2544 Journal of Medicinal Chemistry, 2009, VoL. 52, No 8 Paz et al B Trp279 Tyr70 Ty Tyr334 Trp84 His44 Figure 2. Two representations of the refined structure of the 5h/TcAChE complex.(A) TcAChE is displayed as a beige space-filling surface and sh within the active-site gorge as a magenta stick model. The arrow marks the entrance to the active-site gorge.(B)5h shown as a green stick nodel. and the side chains of selected active- site residues with which it makes contact as red stick models was a homodimer with a nine-carbon spacer (5h)(Figure 1). which displayed ICso values of 3.9+ 1.3 and 10.0+ 3.0 nM CAS GOLD docking simulations of 5h, in which the phenyl groups of both MEP moieties were in equatorial orientations with he azapane rings(ecep), indicated that 5h spans the ctive-site gorge, interacting with both the CAS and PAs of mAChE. The phenyl group of the MEP moiety at the CAs was predicted to make a face-to-face T-stacking interaction wit Trp86(mAChE numbering, Trp84 in TcAChE). The meP The MEP moiety at the Cas and the nonamethylene linker displ moiety at the Pas predicted to make catie almost complete electron density, whereas the MEP moiety at the PAs hydrophobic interactions of its seven-membered azepane ring does not, suggesting that it assumes multiple conformations. The with the indole moiety of Trp286(TcAChE Trp279). In addition, electron density is contoured at 30 h was predicted to form two hydrogen bonds at the CAs,one volving interaction of its hydroxyl group with the main chain Rigid body refinement in CCP4- was based on a previo carbonyl oxygen of His447(TcAChE His440) and the other solved trigonal crystal form of native TcAChE (PDB code lEAS involving interaction of its protonated azepane nitrogen with excluding water molecules and carbohydrates. Initial 2Fo- Fc and Tyr1240-(TcAChE Tyrl21) ata, and the initial Fo Fe map was used, with the aid of the In the following, we present the crystal structure of a complex program Coot,27 to fit 5h into positive density at the CAS of TcAChE structure rather than on the mache structure. as TcAChE, as well as the spacer, and to add 78 water molecules Subsequent restrained refinement rounds with overall B-factor predicted, 5h spans the CAS and the PAS. The crystal structure refinement were performed and 56 carbohydrate atoms were added is compared to those obtained by computer docking of 5h and until convergence to values of Rwork =18.5% and Re to the native crystal structures of TcAChE and mAChE As analyzed by Molprobity, 95.0%o of all residues are in favored regions and 99. 6% are in allowed regions of the ramachandran Materials and Methods plot, the only outliers being Asp380 and Asn457, which are both Crystallization and Data Collection. TcAChe was purifi glycosylated surface residues. Due to the poor electron density at essentially as described by Sussman et al., with the exception of e PAs observed in the 2Fo- Fe map we chose to submit the the affinity column elution which was performed with tetrameth coordinates of 5h without the atoms of the mEP moiety within the lammonium bromide instead of decamethonium bromide. Trigonal PAS. In all figures therein, this mEp moiety is shown for descriptive crystals of the enzyme 4 were soaked for 20 h at 4C in 2 uL of purposes only. Simulated annealing omit maps were constructed I mM dissolved in the crystallization solution(40% PEG 200 PHENIX (v/v)/150 mM MES(Sigma-Aldrich), pH 7.4), employing the Molecular Docking. Molecular simulations were performed on hanging drop procedure. The crystals were then transferred to an R14000 SGI Fuel workstation with the software package SYBYL cryoprotectant oil and flash frozen in liquid nitrogen. 6.9(Tripos Inc, St Louis, MO). Standard parameters were used Data collection was performed at the eSrF in Grenoble, on unless otherwise indicated. The crystal structures of both native beamline ID 14-1. at T=100 K and 2=0.934 A. 180 image TcAChE(PDB code IEA5 )and of the 5h/TCAChE complex were were taken, at an oscillation angle of 1, with an exposure time of used. Heteroatoms and water molecules in the proteins were 6s. DENzo and ScalePacK- were used to integrate and scale removed, and hydrogen atoms were subsequently added. the data. Data were truncated with the CCP4 program TRUN- Three-dimensional structures of sh were generated by connecting CATE, and 5.16% of the reflections were randomly used as test two(-)-nor-MEP units in either equatorial or axial conformations. The equatorial conformation of the(-)-nor-MEP unit was retrieved Structure Determination and Refinement. A model of sh was from the crystal structure of MEP. The axial conformation was constructed using the Gauss View 3.09 pr at using ( Gaussian, Carnegie, generated based on earlier NMR studies. Two sp'Natoms of sh PA)and converted to a PDb file forma Babel. 2b were protonated, and the Gasteiger-Huckel partial charges werewas a homodimer with a nine-carbon spacer (5h) (Figure 1), which displayed IC50 values of 3.9 ( 1.3 and 10.0 ( 3.0 nM toward mouse brain AChE and mouse serum BChE, respec￾tively, and an IC50 of 16.6 µM for inhibition of A
aggregation. GOLD20 docking simulations of 5h, in which the phenyl groups of both MEP moieties were in equatorial orientations with respect to the azapane rings (eCeP), indicated that 5h spans the active-site gorge, interacting with both the CAS and PAS of mAChE. The phenyl group of the MEP moiety at the CAS was predicted to make a face-to-face π-stacking interaction with Trp86 (mAChE numbering, Trp84 in TcAChE). The MEP moiety at the PAS was predicted to make cation-π and hydrophobic interactions of its seven-membered azepane ring with the indole moiety of Trp286 (TcAChE Trp279). In addition, 5h was predicted to form two hydrogen bonds at the CAS, one involving interaction of its hydroxyl group with the main chain carbonyl oxygen of His447 (TcAChE His440) and the other involving interaction of its protonated azepane nitrogen with Tyr124O (TcAChE Tyr121).19 In the following, we present the crystal structure of a complex of 5h with TcAChE and new GOLD models based on the TcAChE structure rather than on the mAChE structure. As predicted, 5h spans the CAS and the PAS. The crystal structure is compared to those obtained by computer docking of 5h and to the native crystal structures of TcAChE and mAChE. Materials and Methods Crystallization and Data Collection. TcAChE was purified essentially as described by Sussman et al.,21 with the exception of the affinity column elution, which was performed with tetramethy￾lammonium bromide instead of decamethonium bromide. Trigonal crystals of the enzyme14 were soaked for 20 h at 4 °C in 2 µL of 1 mM 5h19 dissolved in the crystallization solution (40% PEG 200 (v/v)/150 mM MES (Sigma-Aldrich), pH 7.4), employing the hanging drop procedure.22 The crystals were then transferred to cryoprotectant oil and flash frozen in liquid nitrogen. Data collection was performed at the ESRF in Grenoble, on beamline ID 14-1, at T ) 100 K and λ ) 0.934 Å. 180 images were taken, at an oscillation angle of 1°, with an exposure time of 6 s. DENZO and SCALEPACK23 were used to integrate and scale the data. Data were truncated with the CCP424 program TRUN￾CATE,25 and 5.16% of the reflections were randomly used as test reflections (Table 1). Structure Determination and Refinement. A model of 5h was constructed using the GaussView 3.09 program (Gaussian, Carnegie, PA) and converted to a PDB file format using Babel.26 Rigid body refinement in CCP424 was based on a previously solved trigonal crystal form of native TcAChE (PDB code 1EA5), excluding water molecules and carbohydrates. Initial 2Fo - Fc and Fo - Fc electron density maps were calculated using 40-2.7 Å data, and the initial Fo - Fc map was used, with the aid of the program Coot,27 to fit 5h into positive density at the CAS of TcAChE, as well as the spacer, and to add 78 water molecules. Subsequent restrained refinement rounds with overall B-factor refinement were performed and 56 carbohydrate atoms were added until convergence to values of Rwork ) 18.5% and Rfree ) 23.5%. As analyzed by Molprobity,28 95.0% of all residues are in favored regions and 99.6% are in allowed regions of the Ramachandran plot, the only outliers being Asp380 and Asn457, which are both glycosylated surface residues. Due to the poor electron density at the PAS observed in the 2Fo - Fc map we chose to submit the coordinates of 5h without the atoms of the MEP moiety within the PAS. In all figures therein, this MEP moiety is shown for descriptive purposes only. Simulated annealing omit maps were constructed using PHENIX.29 Molecular Docking. Molecular simulations were performed on an R14000 SGI Fuel workstation with the software package SYBYL 6.9 (Tripos Inc., St. Louis, MO). Standard parameters were used unless otherwise indicated. The crystal structures of both native TcAChE (PDB code 1EA5) and of the 5h/TcAChE complex were used. Heteroatoms and water molecules in the proteins were removed, and hydrogen atoms were subsequently added. Three-dimensional structures of 5h were generated by connecting two (-)-nor-MEP units in either equatorial or axial conformations. The equatorial conformation of the (-)-nor-MEP unit was retrieved from the crystal structure of MEP.30 The axial conformation was generated based on earlier NMR studies.30 Two sp3 N atoms of 5h were protonated, and the Gasteiger-Hu¨ckel partial charges were Figure 2. Two representations of the refined structure of the 5h/TcAChE complex. (A) TcAChE is displayed as a beige space-filling surface and 5h within the active-site gorge as a magenta stick model. The arrow marks the entrance to the active-site gorge. (B) 5h shown as a green stick model, and the side chains of selected active-site residues with which it makes contact as red stick models. Figure 3. Simulated annealing omit map of the 5h/TcAChE complex. The MEP moiety at the CAS and the nonamethylene linker display almost complete electron density, whereas the MEP moiety at the PAS does not, suggesting that it assumes multiple conformations. The electron density is contoured at 3σ. 2544 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 Paz et al