrogen:blue;oxygen:red;and carbon:grey).
CoMFA model.Figure1shows a
和局打一字
CoMFA-predicted and
t.Linearity of the plots show
FA model developed in the study
spiropiperidine analogues.The
contributions57.0%and43.0%to
are appropriate in interpreting
training t.Leave-eight-out
to estimate the extent of chance
The mean(0.795)of the q2values
ferent conclusion from that
The CoMFA model was not
The affinity data for the training
CoMFA models were rebuilt,
The statistical parameters(q2
were compared with tho of the
els in the Y-Randomization test
parameters(not shown),which
FA model bad on the original
chance correlation.
The significance and
checked by predicting the activity
external test t is widely ud
3D-QSAR model.For a reliable
criteria should be satisfied for
0.856k61.15.Herein,k refers
between the experimental and
An excellent correlation between
biological activities is depicted
(q2=0.855,r2=0.912,and k=
cients(q2and r2)and slope k of
ommended criteria.22,23The
confirm that the derived
predictive ability in the external
3D contour maps were
experimental affinity data with
molecular-field contributions,
vel and more effective NOP
CoMFA model are displayed in
static properties are displayed in Figure2c and d.As demonstrated
in Figure2a,P67(K i=0.4nM)orients its long alkyl chain to‘SF1’. The two methyls at the end of the chain enter the green areas, while another two methyls at the4position of the tetrahydro-naphthalene ring are located near the‘SF2’,which leads P67with relatively high affinity.Converly,bulky groups clo to the yel-low contour‘SD1’result in a lower affinity.Accordingly,the prop-argyl group of P18(234nM,the most inactive ligand,e Fig.2b) and the allyl group of P19(203nM)point toward the yellow isop-leths.Compounds with bulky groups near the‘SD1’region(Supple-mentary data Appendix5),such as P29(8.65nM),P30(5nM),and P33(12.1nM),reprent relatively lower NOP binding affinities comparing with tho without bulky groups(P27 2.1nM and P282.55nM).
A condary amine near the30position of the alkyl chain will enhance affinity.The blue and red regions near the30position of the alkyl chain of P67reveal the significance of arranging electro-positive and electronegative groups at this position,which signifi-cantly influence affinity.For example,N at the30position will enhance affinity.According to the MMFF94partial atomic charge calculations,the carbon(0.00|e|)at the3position of the butanyl chain in P20is replaced by nitrogen(À0.90|e|)in P26.The only hydrogen(0.36|e|)linked to the nitrogen in P26is clo to the electropositively favorable region‘EP1’,whereas the lone pair elec-trons of the nitrogen atom are adjacent to the electronegatively favorable area‘EN1’.As a result,higher affinity(4.05nM)of the lat-ter was obrved than that(48.7nM)of the former,as indicated in Supplementary data Appendix5.In addition,the affinity of a com-pound with a condary amine(P480.4nM and P430.7nM)is bet-ter than tho with a primary/tertiary amine(P2547.8nM and P64 6.7nM),which suggests an asymmetry electronic distribution.The reason for the ascending affinity order(P20and P26)can be ud to account for the relatively higher affinities of P48and P43than tho of P25and P64,respectively.
The R1substitute groups have a strong effect on activities.The biological activities of A-substituted and B-substituted compounds are relatively lower(23–234,2.1–56nM,respectively)than tho of the C-substituted and D-substituted compounds(0.4–2.3,0.4–5.4nM,respectively).The contour map point
s out that the biphe-nyl-methyl group substituted at the8th position decreas activ-ity.In Figure2d,blue contours‘EP3’are located above the plane of the upper phenyl of A,which cau energetically unfavorable repulsion and result in low activities.B-Substituted group is quite similar to A-substituted group,except that the chlorine substituted at the cond position which has the electron-withdrawing effect to attenuate the electrostatic/steric repulsion with‘EP3’.Thus the activities of B-substituted compounds have a slightly boost than tho of A-substituted counterparts when own the same R2func-tional groups.In comparison,the volumes of C-substituted and D-substituted groups are relatively smaller tho of A-substituted and B-substituted groups,thus they reduce the energetically unfavorable repulsion with‘EP3’and lead relatively higher acti-vities,which may explain why compounds P28,P43,and P56have Figure3.The docking-binding mode of the NOP(nociceptin/orphanin FQ receptor)/ P67complex after molecular docking procedure.In(a),LHS:extracellular side and RHS:intracellular side,respectively.In(b),the docking po obtained by Surflex-dock is shown in sticks,while the one obtained by CDOCKER is shown in CPK (nitrogen:blue;oxygen:red;and carbon:cyan/grey).Plea refer to the web-version for the alternative color interpretation(nitrogen:blue;oxygen:red;and carbon:green/grey/cyan).
7008
different binding affinities although all of them contain the same i -PrNH(CH 2)2–group.The similar trend could be applied to account for the affinity difference between P34and P48.
A 3D model of NOP built for the rational design of NOP agonists.The Ramachandran Plot of the 3D model is showed in Supplemen-tary data Appendix 6,which is ud to verify predicted torsion an-gles in proteins.It indicates low energy conformations for u (phi)and w (psi),which are ud to reprent the torsion angles on either side of alpha carbons in peptides.In the plot,97.6%residues locate in the preferred and allowed regions,whereas only 2.4%res-idues are in the disallowed regions.Besides,none of the residues within the binding pocket are in the disallowed regions,which cor-roborates the 3D model is acceptable for the molecular docking simulations and suitable for the structure-bad molecular design of NOP agonists.
Salt bridge,hydrogen-bonding,and hydrophobic interactions can be identified as the underlying factors for the high NOP binding affinity of P67.The docking-binding mode of P67in the active site is shown in Figure 4.The protonated piperidine-nitrogen atom forms stable salt bridges with Asp130,which is a crucial anchoring point for agonists,as pointed out by site directed mutagenesis.28,29The D-substituted group takes up the hydrophobic pocket formed by Tyr131,Met134,Trp211,Phe220,Phe224,Trp276,Val279,and Val283.This hydrophobic pocket was pro
pod to hold the Phe1sidechain of nociceptin (the endogenic agonist of NOP),and some of the residues (Tyr131,Phe220,Phe224,and Trp276),which are confirmed to have a significant effect on the binding by previous mutagenesis studies.29,30As described by N.Zaveri,there is a spe-cific lipophilic area that triggers an agonist respon when the site is occupied by an appropriately sized and placed lipophilic moi-ety.30,31Additionally,the long alkyl chain sketchs to another direc-tion to form strong hydrogen bonds with Arg302and Asp110(H1a =2.85Å,H1b =3.12Å,and H2=3.02Å,respectively).The re-sults are in agreement with the phenomenon that the condary amine at the 30position enhances affinity.This nitrogen atom may work as a hydrogen donor when it interacts with Asp110,whereas rves as a hydrogen acceptor when it interacts with
Arg302(Asp 110corresponds to ‘EP2’and Arg302refers to ‘EN1’),which may explain the asymmetry electronic distribution in the 3D contour map (Fig.2c).This result is also consistent with the previous works,which demonstrated that mutating Arg302to a negative charged Asp would result in a great loss of nociceptin binding.32As a result,the energetically favorable interactions stabilize P67in the ligand-binding pocket.
In summary,the resulting CoMFA model performs well in both internal and external consistency in the prent study.The excel-lent correlation between experimental and predicted p K i values for the test
t corroborated the predictive ability of the derived CoMFA model.Bad on the 3D model of NOP built by homology modeling approach,molecular docking simulations reveal that v-eral important residues (Asp110,Asp130,Tyr131,Typ276,and Arg302)exhibit salt bridge,hydrogen-bonding,and hydrophobic interactions with the most active agonist P67,which is consistent with the CoMFA contour maps and the experimental results from the literature.19,30,31The newly obtained 3D model of NOP may rve as a basis for the structure-bad molecular design of novel agonists with enhanced affinity.
Our results highlight that the 3D-QSAR (ligand-bad)and homology modeling/molecular docking (structure-bad)ap-proaches,which were integrated herein,would be uful for the exploratory study of some promising biological systems for drug discovery purpo with respect to affinity enhancement,especially for cas difficult to obtain the crystal structure of a potential protein.
Acknowledgments
The excellent suggestions from the anonymous reviewers are cordially appreciated.We cordially acknowledge the financial sup-port from National Major Projects for science and technology development from Science and Technology Ministry of China (2009ZX09304-003),Fundamental Res
earch Funds for the Central Universities (10ykjc20),Natural Science Foundation of Guangdong Province (9451008901001994),and Rearch Fund for the Doctoral Program of Higher Education of China (20090171120057).Supplementary data
Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.bmcl.2010.09.116.References and notes
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通过
132.
Figure 4.The docking-binding mode of P67in the active site of the nociceptin/orphanin FQ receptor obtained by CDOCKER.Hydrogen bonds are displayed in red dashes (Å).Plea refer to the web-version for the alternative color interpretation (nitrogen:blue;oxygen:red;and carbon:cyan/grey).
Lett.20(2010)7004–70107009
