Supplementary Information for: Conformational Analysis of Peramivir Reveals Critical Differences Between Free and Enzyme-Bound States

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1 Electronic Supplementary Material (ESI) for Medicinal Chemistry Communications. This journal is The Royal Society of Chemistry 2014 Supplementary Information for: Conformational Analysis of Peramivir Reveals Critical Differences Between Free and Enzyme-Bound States MedChemComm 2014 Michele R. Richards, b Michael G. Brant, a Martin J. Boulanger, c Christopher W. Cairo b and Jeremy E. Wulff* a a Department of Chemistry, University of Victoria, Victoria British Columbia V8W V6, Canada. wulff@uvic.ca. b Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton Alberta T6G 2G2, Canada. c Department of Biochemistry and Microbiology, University of Victoria, Victoria British Columbia V8W V6, Canada. Index Experimental Methods Figure S1: Comparison of Structural Properties for Free and Enzyme-Bound Oseltamivir Figure S2: Comparison of Structural Properties for Free and Enzyme-Bound Zanamivir Figure S: Comparison of Structural Properties for Free and Enzyme-Bound Peramivir Figure S4: Structural Properties for Peramivir Bound to Influenza B Neuraminidase Figure S5: Comparison of Simulated and Experimental 1 H NMR Spectra for Peramivir Figure S6: noe Data for Peramivir Figure S7: Total Energy of MD Simulation of Peramivir Figure S8: Histogram of Peramivir Ring Pucker Table S1: Calculated and Experimental Scalar Couplings of Peramivir Table S2: Cartesian Coordinates of Average Peramivir Structure from MD Simulations Table S: Energy and Cartesian Coordinates for DFT-Optimized Bound Peramivir Table S4: Energy and Cartesian Coordinates for DFT-Optimized Unbound Peramivir References for the Supplementary Information: S2 S4 S5 S6 S7 S8 S9 S10 S11 S12 S1 S14 S15 S16 S1

2 Experimental Methods General procedures Peramivir was purchased from D-L Chiral Chemicals. Sources of X-ray diffraction data were from the Cambridge Structure Database (CSD) under CCDC ; S1 or the Protein Data Bank (PDB IDs: 2F10, K7, K9, 1L7F, 1L7G, 1L7H, 2HTU, 4MWV, and 4MX0. S2,S,S4,S5 NMR Spectroscopy The 1D solution-state 1 H NMR spectrum for peramivir was obtained in D 2 O at 27 C on a 700 MHz spectrometer equipped with a cryoprobe. A presaturation pulse sequence was used to reduce the intensity of the residual HOD signal (4.78 ppm at 27 C). The 1 H 1 H coupling constants were determined using a combination of first-order analysis and simulation in SpinWorks S6 as described previously. S7 2D NMR spectra, as well as noe experiments, were performed at 500 MHz. For the noe experiments, the D1 relaxation delay was set to 2 s, and the D8 mixing time was set to 0.65 s. Computational methods Molecular dynamics (MD) simulations on peramivir were run using the Generalized Amber Force Field S8 and the PMEMD module of the AMBER 10 software suite. S9 Peramivir was solvated in a box of 79 TIPP S10 water molecules with an initial size of 28.9 x 28.1 x 25.5 Å. Partial charges were calculated using the previously reported approach for restrained electrostatic potential (RESP) charge fitting, S11,S12,S1 and all aliphatic hydrogens were assigned a charge of zero. The Generalized Amber Force Field did not contain the appropriate dihedral or improper torsion terms for the guanidine group, so these parameters were taken from the ff99sb force field. S14 Prior to the production phase, peramivir was equilibrated in a four-step process. First, the water molecules were minimized while holding peramivir fixed, then the entire system was minimized. For both rounds, 50 steps of steepest decent were followed by 950 step of conjugant gradient minimization. The system was then subjected to simulated annealing, heating from 5 K to 00 K over 50 ps and cooling back to 5 K over 50 ps. Final equilibration was run heating the system from 5 K to 00 K over 100 ps, then running at a constant 00 K for an additional 100 ps. Following equilibration, production dynamics were run for 250 ns at 00 K. The temperature was held constant using the Berendsen thermostat S15 and rescaled every 1 ps. Pressure was held constant at 1 atm. All bonds containing hydrogen were fixed using the SHAKE algorithm. S16 A 2 fs timestep was used for all dynamics steps, and the cutoff for nonbonding interactions was 8 Å. Nonbonded and electrostatic interactions were unscaled. S2

3 For the average coupling constants, 200 conformations were extracted from the MD simulation of peramivir. The Fermi contact term was then calculated for each conformation using BLYP/6-1G(d,p) u+1s S17,S18,S19 in Gaussian0, S20 following the procedure of Bally and Rablen. S21 The resulting J H,H values were averaged over all 200 conformations. S

4 Figure S1: Comparison of Structural Properties for Free and Enzyme-Bound Oseltamivir Figure S1. Comparison of structural properties for free and enzyme-bound oseltamivir, revealing a conserved geometry of the central ring. A: Oseltamivir carboxylate bound in the enzyme active site of H1N1 neuraminidase (from PDB TI6). S22 B: Omit map for the bound ligand of PDB TI6, confirming the quality of the data for the bound ligand. C and D: Top- and side-views of the bound ligand from Figure S1A, showing the twist-boat conformation of the central ring. Colored arrows on the structural drawing correspond to the functional groups in Figure 1. Positions of hydrogen atoms are calculated based on the positions of the other atoms. E: Small-molecule X-ray data for oseltamivir (as the ethyl ester prodrug) from reference S2. Exocyclic bonds are rotated somewhat with respect to the enzyme-bound structure, but the geometry of the central core is largely preserved in the absence of binding. S4

5 Figure S2: Comparison of Structural Properties for Free and Enzyme-Bound Zanamivir Figure S2. Comparison of structural properties for free and enzyme-bound zanamivir, revealing a conserved geometry of the central ring. A: Zanamivir bound in the enzyme active site of H1N1 neuraminidase (from PDB B7E). S24 B: Omit map for the bound ligand of PDB B7E, confirming the quality of the data for the bound ligand. C and D: Top- and side-views of the bound ligand from Figure S2A, showing the twist-boat conformation of the central ring. Colored arrows on the structural drawing correspond to the functional groups in Figure 1. Positions of hydrogen atoms are calculated based on the positions of the other atoms. E: Small-molecule X-ray data for zanamivir (as a dimer) from reference S25. Exocyclic bonds are rotated somewhat with respect to the enzyme-bound structure, but the geometry of the central core is largely preserved in the absence of binding. S5

6 Figure S: Comparison of Structural Properties for Free and Enzyme-Bound Peramivir Figure S. Comparison of structural properties for free and enzyme-bound peramivir, highlighting differences in the geometry of the central ring. A: Peramivir bound in the enzyme active site of H1N9 neuraminidase (from PDB 1L7F). S B: Omit map for the bound ligand of PDB 1L7F, confirming the quality of the data for the bound ligand. C and D: Top- and side-views of the bound ligand from Figure SA, showing the pronounced pseudo-equatorial projections of the carboxylate and guanidinium substituents. Colored arrows on the structural drawing correspond to the functional groups in Figure 1. Positions of hydrogen atoms are calculated based on the positions of the other atoms. E: Small-molecule X-ray data for peramivir from reference S1, showing a substantially different conformation. S6

7 Figure S4: Structural Properties for Peramivir Bound to Influenza B Neuraminidase Figure S4. Structural properties for peramivir bound to influenza B neuraminidase. A: Peramivir bound in the enzyme active site of neuraminidase from influenza B/Perth/211/2001 (from PDB K7). S5 B: Omit map for the bound ligand of PDB K7, confirming the quality of the data for the bound ligand. C and D: Top- and side-views of the bound ligand from Figure S4A, showing a relaxed geometry, relative to that shown in Figure SC and D. Colored arrows on the structural drawing correspond to the functional groups in Figure 1. Positions of hydrogen atoms are calculated based on the positions of the other atoms. E: Overlay of all 18 different bound conformations of peramivir bound to either wild-type B/Perth neuraminidase (from PDB K7) or a D197E mutant (from PDB K9). S5 S7

8 Figure S5: Comparison of Simulated and Experimental 1 H NMR Spectra for Peramivir (D 2 O, 700 MHz) Figure S5. Comparison of simulated and measured 1 H NMR spectra for peramivir. A: The 1 H NMR spectrum for peramivir simulated in SpinWorks. S6 B: The experimental 1 H NMR spectrum for peramivir in D 2 O at 700 MHz. The RMS difference between the two spectra is 0.00 Hz. S8

9 Figure S6: noe Data for Peramivir (D 2 O, 500 MHz) Figure S6. noe data for peramivir in D 2 O at 500 MHz, using a D1 relaxation delay of 2 s, and a D8 mixing time of 0.65 s. Asterisks indicate the signal being irradiated. In each case, the integration of the irradiated signal was set to 100%. S9

10 Figure S7: Total Energy of MD Simulation of Peramivir Figure S7. Total energy plotted over the course of the MD simulation of peramivir. The average energy (red line) is 226 kcal/mol. S10

11 Figure S8: Histogram of Peramivir Ring Pucker Figure S8. Histogram of pseudorotational phase angles, P, from the MD simulation of peramivir. The histogram can be fit to a Gaussian function (blue line), centered at P = 7. S11

12 Table S1: Calculated and Experimental Scalar Couplings of Peramivir Experimental a (Hz) Average from MD simulation (Hz) Average from the X-ray b (Hz) J H1,H ± ± J H1,H5α.9.7 ± ± J H1,H5β ± ± J H2,H ± ± J H2, H5α ± ± J H,H ± ± J H,H ± ± J H,pentyl ± ± J H4,H5α ± ± J H4,H5β ± ± J H5α,H5β ± ± a From the Spinworks S6 simulation (see Figure S5). The error in the experimental values is estimated to be ± 0.2 Hz. b Average across the four conformations visible in the unit cell. S1 c Bound structure from PDB 1L7F. S Bound in H1N9 c (Hz) S12

13 Table S2: Cartesian Coordinates of Average Peramivir Structure from MD Simulations (PDB format) REMARK PDB file generated by ptraj ATOM 1 C1 PER ATOM 2 C PER ATOM O2 PER ATOM 4 O PER ATOM 5 H PER ATOM 6 C4 PER ATOM 7 H4 PER ATOM 8 H5 PER ATOM 9 C5 PER ATOM 10 N1 PER ATOM 11 C6 PER ATOM 12 N2 PER ATOM 1 H6 PER ATOM 14 H7 PER ATOM 15 N PER ATOM 16 H8 PER ATOM 17 H9 PER ATOM 18 H10 PER ATOM 19 H11 PER ATOM 20 C2 PER ATOM 21 O1 PER ATOM 22 H1 PER ATOM 2 H2 PER ATOM 24 C7 PER ATOM 25 H12 PER ATOM 26 C8 PER ATOM 27 H17 PER ATOM 28 C11 PER ATOM 29 C12 PER ATOM 0 C1 PER ATOM 1 H18 PER ATOM 2 H19 PER ATOM H20 PER ATOM 4 H21 PER ATOM 5 H22 PER ATOM 6 H2 PER ATOM 7 C14 PER ATOM 8 C15 PER ATOM 9 H24 PER ATOM 40 H25 PER ATOM 41 H26 PER ATOM 42 H27 PER ATOM 4 H28 PER ATOM 44 N4 PER ATOM 45 H16 PER ATOM 46 C9 PER ATOM 47 C10 PER ATOM 48 H1 PER ATOM 49 H14 PER ATOM 50 H15 PER ATOM 51 O4 PER S1

14 Table S: Energy and Cartesian Coordinates for DFT-Optimized Bound Peramivir (Structure extracted from PDB 2HTU, and populated with hydrogens before minimization) Optimization: DFT/BLYP/6-1G* SCF total energy: hartrees Coordinates: Atom X Y Z C C C C C C C C C C C C O O C C N N C C O O C C N N C C C C C C H H H H H H C C H H H H H H H H H H C C H H H H H H H H C C H H H H H H H H H H H H O O H H H H N N H H H H H H N N H H H H H H H H O O H H S14

15 Table S4: Energy and Cartesian Coordinates for DFT-Optimized Unbound Peramivir (Structure extracted from CCDC ) Optimization: DFT/BLYP/6-1G* SCF total energy: hartrees Coordinates: Atom X Y Z C C N N C C N N C C C C C C C C C C C C C C C C C C C C O O O O H H H H H H H H H H C C H H H H H H C C H H H H H H H H H H C C H H H H H H H H H H H H H H H H H H O O H H H H H H N N N N H H H H O O H H ( H f = 21.2 kj/mol) S15

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