More powerful virus inhibitors from structure-based analysis of

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1 More powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules Luigi De Colibus, Xiangxi Wang, John A. B. Spyrou, James Kelly, Jingshan Ren, Jonathan Grimes, Gerhard Puerstinger, Nicola Stonehouse, Thomas S. Walter, Zhongyu Hu, Junzhi Wang, Xuemei Li, Wei Peng, David Rowlands, Elizabeth E. Fry, Zihe Rao, David I. Stuart. Contents 1. Supplementary Figure 1 2. Supplementary Figure 2 3. Supplementary Figure 3 4. Supplementary Figure 4 5. Supplementary Figure 5 6. Supplementary Figure 6 7. Supplementary Table 1 8. Supplementary Table 2 9. Supplementary Table Supplementary notes 11. Supplementary references

2 1. Supplementary Figures Supplementary Figure 1. Comparison of complexes of EV71 with sphingosine and GPP2. Sphingosine (blue) - EV71 VP1 (blue) (PDBID: 3VBF), superimposed on GPP2 (magenta) - EV71 VP1 (cyan). The RMS difference between all Cα atoms of the icosahedral protomer is 0.4 Å.

3 a 100 b -1 ) -75 Gold Fitness ALD NLD R 2 =0.60 Interaction Energy (kcal mol NLD ALD R 2 = c -22 NLD -20 pic 50 d -45 pic 50 NLD interface_delta ALD R 2 =0.29 G of binding ALD R 2 = pic50 50 pic 50 Supplementary Figure 2. Predicted binding affinity (GOLD score or ΔG) vs. experimental pic 50 values (-log(ic 50 )). The compounds are the 3-(-4-Pyridyl)-2- imidazolidinone derivatives listed in Supplementary Table 1. The calculated points for ALD and NLD are in red whilst the experimental values are reported in yellow on the plot. Plot (a) shows results for GOLD (with GOLD fitness scores), (b) for Glide (energy of interaction expressed as van der Waals plus electrostatic interactions) (c) for Rosetta (interface_delta), (d) GOLD/AMBER (ΔG of binding). For details of the protocols see the methods section of the main text. The ligand 42-8e reported in the Supplementary Table1 could not be docked into the crystal structure using Glide. The following ligands: 2-d-21, 10-26, 4-24 reported in the Supplementary Table 1 were excluded from the Rosetta plot. They represent outlier points, because these molecules were partially docked in the binding pocket resulting in spuriously large negative values of interface_delta.

4 Supplementary Figure 3. Stereo diagrams comparing the results of the various docking protocols for GPP2. The side chain of Ile113 is not shown, to reveal the

5 hydrogen bond, drawn as dotted lines, between the main chain nitrogen of this residue and the ligand oxygen. Residues from VP1 are shown in blue and Ile24 from VP3 in orange. (a) Overlay of the docked ligand GPP2 (light green) by QMPLD method and the structurally determined conformation of GPP2 (magenta) (RMSD for all inhibitor atoms 1.3 Å). (b) Overlay of the docked ligand GPP2 by GOLD (violet) and the structurally determined conformation of GPP2 (magenta) (RMSD for all inhibitor atoms 0.9 Å). (c) Overlay of the docked ligand GPP2 by Rosetta (gray) and the structurally determined conformation of GPP2 (magenta) (note GPP2 is docked upside down).

6 Supplementary Figure 4. Docking of GPP3 and NLD molecules in HEV71 crystal structures. (a) Docking of GPP3 by QMPLD into the 2.7 Å crystal structure reported by Plevka et al (reference 15 in main text, PDBID:3ZFE). In cyan the sphingosine molecule found in the 3ZFE crystal structure and in grey GPP3 docked by QMPLD. The dashed line represents the hydrogen bond between GPP3 and Ile113 of VP1. (b) In magenta the GPP3 molecule found in the crystal structure and in grey GPP3 molecule docked by QMPLD. The dashed line represents the hydrogen bond between GPP3 and Ile113 of VP1. The residue Phe135 is at the front whereas residue Phe155 is towards the back of the figure. Residue Ile24 of VP3 is coloured orange. The RMSD between the two virus structures (all atoms) is 0.8 Å, whilst the RMSD between the two GPP3 positions is 1.6 Å. (c) Docking of GPP3 by QMPLD into the 3.7 Å crystal structure reported by Plevka et al (reference 14 in main text, PDBID:4AED) with the experimentally observed conformation. In gray GPP3 molecule docked by QMPLD into the 4AED crystal structure and in magenta

7 the experimentally observed conformation of GPP3 (obtained by superposition of our complex onto the 4AED structure). (d) In grey the protonated form of the GPP3 molecule docked by QMPLD into the 4AED crystal structure and in magenta the experimentally observed conformation of GPP3. The dashed line represents the hydrogen bond established by GPP3 and residue Ile113 of VP1. Residue Phe135 is at the front whereas residue Phe155 is towards the back of the figure. The RMSD between the two virus structures (4AED and our complex, all atoms) is 1.1 Å, whilst the RMSD between the two GPP3 positions shown in (c) is 13.3 Å (GPP3 is docked upside down) and that between the two GPP3 positions in shown in (d) is 1.8 Å. (e) Docking of NLD by QMPLD into the 2.7 Å crystal structure reported by Plevka et al (reference 15 in main text, PDBID:3ZFE) with the experimentally observed conformation. In cyan the sphingosine molecule found in the 3ZFE crystal structure and in grey NLD molecule docked by QMPLD. The dashed line represents the hydrogen bond established by GPP3 and residue Ile113 of VP1. (f) In magenta the NLD molecule found in the crystal structure and in grey NLD docked by QMPLD. The dashed line is the hydrogen bond between NLD and Ile113 and Gln202 of VP1. Phe135 is towards the front and Phe155 towards the back of the figure. Residue Ile24 of VP3 is coloured orange. The RMSD between the two virus structures (all atoms) is 0.8 Å, whilst the RMSD between the two NLD positions is 1.6 Å. a

8 b Supplementary Figure 5. Mass spectrum of ALD and NLD compounds. (a) Upper panel, experimental peaks with the associated molecular mass values. The main species in the sample has a measured molecular mass of m/z corresponding to the introduction of an amide group on the pyridine moiety of 3-(4- pyridyl)-2-imidazolidinone. The lower panel shows the theoretical molecular mass for this compound. (b) Upper panel shows the experimental peaks with the associated molecular mass values. The main specie in the sample has a measured molecular mass

9 of m/z corresponding to the introduction of an amine group on the pyridine moiety of 3-(4-pyridyl)-2-imidazolidinone. The lower panel shows the theoretical molecular mass for this compound.

10 Supplementary Figure 6. Structural comparison of key residues involved in forming a hydrophobic trap for pocket binders. Structurally equivalent residues - within VP1 pocket of EV71 (blue), Poliovirus type 2 (yellow, PDBID:1EAH) and rhinovirus 14 (orange, PDBID:1NA1). These residues are critical for positioning the compounds within the pocket.

11 7. Supplementary Tables Compound -log(ic 50) 28b a b b d a a c c d f d b i h e 4.77 Supplementary Table 1. List of compounds used in evaluation docking methods. This set of HEV71 inhibitors was assembled merging the list of molecules reported in Ke et al. (2006), Shia et al. (2002) and Chang et al. (2002).

12 Supplementary Table 2. ADME-tox values calculated with QikProp v3.6 for GPP3, ALD and NLD. GPP3 ALD NLD Predicted brain/blood partition coefficient ( ) Predicted apparent Caco-2 cell a permeability in nm/sec (<25 poor, >500 great) Predicted apparent MDCK cell b permeability in nm/sec (<25 poor, >500 great) Predicted skin permeability, log Kp (Kp in cm/h) Lipinski Rule of 5 violations (maximum is 5) Jorgensen Rule of 3 Violations (maximum is 3) % Human Oral Absorption (<25 % is poor) Predicted qualitative human oral absorption (> 80% is high) High Low Low HERG K + Channel Blockage, logic50 (concern below -5) of 1712 molecules most similar to: GPP3 ALD NLD Molecule Similarity(%) Molecule Similarity(%) Molecule Similarity(%) Amtolmetin Valsartan Valsartan Bulaquine Cisapride Eprosartan Tribuzone Glisentide Glisentide Nafronyl Suxibuzone Losartan Eprosartan Rosuvastatin Cisapride a Caco-2 cells are a model for the gut-blood barrier. b MDCK cells are considered to be a good mimic for the blood brain Barrier

13 Supplementary Table 3. IC50 of NLD and ALD against various Enteroviruses. Enterovirus Subtypes IC50(pM) NLD ALD HEV71 A HEV71 B HEV71 C CVA16 A CVA16 B

14 SUPPLEMENTARY NOTES Docking methods tested We evaluated several in silico methods for docking and binding affinity calculations GOLD 1 (with and without molecular dynamics), Rosetta Ligand Dock 2 and Glide In GOLD docking 1 calculations, the docking site was based on the position of C5 of GPP2 in the crystal structure (the radius of the cavity was between 10 and 30 Å depending on the size of the compound). High-efficiency docking was performed using the default settings and the GoldScore scoring function. The poses with highest scores were kept. The topology and parameters files for each ligand were generated by the ANTECHAMBER program distributed with AMBER TOOLS 4. All VP1- ligand complexes were subjected to minimization in AMBER11 4. The energyminimized structures were then used as an initial reference for molecular dynamics simulations. Due to the known rigidity of the structure after docking, positional restraints were applied to all Cα atoms. The simulation was carried out at 310K, with constant volume periodic boundary and an integration step of 2fs. Long-range electrostatics were included using the PME (Particle Mesh Ewald) method. Prior to simulation the structures were fully TIP3 solvated, using a 10Å box of explicit water, and Cl - ions were used to neutralize the surface charge. Hydrogen bonds were constrained using SHAKE 5. Each solvated assembly was simulated for 1ns, with a snapshot taken every 10ps. The ΔG of binding was calculated from snapshots using the MMPSA.py 6 program as implemented in AMBER11 package 4. All calculations were performed on a computer cluster consisting 672 cores, made up of two types of nodes, AMD nodes with 2.67 GB/core and intel nodes with 8GB/core. Each MD simulation was run in parallel distributed over 12 cores. It took on average 42h to complete a single MD run (47 being required for a full benchmark set). Docking with Rosetta ligand docking 2 used the position of C5 of GPP2 to define the target position of the ligand. Experiments were performed with the following modification to the default protocol. To search for rotations that maximize the attractive and repulsive score the ligands were randomly rotated 1000 times. The final method tested was that embodied in the Schrödinger software. Docking into the rigid binding pocket of HEV71 was performed with the Glide SP 3 (Glide5.5 standard precision) procedure. For ligand generation and docking parameters see the On Line Methods. For GOLD 1, the GOLD score provides a ranking, and this showed reasonable correlation with the measured IC 50 (CC=0.60, Supplementary Fig. 2). Perhaps surprisingly when the ligand poses were subjected to further molecular dynamics runs to calculate the ΔG binding using AMBER 4, the overall correlation between the IC 50 and ΔG binding was very poor (CC=0.26, Supplementary Fig. 2). A similarly poor level of agreement (CC=0.29, Supplementary Fig. 2) was obtained with Rosetta Ligand Dock 2, which allows ligand flexibility by minimizing ligand torsion angles (Supplementary Fig. 2). Energy values generated by Glide scored better with a correlation coefficient of 0.67 (Supplementary Fig. 2).

15 SUPPLEMENTARY REFERENCES 1 Jones, G., Willett, P., Glen, R. C., Leach, A. R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. Journal of molecular biology 267, (1997). 2 Davis, I. W. & Baker, D. RosettaLigand docking with full ligand and receptor flexibility. Journal of molecular biology 385, (2009). 3 Friesner, R. A. et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of medicinal chemistry 47, (2004). 4 AMBER 11 (University of California, San Francisco, 2010). 5 Ryckaert, J. P., Cicciotti, G. & Berendsen, H. J. C. Numerical integration of the cartesian equations of motion of a system with constraints : Molecular dynamics of n-alkanes. J Comput Phys 23, (1977). 6 Miller, B. R. et al. MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J Chem Theory Comput 8, (2012).