W. Li et al./ Bioorg. Med. Chem. 14(2006)601-610 2.5. Homology modeling of kappa opioid receptor The sequence of human kappa opioid receptor was re trieved from the Swiss Prot database(Accession N P41 145). 3The sequences of bovine rhodopsin and hu Pror k and 8 opioid receptors were obtained from Swiss- L,too,for sequence alignments(see Fig. 3). The crystal structure of bovine rhodopsin was retrieved from Protein Data Bank(PDB entry code 1F88),24 which served as the template to generate the structural of kappa opioid receptor. At first the 7 TM fra were constructed by mutating the corresponding m in the template into target residue in kappa receptor Residue Ala106 was inserted into the target structure The extracellular loop 2(EL2), connecting TM4 and TM5, was built on the basis of EL2 of rhodopsin. The 2. Superposition of 39 molecules including compounds in the other extra- and intra-cellular loop regions were built set and test set based on the template of nor-BNI, a potent and with loop search function of sYBYL/Biopolymer module ctive antagonist. (The structure of nor-BNI was removed for The N-and C-terminal regions were extended from the purposes of clarity. ransmembrane regions for 10 residues, not completely built.a disulfide bond was formed between the side was used for model validation. Similar to cross-validat chains of residues Cys131 and Cys210. After all done, ed g values of LOO method, the predictive performance adding all side chains and hydrogen atoms and loading of models on the test set was estimated by predictive Kollman All-Atom charges, the initial structure was values, which is expressed in the following equation energy minimized for 5000 steps with Kollman All-At predictive 2 SSD-press om force field. 25 SSD The protonated GNTi was docked into the minimized where SSD is the sum of squared deviation between the structure of kappa receptor manually, by putting the pK, values of test set molecules and PRESS is the sum of protonated nitrogen toward the side chain of residue squared deviations between the observed and the pre- Asp138 and the guanidine group close to the side chain dicted pKi values of Glu297. The whole complex structure was then min- bRo 35 WQFS hKOR 57 AIPV hMOK68⊥A⊥ h DOR 47 ALAI IL2 SLHGYEVEGPTG FEATLGGEIA LAIERYVVVCKPMSNFRFG-ENH 152 hKOR ISIDYYNMETS HPVKALDERTPLK 174 hMOR ISIDYYNMETS HPVKALDERTPRN 185 LMETWPFGEL Ls工 DYYNMETS IAVCHPVKALDERTPAK 164 EL2 Hairpin bOho 153 WSRYI PEGMQC-SCGIDYYTPHEET 211 hKOR 175 KVREDVDVIECSLQFPDDDXSWWD 234 186 TTKYRQG-S-IDCT'LTESHPTW hDOR 165 MAVTRPRDGA-VV-CMLQFPSPSW 工L3 bRno 212 KEAAAQQQESATTQKAEKEVTRMVIIMVIAFT hKoR 235 RLKSVRLLSG-SREKDRN TRLVLVVVAV 293 hMOR 243 SVRMLSG-SKEKDRNLRRITRMVLVVVAVE h DOR 221 LRSVRLLSG-SKEKDRS 280 bRo 272 328 hKOR 294 ILYA RCERDEC FPLKMRM 352 hMOR 302 RCEREFC 360 RCERQLCRKPCGRP 340 Sequence alignments of three subtypes of human opioid receptors with bovine rhodopsin(N- and C-terminals omitted). The among them were highlight- b(EL), and intra-cellular loop (IL)regions were labeled correspondingly. In transmembrane regions,identical mbrane(TM)extracellular in dark blue, while residues in opioid receptors analogous to those in rhodopsin were colored in red.was used for model validation. Similar to cross-validat￾ed q2 values of LOO method, the predictive performance of models on the test set was estimated by predictive r 2 values, which is expressed in the following equation: predictive r 2 ¼ SSD
PRESS SSD where SSD is the sum of squared deviation between the pKi values of test set molecules and PRESS is the sum of squared deviations between the observed and the pre￾dicted pKi values. 2.5. Homology modeling of kappa opioid receptor The sequence of human kappa opioid receptor was re￾trieved from the SwissProt database (Accession No. P41145).23 The sequences of bovine rhodopsin and hu￾man l and d opioid receptors were obtained from Swiss￾Prot, too, for sequence alignments (see Fig. 3). The crystal structure of bovine rhodopsin was retrieved from Protein Data Bank (PDB entry code 1F88),24 which served as the template to generate the structural model of kappa opioid receptor. At first the 7 TM fragments were constructed by mutating the corresponding residue in the template into target residue in kappa receptor. Residue Ala106 was inserted into the target structure. The extracellular loop 2 (EL2), connecting TM4 and TM5, was built on the basis of EL2 of rhodopsin. The other extra- and intra-cellular loop regions were built with loop search function of SYBYL/Biopolymer module. The N- and C-terminal regions were extended from the transmembrane regions for 10 residues, not completely built. A disulfide bond was formed between the side chains of residues Cys131 and Cys210. After all done, adding all side chains and hydrogen atoms and loading Kollman All-Atom charges, the initial structure was energy minimized for 5000 steps with Kollman All-At￾om force field.25 The protonated GNTI was docked into the minimized structure of kappa receptor manually, by putting the protonated nitrogen toward the side chain of residue Asp138 and the guanidine group close to the side chain of Glu297. The whole complex structure was then min￾Figure 2. Superposition of 39 molecules including compounds in the training set and test set based on the template of nor-BNI, a potent and j selective antagonist. (The structure of nor-BNI was removed for purposes of clarity.) Figure 3. Sequence alignments of three subtypes of human opioid receptors with bovine rhodopsin (N- and C-terminals omitted). The transmembrane (TM) extracellular loop (EL), and intra-cellular loop (IL) regions were labeled correspondingly. In transmembrane regions, identical residues among them were highlighted in dark blue, while residues in opioid receptors analogous to those in rhodopsin were colored in red. 604 W. Li et al. / Bioorg. Med. Chem. 14 (2006) 601–610