Supplementary Information. Subtly modulating GSK-3: allosteric inhibitors development. and their potential for the treatment of chronic diseases

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1 Supplementary Information Subtly modulating GSK-3: allosteric inhibitors development and their potential for the treatment of chronic diseases Valle Palomo, a,# Daniel I. Perez, a,# Carlos Roca, a Cara Anderson, b Natalia Rodríguez- Muela, c Concepción Perez, d Jose A. Morales-Garcia, e,f Julio A. Reyes, d Nuria E. Campillo, a Ana M. Perez-Castillo, e,f Lee L. Rubin, c LubovTimchenko, b Carmen Gil, a and Ana Martinez, a, * a IPSBB Unit, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, Madrid, Spain b Department of Pediatrics, Division of Neurology, Cincinnati Children's Hospital, Cincinnati, OH 45219, USA c Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA. Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA. d Instituto de Quimica Médica-CSIC, Juan del Cierva 3, Madrid, Spain e Instituto de Investigaciones Biomedicas-CSIC, Arturo Duperier 4, Madrid, Spain f Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. Correspondence to: Ana Martinez CIB-CSIC Ramiro de Maetzu Madrid ana.martinez@csic.es # these authors contribute equally to this work; *to whom correspondence should be addressed S1

2 Contents Table S1. Elemental analysis of new synthesized compounds... pag S3 Figure S1. Chemical structures for compound 1 tautomeric forms A (top) and B (down) at physiological ph, image of their respective optimized geometries and Gibbs energy values pag S5 Figure S2. Docking pose for compounds 32 (top), 63 (middle) and 65 (down). Interactions between ligand and Arg209 are present in the three poses, but the interactions with Ser236 and the hydrophobic region disappears, explaining the loss of inhibitory activity for these quinolines. pag S6 Figure S3. Docking pose for compounds 40 (top left), 39 (top right), 61 (down left), 62 (down right). Quinolines with bigger substituents in R 2 flip the quinoline ring position, but maintain the interactions with Arg209 due to cation-π (compounds 61, 62) pag S7 Figure S4. LogP versus pic50 of the inhibitors. Inactive compounds are coloured red. Active compounds are coloured blue.. pag S8 Figure S5. RMSD values of backbone atoms in the three trajectories studied: GSK3β without ligand (top), GSK3β complexed with compound 1 (middle) and GSK3β complexed with compound 63 (down). GSK3β natural movement is restricted by the presence of compound 1 due to the decrease of the backbone movement, but compound 63 does not restrict the GSK3β movement... pag S9 Figure S6. Root Mean Square Fluctuation profile for the trajectory of GSK-3β (black) against the complex GSK-3β-Comp 1. This graphic shows different behavior of Arg209, with less movement, due to the interaction of compound 1... pag S10 Figure S7. Distance between atom NZ of Lys205 and atom OE1 of Glu211 along the three molecular dynamics trajectories. As normal behavior, distance between these atoms oscillates between 5 Å and 7Å, due to the flexibility of both residues. When compound 1, interacts with the target, the distance decrease, allowing the hydrogen bond interaction along the half of the simulation. Compound 63 does no modify the behavior of these residues.. pag S11 S2

3 Table S1. Elemental analysis of new synthesized compounds Compound Molecular Formula Calculated Found %C %H %N %C %H %N 1 C 24 H 35 N 3 O C 12 H 13 NO C 10 H 8 BrNO C 10 H 8 ClNO C 16 H 19 NO C 17 H 19 NO C 19 H 17 NO C 14 H 14 ClNO C 13 H 12 BrNO C 14 H 14 BrNO C 13 H 12 ClNO C 14 H 14 ClNO C 19 H 16 ClNO C 22 H 31 N 3 O C 18 H 23 N 3 O C 23 H 33 N 3 O C 19 H 25 N 3 O C 27 H 41 N 3 O C 18 H 15 N 3 O C 13 H 13 N 3 O C 19 H 17 N 3 O C 22 H 20 N 4 O C 20 H 27 N 3 O C 23 H 22 N 4 O C 21 H 21 N 3 O C 26 H 39 N 3 O S3

4 39 C 27 H 39 N 3 O C 23 H 31 N 3 O C 26 H 37 N 3 O C 29 H 37 N 3 O C 22 H 30 FN 3 O C 18 H 22 FN 3 O C 22 H 30 ClN 3 O C 23 H 32 ClN 3 O C 19 H 24 ClN 3 O C 24 H 34 ClN 3 O C 20 H 26 ClN 3 O C 22 H 30 BrN 3 O C 23 H 32 BrN 3 O C 19 H 24 BrN 3 O C 24 H 34 BrN 3 O C 20 H 26 BrN 3 O C 22 H 30 ClN 3 O C 18 H 22 ClN 3 O C 23 H 32 ClN 3 O C 19 H 24 ClN 3 O C 24 H 34 ClN 3 O C 20 H 26 ClN 3 O C 29 H 36 ClN 3 O C 25 H 28 ClN 3 O C 17 H 22 N 2 O C 22 H 21 N 2 O C 14 H 14 N 4 O S4

5 Figure S1. Chemical structures for compound 1 tautomeric forms A (top) and B (bottom) at physiological ph, image of their respective optimized geometries and Gibbs energy values. S5

6 Figure S2. Docking pose for compounds 32 (top), 63 (middle) and 65 (bottom). Interactions between ligand and Arg209 are present in the three poses, but the interactions with Ser236 and the hydrophobic region disappears, explaining the loss of inhibitory activity for these quinolines. Arg209 Thr235 Ser236 Arg328 Val240 Pro331 Leu169 Thr330 Arg209 Thr235 Ser236 Arg328 Val240 Pro331 Leu169 Thr330 S6

7 Figure S3. Docking pose for compounds 40 (top left), 39 (top right), 42 (bottom left), 62 (bottom right). Quinolines with bigger substituents in R2 flip the quinoline ring position, but maintain the interactions with Arg209 due to cation-π (compounds 42, 62) Arg209 Thr235 Ser236 Arg328 Val240 Pro331 Thr330 Leu169 S7

8 Figure S4. LogP versus pic50 of the inhibitors. Inactive compounds are coloured red. Active compounds are coloured blue S8

9 Figure S5. RMSD values of backbone atoms in the three trajectories studied: GSK3β without ligand (top), GSK3β complexed with compound 1 (middle) and GSK3β complexed with compound 63 (bottom). GSK3β natural movement is restricted by the presence of compound 1 due to the decrease of the backbone movement, but compound 63 does not restrict the GSK3β movement. GSK3β GSK3β-Comp1 GSK3β-Comp63 S9

10 Figure S6. Root Mean Square Fluctuation profile for the trajectory of GSK-3β (black) against the complex GSK-3β-Comp 1. This graphic shows different behavior of Arg209, with less movement, due to the interaction of compound 1. S10

11 Figure S7. Distance between atom NZ of Lys205 and atom OE1 of Glu211 along the three molecular dynamics trajectories. As normal behavior, distance between these atoms oscillates between 5 Å and 7Å, due to the flexibility of both residues. When compound 1, interacts with the target, the distance decreases, allowing the hydrogen bond interaction around 12nsof the simulation. Compound 63 does no modify the behavior of these residues. S11