Journal of chemical Information and modeling Article Figure 6. Comparison of superimposed structures inactive S-HTIAR structure( blue cartoon)and agonist(FWOl, yellow ball and sticks )-bound active S-HTIAR structure(orange cartoon). a, Extracellular view showing outward movements of TM2 and TM7 by ca. 4.48 A and 8.3 A respectively, and modest structural changes of other TMs. b, Cytoplasmic view showing large outward movement of TM6, ca. 17 A expected, they are all turned out to be S-HTIAR agonists(Table( Figure 6). The extracellular part of TM2 and TM7 undergoe large-scale displacements-a 4.48 A outward movement of Through chemical structure analysis, these identified TM2 and an 8.3 A outward movement of TM7(E potential S-HTIAR agonists all contain common sp nitrogen, which are demonstrated in current available crystal which is universe among GPCRs ligands, and aromatic rings structures of GPCRs. The most significant outward displace attached to different match our proposed dynamic pharmacy of scaffolds. Accordingly, these ment, as much as 17A(Figure 6b), is found at the cytoplasm end of TM6 in concert with a slight inward tilt of the phore model. The most potential hit FWOl(ECso=7 nM) extracellular segment( Figure 6a), which is in line with the attracted our interest, which turns out to display comparable crystal structures of P,AR, but only partly conforms to the intrinsic activity to S-HT; therefore, it was chosen as the lead previously proposed common activation mechanism of the compound for the analogue optimization, and a series of strong Rhodopsin family "global toggle switch mechanism")which agonists with K 10 nM was already obtained (unpublished suggests a"vertical"seesaw movement of TM6. In the cytoplasmic face, an inward motion of TM7 and an upward Binding of FWo1 with 5-HT1AR. To get structural insights shift of TM3 are observed as expected (Figure 6b) into the strong agonistic mechanism of Fwol, its binding mode Comparison among crystal structures of different GPCrs with 5-HTiAR was predicted by molecular docking( Figure Sa), suggests that the extent of the TM6 motion varies, and the nd the obtained complex was subjected to 100 ns molecular movements of TM3 and TM7 depend on particular receptor ynamics simulation. Fwol, with a stretched scaff in and the binding of different stem. the the same pocket as R-8-OH-DPAT. The conserved salt bridge binding of FwOl contributes to the conserved but also unique interaction with D3. 32 and hydrogen bond with Ser5.42 conformational changes of S-HTIAR First, the side chain carbonyl group can be both potentially involved as an acceptor nitrogen of the Fwol, which might count for the upshift of in the hydrogen bond with Y7. 43. Besides, adjacent residues TM3. The carbonyl group in Fwol is engaged in the hydrogen like Y5.38 and K191 located in ECL2 form favorable T- and bond with N7.39(or Y7.43 at first). As a result, the direct salt cation- interactions with the indole ring, respectively. In bridge between D3.32 and Y7. 43 present in the inactive 5- ddition, the benzene ring in the tail is sandwiched between the HTIAR is interrupted and mediated by FwOl instead Specially side chains of F3. 28 and Y2. 64 the latter involvement of N7. 39 in the stronger hydrogen bond During the simulation time, the salt bridge between D3.32 with the carbonyl group of the ligand leads to the outward nd Fwol remains stable(Figure Sb). S5.42 interacts tightl leaning of the extracellular segment of TM7. TM2, which was with the nitrogen atom of the indole ring(Figure Sb). While supposed to have little displacement, moves outward to the other hydrogen bond between Y7.43 and the carbonyl accommodate the bulky tail of Fwo1-phenyl and cyclohexyl group fluctuates and disappears after 31 ns(Figure 5b). groups, so that the phenyl ring forms favorable T-3 N7. 39, which locates one turn above y7. 43, was involved in as a interactions with y2. 64 and F3. 28. Likewise the substantial also play an important ro( figure Sa). Thus N7.39 might out-swing of TM6 is also facilitated by agonist binding.The competitive H-bond par tner conformation of 5-HT1AR The results suggest that residues monoamine receptor agonists and has been proved to be Y2.64, F3. 28, Y5.38, Y7.39, Y7. 43, and K191 also contribute to important for agonist binding and activation. 4 Given this the binding of the agonist and activation of the receptor. the stable hydrogen bond between the nitrogen atom in the Agonist-Induced Structural Changes in 5-HT1AR. We indole ring and $5.42 results in an inward bulge and clockwise next compared the structure of active and inactive 5-HTIAR rotation of TMS and then triggers concerting rearrangements Several major conformational changes are found both at the of nearby residues like what is observed in P2AR(Figure 7, cytoplasmic face and the extracellular side of the receptor R"). The hydrophobic packing interaction, which stabilizes the dx dolor/10.1021/c400481plJ Chem Inf Model. 2013, 53, 3202-3211expected, they are all turned out to be 5-HT1AR agonists (Table 2). Through chemical structure analysis, these identified potential 5-HT1AR agonists all contain common sp3 nitrogen, which is universe among GPCRs ligands, and aromatic rings attached to different types of scaffolds. Accordingly, these compounds can well match our proposed dynamic pharmaco￾phore model. The most potential hit FW01 (EC50 = 7 nM) attracted our interest, which turns out to display comparable intrinsic activity to 5-HT; therefore, it was chosen as the lead compound for the analogue optimization, and a series of strong agonists with Ki < 10 nM was already obtained (unpublished work). Binding of FW01 with 5-HT1AR. To get structural insights into the strong agonistic mechanism of FW01, its binding mode with 5-HT1AR was predicted by molecular docking (Figure 5a), and the obtained complex was subjected to 100 ns molecular dynamics simulation. FW01, with a stretched scaffold, lies in the same pocket as R-8-OH-DPAT. The conserved salt bridge interaction with D3.32 and hydrogen bond with Ser5.42 were kept. The other nitrogen atom in the\ piperazine ring and the carbonyl group can be both potentially involved as an acceptor in the hydrogen bond with Y7.43. Besides, adjacent residues like Y5.38 and K191 located in ECL2 form favorable π−π and cation-π interactions with the indole ring, respectively. In addition, the benzene ring in the tail is sandwiched between the side chains of F3.28 and Y2.64. During the simulation time, the salt bridge between D3.32 and FW01 remains stable (Figure 5b). S5.42 interacts tightly with the nitrogen atom of the indole ring (Figure 5b). While the other hydrogen bond between Y7.43 and the carbonyl group fluctuates and disappears after ∼31 ns (Figure 5b). N7.39, which locates one turn above Y7.43, was involved in as a competitive H-bond partner (Figure 5a). Thus N7.39 might also play an important role in facilitating the agonistic conformation of 5-HT1AR. The results suggest that residues Y2.64, F3.28, Y5.38, Y7.39, Y7.43, and K191 also contribute to the binding of the agonist and activation of the receptor. Agonist-Induced Structural Changes in 5-HT1AR. We next compared the structure of active and inactive 5-HT1AR. Several major conformational changes are found both at the cytoplasmic face and the extracellular side of the receptor (Figure 6). The extracellular part of TM2 and TM7 undergoes large-scale displacementsa 4.48 Å outward movement of TM2 and an 8.3 Å outward movement of TM7 (Figure 6a), which are not demonstrated in current available crystal structures of GPCRs. The most significant outward displace￾ment, as much as 17 Å (Figure 6b), is found at the cytoplasmic end of TM6 in concert with a slight inward tilt of the extracellular segment (Figure 6a), which is in line with the crystal structures of β2AR,48 but only partly conforms to the previously proposed common activation mechanism of the Rhodopsin family (“global toggle switch mechanism”) which suggests a “vertical” seesaw movement of TM6.49 In the cytoplasmic face, an inward motion of TM7 and an upward shift of TM3 are observed as expected (Figure 6b). Comparison among crystal structures of different GPCRs suggests that the extent of the TM6 motion varies, and the movements of TM3 and TM7 depend on particular receptor and the binding of different ligands.50 In our system, the binding of FW01 contributes to the conserved but also unique conformational changes of 5-HT1AR. First, the side chain of D3.32 bends upward to form a hydrogen bond with protonated nitrogen of the FW01, which might count for the upshift of TM3. The carbonyl group in FW01 is engaged in the hydrogen bond with N7.39 (or Y7.43 at first). As a result, the direct salt bridge between D3.32 and Y7.43 present in the inactive 5- HT1AR is interrupted and mediated by FW01 instead. Specially, the latter involvement of N7.39 in the stronger hydrogen bond with the carbonyl group of the ligand leads to the outward leaning of the extracellular segment of TM7. TM2, which was supposed to have little displacement, moves outward to accommodate the bulky tail of FW01phenyl and cyclohexyl groups, so that the phenyl ring forms favorable π−π interactions with Y2.64 and F3.28. Likewise, the substantial out-swing of TM6 is also facilitated by agonist binding. The hydrogen bond interaction with S5.42 is common among monoamine receptor agonists and has been proved to be important for agonist binding and activation.51,52 Given this, the stable hydrogen bond between the nitrogen atom in the indole ring and S5.42 results in an inward bulge and clockwise rotation of TM5 and then triggers concerting rearrangements of nearby residues like what is observed in β2AR48 (Figure 7, R″). The hydrophobic packing interaction, which stabilizes the Figure 6. Comparison of superimposed structures - inactive 5-HT1AR structure (blue cartoon) and agonist (FW01, yellow ball and sticks)-bound active 5-HT1AR structure (orange cartoon). a, Extracellular view showing outward movements of TM2 and TM7 by ca. 4.48 Å and 8.3 Å, respectively, and modest structural changes of other TMs. b, Cytoplasmic view showing large outward movement of TM6, ca. 17 Å. Journal of Chemical Information and Modeling Article 3208 dx.doi.org/10.1021/ci400481p | J. Chem. Inf. Model. 2013, 53, 3202−3211