Platinum(II) and ruthenium(II) coordination complexes equipped with an anchoring site for binding the protein kinase enzyme pockets: synthesis, molecular docking and biological assays.

Supplementary Information (SI) for Dalton Transactions. This journal is © The Royal Society of Chemistry 2025 Platinum(II) and Ruthenium(II) Coordination complexes equipped with an anchoring site for binding the protein kinase enzyme pockets: synthesis, molecular docking and biological assays. Matthieu Scarpi-Luttenauer, 1 Katia Galentino,2 Christophe Orvain3,4, Audrey Fluck, 1 Marco Cecchini,2 Georg Mellitzer3,4, Christian Gaiddon,3,5, * Pierre Mobian*1 1 Université de Strasbourg, CNRS, UMR 7140, F-67000 Strasbourg, France 2 Université de Strasbourg, CNRS, UMR 7177, F-67000 Strasbourg, France 3 Inserm U1113 IRFAC, Team STREINTH, Strasbourg, France 4 present address: INSERM, UMR 1260, CRBS, Regenerative Nanomedicine, “GP_SMIT” Laboratory, CRBS; 1 Rue Eugène Boeckel, 67085 Strasbourg, France 5 present address: UMR7242, Biotechnology et Signalisation Cellulaire, group STREINTH, 300 Bld S. Brant, FR-67412 Illkirch Cedex, France Figure S1. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(1) Figure S2. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(1) Figure S3. ESI-MS spectrum + simulated spectrum and close-up of Ru(1) Figure S4. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(2) Figure S5. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(2) Figure S6. ESI-MS spectrum + simulated spectrum and close-up of Ru(2) Figure S7. 1H NMR spectrum (CDCl3, 500 MHz) of Ru(3) Figure S8. 13C NMR spectrum (CDCl3, 126 MHz) of Ru(3) Figure S9. ESI-MS spectrum + simulated spectrum and close-up of Ru(3) Figure S10. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(4) Figure S11. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(4) Figure S12. ESI-MS spectrum + simulated spectrum and close-up of Ru(4) Figure S13. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(5) Figure S14. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(5) Figure S15. ESI-MS spectrum + simulated spectrum and close-up of Ru(5) Figure S16. 1H NMR spectrum (CD3CN, 500 MHz) of Pt(1) Figure S17. 13C NMR spectrum (CD3CN, 126 MHz) of Pt(1) Figure S18. HR-MS spectrum + simulated spectrum and close-up of Pt(1) Figure S19. 1H NMR spectrum (D2O, 500 MHz) of Pt(2) Figure S20. 13C NMR spectrum (D2O, 126 MHz) of Pt(2) Figure S21. HR-MS spectrum + simulated spectrum and close-up of Pt(2) Figure S22. 1H NMR spectrum (MeOD, 500 MHz) of Pt(3) Figure S23. 13C NMR spectrum (MeOD, 126 MHz) of Pt(3) Figure S24. HR-MS spectrum + simulated spectrum and close-up of Pt(3) Figure S25. 1H NMR spectrum (CD3CN, 500 MHz) of Pt(4) Figure S26. 13C NMR spectrum (CD3CN, 126 MHz) of Pt(4) Figure S27. HR-MS spectrum + simulated spectrum and close-up of Pt(4) Figure S28. Hydrolysis kinetics of Ru(1-5) Figure S29. Hydrolysis kinetics of Pt(1-4) Figure S30. HPLC-MS analysis of Ru(3) Figure S31. Re-docking of known MST2 and S6K1 inhibitors Figure S32. Ellipsoid plot of the Ru(2) crystal structure. Figure S33. Ellipsoid plot of the Pt(4) crystal structure. Figure S34. Western Blots Table S1. Analysis of the interaction pattern of the ruthenium and platinum compounds at S6K1 as predicted by docking Table S2. Analysis of the interaction pattern of the ruthenium and platinum compounds at MST2 Antibodies list 1H, 13C NMR spectra and ESI-MS spectra of the synthesized complexes 2+ 2 PF6N N N Ru N N N N H O Figure S1. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(1) Figure S2. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(1) Figure S3. ESI-MS spectrum + simulated spectrum and close-up of Ru(1) 2+ 2 PF6N N N Ru N N N Figure S4. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(2) 2+ 2 PF6N N N Ru N N Figure S5. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(2) N NH O NH O Figure S6. ESI-MS spectrum + simulated spectrum and close-up of Ru(2) + PF6- Me2N N N Ru N N NH O Ru(3) Figure S7. 1H NMR spectrum (CDCl3, 500 MHz) of Ru(3) Figure S8. 13C NMR spectrum (CDCl3, 126 MHz) of Ru(3) Figure S9. ESI-MS spectrum + simulated spectrum and close-up of Ru(3) + PF6N N N Ru N N NH O Ru(4) Figure S10. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(4) Figure S11. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(4) Figure S12. ESI-MS spectrum + simulated spectrum and close-up of Ru(4) + PF6N Ru Cl N NH O Ru(5) Figure S13. 1H NMR spectrum (CD3CN, 500 MHz) of Ru(5) Figure S14. 13C NMR spectrum (CD3CN, 126 MHz) of Ru(5) Figure S15. ESI-MS spectrum + simulated spectrum and close-up of Ru(5) 2+ 2 Cl NH2 N Pt NH2 N NH O Pt(2) Figure S16. 1H NMR spectrum (D2O, 500 MHz) of Pt(1) Figure S17. 13C NMR spectrum (D2O, 126 MHz) of Pt(1) Figure S18. ESI-MS spectrum + simulated spectrum and close-up of Pt(1) O NH2 N Pt NH2 N 2+ 2 Cl - NH Figure S19. 1H NMR spectrum (CD3CN, 500 MHz) of Pt(2) O NH2 N Pt NH2 N 2+ 2 Cl - NH Figure S20. 13C NMR spectrum (CD3CN, 126 MHz) of Pt(2) Figure S21. HR-MS spectrum + simulated spectrum and close-up of Pt(2) 2+ 2 NO3N HN H2N Pt N O H2N Figure S22. 1H NMR spectrum (MeOD, 500 MHz) of Pt(3) 2+ 2 NO3HN O N H2N Pt N H2N Figure S23. 13C NMR spectrum (MeOD, 126 MHz) of Pt(3) Figure S24. HR-MS spectrum + simulated spectrum and close-up of Pt(3) 2+ 2 PF6NH2 N Pt NH N O NH2 Figure S25. 1H NMR spectrum (CD3CN, 500 MHz) of Pt(4) Figure S26. 13C NMR spectrum (CD3CN, 126 MHz) of Pt(4) Figure S27. HR-MS spectrum + simulated spectrum and close-up of Pt(4) 2,0 a) a.u.) Absorbance ((u.a.) 1,6 1,2 0,8 0,4 0,0 300 350 400 450 500 Longueur d'onde (nm) 550 600 Wavelength (nm) 1,0 2,0 b) c) 0,8 (a.u.) Absorbance (u.a.) (a.u.) Absorbance (u.a.) 1,6 1,2 0,8 0,6 0,4 0,2 0,4 0,0 300 350 400 450 500 550 0,0 250 600 300 350 Longueur d'onde (nm) Wavelength (nm) d) e) 2,00 400 450 500 Longueur d'onde Wavelength (nm)(nm) 550 600 350 400 450 Longueur d'onde(nm) (nm) Wavelength 550 600 2,0 1,75 1,6 1,25 (a.u.) Absorbance (u.a.) (a.u.) Absorbance (u.a.) 1,50 1,00 0,75 0,50 1,2 0,8 0,4 0,25 0,00 250 300 350 400 450 500 550 Longueur d'onde (nm) Wavelength (nm) 600 0,0 250 300 500 Figure S28: Hydrolysis kinetics of Ru(1-5) measured by UV-Vis absorption in a PBS buffer for 24 h (one spectrum every hour). A slight drop in intensity was observed for the analyzed complexes, which was explained by the medium evaporation. a) Ru(1), b) Ru(2), c) Ru(3), d) Ru(4), e) Ru(5). 0,40 a) 1,0 b) 0,35 0,8 (a.u.) Absorbance (u.a.) Absorbance (a.u.) (u.a.) 0,30 0,25 0,20 0,15 0,10 0,4 0,2 0,05 0,00 200 0,6 250 300 350 400 450 0,0 500 250 Wavelength (nm) Longueur d'onde (nm) 450 500 550 600 1,6 0,4 Absorbance (a.u.) (u.a.) (a.u.) Absorbance (u.a.) 400 2,0 d) 0,5 0,3 0,2 1,2 0,8 0,4 0,1 0,0 250 350 Wavelength (nm) Longueur d'onde (nm) 0,6 c) 300 0,0 300 350 400 450 500 550 600 250 Wavelength (nm) Longueur d'onde (nm) 300 350 400 450 500 Longueur d'onde (nm) Wavelength (nm) 3,0 e) 2,5 Figure S29: Hydrolysis kinetics of Pt(1-4) measured by UV-Vis absorption in a PBS 2,0 Absorbance (a.u.) (u.a.) buffer for 24 h (one spectrum every hour). A slight drop in intensity was observed for 1,5 the analyzed complexes, which was explained by the medium evaporation. a) Pt(1), b) Pt(2), c) Pt(3), d) Pt(4).1,0 0,5 0,0 250 300 350 400 Longueur d'onde (nm) Wavelength (nm) 450 500 3.16 65000 60000 55000 50000 3DFIELD 45000 40000 35000 30000 4.12 3.87 25000 4.54 4.70 4.86 5.00 5.15 5.27 5.34 5.60 7.02 6.67 6.88 5.96 6.23 6.36 6.49 7.20 7.34 7.51 7.59 7.78 8.40 8.07 8.24 8.52 8.68 8.84 8.99 NL: 6.79E4 Spectrum Maximum nm=190.0-802.0 PDA RuIII_10uM_vinj50 uL_Met2_withPos MS 3.95 20000 15000 10000 5000 a) 0 NL: 2.81E4 3DFIELD PDA RuIII_10uM_vinj50 uL_Met2_withPos MS 3.16 25000 20000 3DFIELD 3.87 4.11 15000 4.70 4.97 5.09 10000 3.61 4.40 3.95 5.22 5.33 5.60 4.53 5.67 5.76 5.95 6.18 6.26 6.44 6.59 5000 0 b) NL: 2.44E9 TIC MS RuIII_10uM_vinj50 uL_Met2_withPos MS 3.18 3.20 100 90 Relative Abundance 80 70 60 50 1.07 3.25 40 30 1.15 1.20 1.56 1.57 1.79 3.14 2.48 3.26 3.60 3.62 3.90 4.15 4.20 4.67 4.68 5.06 5.29 5.33 5.36 5.63 5.85 5.96 20 10 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time (min) 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Figure S30: a) UV-Vis (260 nm) of the HPLC analysis of Ru(3) after 24 h in PBS. b) Total Ion Chromatogram. a) b) Figure S31: Panel A. re-docking of 72B in S6K1 (PDB code: 4rlp.pdb). In cyan is depicted the co-crystallized ligand and in orange the re-docked molecule. In green are highlighted the hydrophobic interactions and in yellow the hydrogen bonds. The docking score of this binding mode is -97.17. 72B in this binding pose establishes the same 3 H-bonds (with LEU75, GLU73 and LYS15) and hydrophobic interactions (with THR135, LEU13 and VAL21) as the reference in the crystal structure. Panel B. redocking of 5BS in MST2 (PDB code: 5dh3.pdb). In blue is depicted the co-crystallized ligand and in magenta the re-docked molecule. In green are highlighted the hydrophobic interactions and in yellow the hydrogen bonds. The docking score of this binding mode is -76.72. In this binding pose 5BS establishes both the same H-bonds (with CYS102, LYS298 and ASP109) and hydrophobic interactions (with LEU33 and TYR101) as the reference in the crystal structure. Datablock e4820a_a - ellipsoid plot Figure S32: Ellipsoid plot of the Ru(2) crystal structure. Datablock en_a_sq - ellipsoid plot Figure S33: Ellipsoid plot of the Pt(4) crystal structure. pS6 p-S235/236 Ctrl Ru(3) 2 μM Ru(5) 25 μM 2 μM Pt(3) 25 μM 2 μM Pt(4) 25 μM 2 μM 25 μM pS6 p-S235/236 2 Ctrl Ru(3) 2 μM Ru(5) 25 μM 2 μM Pt(3) 25 μM 2 μM Pt(4) 25 μM 2 μM 25 μM pS6 p-S235/236 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM pYAP p-S127 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM pYAP p-S127 2 Ctrl Ru(3) 2 μM Ru(5) 25 μM 2 μM Pt(3) 25 μM 2 μM Pt(4) 25 μM 2 μM pYAP p-S127 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM 25 μM S6 2 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM S6 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM Pt(4) 2 μM 25 μM YAP 2 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM Pt(3) 25 μM 2 μM Pt(4) 25 μM 2 μM 25 μM YAP 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM Actin from S6 2 Ctrl Ru(3) 2 μM Ru(5) 25 μM 2 μM Pt(3) 25 μM 2 μM Pt(4) 25 μM 2 μM 25 μM Actin from S6 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM 25 μM Pt(4) 2 μM 25 μM Actin from YAP 2 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM Pt(4) 25 μM 2 μM 25 μM Actin from YAP 3 Ctrl Ru(3) 2 μM 25 μM Ru(5) 2 μM 25 μM Pt(3) 2 μM Pt(4) 25 μM 2 μM 25 μM Figure S34: Western Blots Residue Residue Interaction (PDB: (this paper) type Pt(3) Pt(4) Ru(3) Ru(5) 4RLP) E173/O GLU-73 H bond - - - - L175/N LEU-75 H bond ✓ - - - K99/O LYS-99 H bond - - - - Y174/OH TYR74 H bond - ✓ - - L97 LEU-13 VdW ✓ ✓ - - V105 VAL-21 VdW ✓ - ✓ ✓ A121 ALA-37 VdW ✓ ✓ - ✓ T235 THR-135 VdW ✓ - ✓ ✓ Table S1. Analysis of the interaction pattern of the ruthenium and platinum compounds at S6K1 as predicted by docking relative to the reference compounds FL772 solved in complex with the protein (PDB:4RLP). The interactions were analyzed using the software PLIP.42 Green checks highlight when the interaction was detected in the lowest-energy binding mode by docking. Residue Residue Interaction (PDB: (this paper) type Pt(3) Pt(4) Ru(3) Ru(5) C102/O CYS-102 H bond ✓ ✓ - - C102/N CYS-102 H bond ✓ - - - D109/OD1 ASP-109 H bond ✓ - - - L33 LEU-33 VdW ✓ ✓ - ✓ V41 VAL-41 VdW - - - ✓ Y101 TYR-101 VdW - - - ✓ 5DH3) L153 LEU-153 VdW ✓ - ✓ ✓ Table S2. Analysis of the interaction pattern of the ruthenium and platinum compounds at MST2 as predicted by docking relative to the reference compounds XMU-MP-1 solved in complex with the protein (PDB:5DH3). The interactions were analyzed using the software PLIP.42 Green checks highlight when the interaction was detected in the lowest-energy binding mode by docking. Antibodies List β-Actin (anti-mouse) from Merck Millipore, anti-mouse (7076), anti-rabbit (7074), pS235/236-S6 Ribosomal Protein (anti-rabbit, D57.2.2E), S6 Ribosomal Protein (antirabbit, 5G10) and pS127-YAP (anti-rabbit, S127-D9W2I) from Cell Signaling technology and YAP (anti-mouse, sc-101190) from Santa Cruz Biotechnology and all antibodies were used at 1:1000 dilutions except for pS6 (1:2000), actin (1:15000), antimouse (1:2000), and anti-rabbit (1:10000).