Supporting Information Selective DYRK1A inhibitor for the treatment of Type 1 Diabetes: Discovery of 6-azaindole derivative GNF2133 Yahu A. Liu*, Qihui Jin, Yefen Zou, Qiang Ding, Shanshan Yan, Zhicheng Wang, Xueshi Hao, Bao Nguyen, Xiaoyue Zhang, Jianfeng Pan, Tingting Mo, Kate Jacobsen, Thanh Lam, Tom Y.-H. Wu, H. Michael Petrassi, Badry Bursulaya, Michael DiDonato, W. Perry Gordon, Bo Liu, Janine Baaten, Robert Hill, Vân Nguyen-Tran, Minhua Qiu, You-Qing Zhang, Anwesh Kamireddy, Sheryll Espinola, Lisa Deaton, Sukwon Ha, George Harb, Yong Jia, Jing Li, Weijun Shen, Andrew M. Schumacher, Karyn Colman, Richard Glynne, Shifeng Pan, Peter McNamara, Bryan Laffitte, Shelly Meeusen, Valentina Molteni, Jon Loren* Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, California 92121, United States E-mail: yliu2@gnf.org; Phone: +01 858-812-1838 E-mail: jloren@gnf.org; Phone: +01 858-332-4439 S1
Table of Contents Part I 1 H, 13 C and 2D NMR of the compounds in Experimental Section S5-S30 Figure S1. 1 H NMR of 2 in DMSO-d 6 (500 MHz) Figure S2. 1 H NMR of 3e in DMSO-d 6 (500 MHz) Figure S3. 1 H NMR of 5e in DMSO-d 6 (500 MHz) Figure S4. 1 H NMR of 5f in DMSO-d 6 (400 MHz) Figure S5. HMQC NMR of 5f in DMSO-d 6 (400 MHz) Figure S6. COSY NMR of 5f in DMSO-d 6 (400 MHz) Figure S7. NOESY NMR of 5f in DMSO-d 6 (400 MHz) Figure S8. 1 H NMR of 5g in DMSO-d 6 (500 MHz) Figure S9. 1 H NMR of 6 in DMSO-d 6 (500 MHz) Figure S10. 1 H NMR of 7 in DMSO-d 6 (400 MHz) Figure S11. 1 H NMR of 8a in DMSO-d 6 (400 MHz) Figure S12. 1 H NMR of 8b in MeOH-d 4 (400 MHz) Figure S13. 1 H NMR of 8c in DMSO-d 6 (600 MHz) Figure S14. 1 H NMR of 8d in MeOH-d 4 (400 MHz) Figure S15. 1 H NMR of 8e in DMSO-d 6 (400 MHz) Figure S16. 1 H NMR of 8f, in MeOH-d 4 (400 MHz) Figure S17. NMR analysis of 8f in DMSO-d 6 (600 MHz) Figure S18. 1 H NMR of 8f in DMSO-d 6 (600 MHz) Figure S19. HMQC of 8f in DMSO-d 6 (600 MHz) Figure S20. COSY of 8f in DMSO-d 6 (600 MHz) Figure S21. HMBC of 8f in DMSO-d 6 (600 MHz) Figure S22. ROESY of 8f in DMSO-d 6 (600 MHz) Figure S23. NMR analysis of 8f in DMSO-d 6 (600 MHz) Figure S24. 1 H NMR of 8f in DMSO-d 6 (600 MHz) Figure S25. HMQC of 8f in DMSO-d 6 (600 MHz) Figure S26. COSY of 8f in DMSO-d 6 (600 MHz) Figure S27. HMBC of 8f in DMSO-d 6 (600 MHz) Figure S28. ROESY of 8f in DMSO-d 6 (600 MHz) Figure S29. 1 H NMR of 8g in DMSO-d 6 (400 MHz) Figure S30. 1 H NMR of 9e in DMSO-d 6 (500 MHz) Figure S31. 1 H NMR of 10e in MeOH-d 4 (400 MHz) Figure S32. 1 H NMR of 10g in DMSO-d 6 (400 MHz) Figure S33. 1 H NMR of 11a in MeOH-d 4 (400 MHz) Figure S34. 1 H NMR of 12e in DMSO-d 6 (500 MHz) Figure S35. 1 H NMR of 12g in DMSO-d 6 (500 MHz) S2 S5 S5 S6 S6 S7 S8 S9 S10 S11 S11 S12 S12 S13 S13 S14 S14 S15 S15 S16 S16 S17 S17 S18 S18 S19 S19 S20 S20 S21 S21 S22 S22 S23 S23 S24
Figure S36. 1 H NMR of 13a in DMSO-d 6 (500 MHz) Figure S37. 13 C NMR of 13a in DMSO-d 6 (125 MHz) Figure S38. 1 H NMR of 13b in DMSO-d 6 (400 MHz) Figure S39. 1 H NMR of 13c in MeOH-d 4 (400 MHz) Figure S40. 1 H NMR of 13d in DMSO-d 6 (400 MHz) Figure S41. 13 C NMR of 13d in DMSO-d 6 (125 MHz)) Figure S42. 1 H NMR of 13e in DMSO-d 6 (400 MHz) Figure S43. 13 C NMR of 13e in DMSO-d 6 (125 MHz)) Figure S44. 1 H NMR of 13f in DMSO-d 6 (600 MHz) Figure S45. 13 C NMR of 13f in DMSO-d 6 (125 MHz) Figure S46. 1 H NMR of 13g in DMSO-d 6 (400 MHz) Figure S47. 13 C NMR of 13g in DMSO-d 6 (100 MHz) Figure S48. 1 H NMR of 13g (HCl salt) in DMSO-d 6 (400 MHz) S24 S25 S25 S26 S26 S27 S27 S28 S28 S29 S29 S30 S30 Part II Structure-activity relationship (SAR) of the core framework S31-S45 Table S1. Structure-activity relationship (SAR) of the core framework Table S2. Analytical data of the compounds listed in Table S1 S31 S33 Figure S49-S70. 1 H, 13 C and 2D NMR of the compounds in Table S1 S35-S45 Figure S49. 1 H NMR of 8h in DMSO-d 6 (600 MHz) Figure S50. 1 H NMR of 9g in DMSO-d 6 (400 MHz) Figure S51. 1 H NMR of 13h in MeOH-d 4 (500 MHz) Figure S52. 1 H NMR of 14 in CDCl 3 (400 MHz) Figure S53. 1 H NMR of 15 in MeOH-d 4 (400 MHz) Figure S54. 1 H NMR of 16 in MeOH-d 4 (400 MHz) Figure S55. 1 H NMR of 17 in CDCl 3 (400 MHz) Figure S56. 1 H NMR of 17 in DMSO-d 6 (600 MHz) Figure S57. 13 C NMR of 17 in DMSO-d 6 (600 MHz) Figure S58. HMQC NMR of 17 in DMSO-d 6 (600 MHz) Figure S59. COSY NMR of 17 in DMSO-d 6 (600 MHz) Figure S60. HMBC NMR of 17 in DMSO-d 6 (600 MHz) Figure S61. ROESY NMR of 17 in DMSO-d 6 (600 MHz) Figure S62. 1 H NMR of 22 in DMSO-d 6 (400 MHz) Figure S63. 1 H NMR of 23 (TFA salt) in MeOH-d 4 (400 MHz) Figure S64. 1 H NMR of 24 in CDCl 3 (400 MHz0 Figure S65. 1 H NMR of 25 in MeOH-d 4 (400 MHz) Figure S66. 1 H NMR of 26 (TFA salt) in DMSO-d 6 (400 MHz) Figure S67. 1 H NMR of 27 in DMSO-d 6 (400 MHz) S35 S35 S36 S36 S37 S37 S38 S38 S39 S39 S40 S40 S41 S41 S42 S42 S43 S43 S44 S3
Figure S68. 1 H NMR of 28 in MeOH-d 4 (400 MHz) Figure S69. 1 H NMR of 29 in MeOH-d 4 (400 MHz) Figure S70. 1 H NMR of 30 in MeOH-d 4 (400 MHz) S44 S45 S45 Part III Images and dose repsonse of rat and human cell proliferation S46-S47 Figure S71. Images of rat and human primary β-cell replication Figure S72. Dose response of rat and human β-cell proliferation S46 S47 Part IV X-ray co-crystal structure of DYRK1A and GNF2133 S48-S50 Protein expression and purification S48 Crystallization and data collection S48 Structure Determination and refinement S48 Figure S73. Resolution of X-ray co-crystal structure S49 Table S3. Crystallographic data and refinement statistics S50 Part V Two-week in vivo safety study of GNF2133 S51 Part VI Kinase inhibition profiling and Ba/F3 cellular profiling of GNF2133 S52-S56 Table S4. GNF2133 in enzymatic cell free Caliper microfluidic kinase assay S52 Table S5. GNF2133 in Ba/F3 cellular kinase Profiling S56 References S57 S4
Part I. 1 H, 13 C and 2D NMR of the compounds in Experimental Section Figure S1. 1 H NMR of 2 in DMSO-d6 (500 MHz) Figure S2. 1 H NMR of 3e in DMSO-d6 (500 MHz) S5
Figure S3. 1 H NMR of 5e in DMSO-d6 (500 MHz) Figure S4. 1 H NMR of 5f in DMSO-d6 (400 MHz) S6
Figure S5. HMQC NMR of 5f in DMSO-d6 (400 MHz) S7
Figure S6. COSY NMR of 5f in DMSO-d6 (400 MHz) S8
Figure S7. NOESY NMR of 5f in DMSO-d6 (400 MHz) (continued on next page) S9
(continued from previous page) Figure S7. NOESY NMR of 5f in DMSO-d6 (400 MHz) Figure S8. 1 H NMR of 5g in DMSO-d6 (500 MHz) S10
Figure S9. 1 H NMR of 6 in DMSO-d6 (500 MHz) Figure S10. 1 H NMR of 7 in DMSO-d6 (400 MHz) S11
Figure S11. 1 H NMR of 8a in DMSO-d6 (400 MHz) Figure S12. 1 H NMR of 8b in DMSO-d6 (400 MHz) S12
Figure S13. 1 H NMR of 8c in DMSO-d6 (600 MHz) Figure S14. 1 H NMR of 8d in MeOH-d4 (400 MHz) S13
Figure S15. 1 H NMR of 8e in DMSO-d6 (400 MHz) Figure S16. 1 H NMR of 8f in MeOH-d4 (400 MHz) S14
Figure S17. NMR analysis of 8f in DMSO-d6 (600 MHz) Figure S18. 1 H NMR of 8f in DMSO-d6 (600 MHz) S15
Figure S19. HMQC of 8f in DMSO-d6 (600 MHz) Figure S20. COSY of 8f in DMSO-d6 (600 MHz) S16
Figure S21. HMBC of 8f in DMSO-d6 (600 MHz) Figure S22. ROESY of 8f in DMSO-d6 (600 MHz) S17
Figure S23. NMR analysis of 8f in DMSO-d6 (600 MHz) Figure S24. 1 H NMR of 8f in DMSO-d6 (600 MHz) S18
Figure S25. HMQC of 8f in DMSO-d6 (600 MHz) Figure S26. COSY of 8f in DMSO-d6 (600 MHz) S19
Figure S27. HMBC of 8f in DMSO-d6 (600 MHz) Figure S28. ROESY of 8f in DMSO-d6 (600 MHz) S20
Figure S29. 1 H NMR of 8g in DMSO-d6 (400 MHz) Figure S30. 1 H NMR of 9e in DMSO-d6 (500 MHz) S21
Figure S31. 1 H NMR of 10e in DMSO-d6 (400 MHz) Figure S32. 1 H NMR of 10g in DMSO-d6 (400 MHz) S22
Figure S33. 1 H NMR of 11a in MeOH-d4 (400 MHz) Figure S34. 1 H NMR of 12e in DMSO-d6 (500 MHz) S23
Figure S35. 1 H NMR of 12g in DMSO-d6 (500 MHz) Figure S36. 1 H NMR of 13a in DMSO-d6 (500 MHz) S24
Figure S37. 13 C NMR of 13a in DMSO-d6 (125 MHz) Figure S38. 1 H NMR of 13b in DMSO-d6 (400 MHz) S25
Figure S39. 1 H NMR of 13c MeOH-d4 (400 MHz) Figure S40. 1 H NMR of 13d in DMSO-d6 (400 MHz) S26
Figure S41. 13 C NMR of 13d in DMSO-d6 (125 MHz) Figure S42. 1 H NMR of 13e in DMSO-d6 (400 MHz) S27
Figure S43. 13 C NMR of 13e in DMSO-d6 (125 MHz) Figure S44. 1 H NMR of 13f in DMSO-d6 (600 MHz) S28
Figure S45. 13 C NMR of 13f in DMSO-d6 (125 MHz) Figure S46. 1 H NMR of 13g in DMSO-d6 (400 MHz) S29
Figure S47. 13 C NMR of 13g in DMSO-d6 (100 MHz) Figure S48. 1 H NMR of 13g (HCl salt) in DMSO-d6 (400 MHz) S30
Part II. Structure-activity relationship (SAR) of the core framework Table S1. Structure-activity relationship (SAR) of the core framework compound R 1 R 2 J L M Q T X Y Z a IC 50 (µm) DYRK1A GSK3 IC 50 a (µm) 8h CH CH N CH C N CH C 0.382 9.8 14 N CH N CH C N CH C 17.03 >50 8g CH CH N CH C N CH C 0.013 >8.3 15 CH CH N CH N C N C 33.84 >50 16 CH CH N CH C N CH C >16 >50 8a CH CH N CH C N CH C 0.043 >8.3 17 CH CH N CH C N N C 0.361 27.13 18 CH C-Cl CH CH C N N C >50 >50 19 CH C-OMe CH CH C N N C 3.598 >50 20 CH C-Cl CH N C N N C >14 >50 21 CH C-OMe CH N C N N C >50 >50 (continued on next page) S31
Table S1. (continued) compound R 1 R 2 J L M Q T X Y Z a IC 50 (µm) DYRK1A GSK3 IC 50 a (µm) 9g CH CH N CH C N CH C 0.0218 >50 22 CH CH N CH C N C=O N >50 >50 23 CH C-OH N CH C N CH C 14.72 >50 24 CH C-Cl N CH C N CH C 4.85 4.17 25 CH C-OMe N CH C N CH C >50 >50 10e CH H N CH C N CH C 0.0192 >50 26 CH H N C-Me C N CH C 0.371 >50 27 CH H N C-Cl C N CH C 0.203 >50 13h CH CH N CH C N CH C 0.0246 >50 28 C-Me CH N CH C N CH C 6.35 >50 29 C-Cl CH N CH C N CH C >50 >50 30 C-CN CH N CH C N CH C >50 >50 a Obtained from three or more independent experiments. S32
Table S2. Analytical data of the compounds listed in Table S1 compound 1 H NMR LCMS m/z 8h (600 MHz, DMSO-d 6 ) δ 5.60 (s, 2H), 7.16-7.20 (2H), 7.42-7.46 (2H), 7.74 (d, J = 6.1 Hz, 2H), 7.98 (d, J = 5.4 Hz, 1H), 8.27 (d, J = 5.4 Hz, 1H), 8.50 (s, 1H), 8.57 (d, J = 6.1 Hz, 2H), 8.99 (s, 1H). 304 (MH + ) 14 (400 MHz, CDCl 3 ) δ 5.43 (s, 2H), 7.07-7.11 (2H), 7.19-7.23 (2H), 7.90 (s, 1H), 8.02-8.04 (2H), 8.63-8.65 (2H), 8.82 (s, 1 H), 9.13 (s, 1 H). 305 (MH + ) 8g See Experimental Section 15 (400 MHz, MeOH-d 4 ) δ 7.44 (m, 1H), 7.52-7.57 (2H), 7.69 (d, J = 5.2 Hz, 1H), 7.98-8.04 (4H), 8.53 (dd, J = 5.2, 1.6 Hz, 1H), 8.77 (dd, J = 4.6, 1.6 Hz, 2H), 9.53 (d, J = 1.6 Hz, 1H). 16 (400 MHz, MeOH-d 4 ) δ 7.29 (m, 1H), 7.39-7.43 (2H), 7.61-7.63 (2H), 7.68-7.70 (2H), 7.84 (d, J = 5.2 Hz, 1H), 8.06 (s, 1H), 8.23 (d, J = 5.2 Hz, 1H), 8.64 (d, J = 5.0 Hz, 2H), 9.00 (s, 1H). 273 (MH + ) 272 (MH + ) 8a See Experimental Section 17 (400 MHz, CDCl 3 ) δ 1.00 (d, J = 6.7 Hz, 6H), 2.44 (m, 1H), 4.35 (d, J = 7.3 Hz, 2H), 7.89-7.91 (2H), 7.92 (dd, J = 5.8, 1.2 Hz, 1H), 8.43 (d, J = 5.6 Hz, 1H), 8.72-9.76 (2H), 9.04 (d, J = 1.0 Hz, 1H). 253 (MH + ) 18 285 (MH + ) 19 281 (MH + ) 20 286 (MH + ) 21 282 (MH + ) 9g (400 MHz, DMSO-d 6 ) δ 1.51 (s, 9H), 7.48 (dd, J = 5.2, 1.6 Hz, 1H), 7.52 (m, 1H), 7.63-7.68 (2H), 7.77-7.81 (2H), 7.99 (dd, J = 5.6, 1.0 Hz, 1H), 7.26-7.30 (2H), 8.53 (s, 1H), 8.96 (s, 1H), 9.79 (s, 1H). 287 (M-100+H + ) 22 (400 MHz, DMSO-d 6 ) δ 1.48 (s, 9H), 7.35-7.39 (2H), 7.51 (m, 1H), 7.60-7.70 (4H), 8.14 (d, J = 1.8 Hz, 1H), 8.32 (s, 1H), 8.37 (d, J = 5.3 Hz, 1H), 8.46 (d, J = 5.3 Hz, 1H), 10.13 (s, 1H). 23 (400 MHz, MeOH-d 4 ) δ 0.98 (d, J = 6.6 Hz, 6H), 2.27 (m, 1H), 4.08 (d, J = 7.6 Hz, 2H), 7.14 (s, 1H), 8.16 (d, J = 6.8 Hz, 2H), 8.28 (s, 1H), 8.61 (d, J = 6.8 Hz, 2H), 8.66 (s, 1H). 304 (M-100+H + ) 268 (MH + ) (continued on next page) S33
Table S2. (continued) compound 1 H NMR LCMS m/z 24 (400 MHz, CDCl 3 ) δ 0.98 (d, J = 6.6 Hz, 6H), 2.26 (m, 1H), 4.08 (d, J = 7.3 Hz, 2H), 7.48-7.55 (2H), 7.60 (s, 1H), 7.85 (s, 1H), 8.58 (s, 1H), 8.58-8.77 (2H). 25 (400 MHz, MeOH-d 4 ) δ 0.99 (d, J = 6.6 Hz, 6H), 2.31 (m, 1H), 4.05 (s, 3H), 4.22 (d, J = 7.5 Hz, 2H), 7.53 (s, 1H), 8.30 (d, J = 6.9 Hz, 2H), 8.62-8.66 (4H). 286 (MH + ) 282 (MH + ) 10e See Experimental Section 26 (400 MHz, DMSO-d 6 ) δ 2.08-2.25 (4H), 3.18 (s, 3H), 3.57-.364 (2H), 4.03-4.07 (2H), 5.10 (m, 1H), 7.37 (d, J = 6.8 Hz, 1H), 7.41 (s, 1H), 7.99 (br s, 2H, NH 2 ), 8.04 (d, J = 6.8 Hz, 1H), 8.18 (d, J = 6.4 Hz, 1H), 8.42 (d, J = 6.4 Hz, 1H), 9.08 (s, 1H). 27 (400 MHz, DMSO-d 6 ) δ 2.04-2.10 (2H), 2.10-2.07 (2H), 3.53-3.59 (2H), 4.03-4.07 (2H), 5.47 (m, 1H), 7.02-7.15 (3H), 7.28 (br, 1H), 7.93-7.96 (2H), 8.12 (d, J = 5.5 Hz, 1H), 8.57 (s, 1H). 13h (500 MHz, MeOH-d 4 ) δ 1.27 (d, J = 6.6 Hz, 6H), 4.00 (m, 1H), 7.38 (dd, J = 5.7, 1.6 Hz, 1H), 7.54 (m, 1H), 7.63-7.70 (5H), 8.10 (dd, J = 5.7, 1.0 Hz, 1H), 8.24 (d, J = 5.7 Hz, 1H), 8.31 (s, 1H), 8.35 (d, J = 5.7 Hz, 1H), 8.85 (s, 1H). 28 (400 MHz, MeOH-d 4 ) δ 1.25 (d, J = 6.6 Hz, 6H), 2.42 (s, 3H), 3.99 (m, 1H), 7.08 (dd, J = 5.3, 1.0 Hz, 1H), 7.29 (s, 1H), 7.47 (m, 1H), 7.56-7.63 (4H), 7.75 (s, 1H), 8.01 (s, 1H), 8.21 (d, J = 5.3 Hz, 1H), 8.67 (s, 1H). 29 (400 MHz, MeOH-d 4 ) δ 1.25 (d, J = 6.6 Hz, 6H), 3.99 (m, 1H), 7.08 (dd, J = 5.4, 1.5 Hz, 1H), 7.32 (s, 1H), 7.53 (m, 1H), 7.60-7.67 (4H), 7.91 (s, 1H), 8.20 (d, J = 5.4 Hz, 1H), 8.23 (s, 1H), 8.74 (s, 1H). 30 (400 MHz, MeOH-d 4 ) δ 1.25 (d, J = 6.6 Hz, 6H), 3.99 (m, 1H), 7.24 (d, J = 5.4 Hz, 1H), 7.36 (s, 1H), 7.56 (m, 1H), 7.60-7.67 (4H), 5.12 (s, 1H), 8.26 (d, J = 5.4 Hz, 1H), 8.65 (s, 1H), 9.01 (s, 1H). 309 (MH + ) 329 (MH + ) 372 (MH + ) 386 (MH + ) 406 (MH + ) 397 (MH + ) S34
1 H, 13 C and 2D NMR of the compounds in Table S1 Figure S49. 1 H NMR of 8h in DMSO-d6 (600 MHz) Figure S50. 1 H NMR of 9g in DMSO-d6 (400 MHz) S35
Figure S51. 1 H NMR of 13h in MeOH-d4 (500 MHz) Figure S52. 1 H NMR of 14 in CDCl3 (400 MHz) S36
Figure S53. 1 H NMR of 15 in MeOH-d4 (400 MHz) Figure S54. 1 H NMR of 16 in MeOH-d4 (400 MHz) S37
Figure S55. 1 H NMR of 17 in CDCl3 (400 MHz) Figure S56. 1 H NMR of 17 in DMSO-d6 (600 MHz) S38
Figure S57. 13 C NMR of 17 in DMSO-d6 (150 MHz) Figure S58. HMQC NMR of 17 in DMSO-d6 (600 MHz) S39
Figure S59. COSY NMR of 17 in DMSO-d6 (600 MHz) Figure S60. HMBC NMR of 17 in DMSO-d6 (600 MHz) S40
Figure S61. ROESY NMR of 17 in DMSO-d6 (600 MHz) Figure S62. 1 H NMR of 22 in DMSO-d6 (400 MHz) S41
Figure S63. 1 H NMR of 23 (TFA salt) in MeOH-d4 (400 MHz) Figure S64. 1 H NMR of 24 in CDCl3 (400 MHz) S42
Figure S65. 1 H NMR of 25 in MeOH-d4 (400 MHz) Figure S66. 1 H NMR of 26 (TFA salt) in DMSO-d6 (400 MHz) S43
Figure S67. 1 H NMR of 27 in DMSO-d6 (400 MHz) Figure S68. 1 H NMR of 28 in MeOH-d4 (400 MHz) S44
Figure S69. 1 H NMR of 29 in MeOH-d4 (400 MHz) Figure S70. 1 H NMR of 30 in MeOH-d4 (400 MHz) S45
Figure S71. Images of rat and human primary β-cell replication. Images of dispersed rat (A) and human (B) islets treated with DMSO, 13f, 13g, or harmine, and stained with EdU (red), anti-insulin (green), or/and DAPI (blue). Images were processed in parallel and cropped. S46
Figure S72. Dose response of harmine, 5-IT, 13f and 13g-induced rat (A) and human (B) β-cell proliferation. The assay was performed in triplicate in each experiment. Error bars represent standard deviation. Asterisks indicate significance testing compared to DMSO-treated wells: * p < 0.05; ** < 0.01; *** p < 0.001; **** p < 0.0001. S47
Part IV. X-ray co-crystal structure of DYRK1A and GNF2133 Protein expression and purification The DYRK1A kinase domain (residues 118-476) was expressed in E. coli (BL21(DE3) CodonPlus-RIPL, Agilent Technologies) with a cleavable N-terminal 6xHIS tag. Briefly, 14 ml of O/N culture was seeded into 1L TB media (Teknova #T7060) supplemented with the appropriate antibiotics and incubated at 37 C until an OD600 of ~0.4. The temperature was decreased to 18 until an OD600 of ~0.6 was reached at which point expression was induced with 1 mm IPTG. The induced culture was then incubated overnight at 18 C. The next morning cells were harvested by centrifugation (4000xg, 30 min, 4 ). The bacterial pellet was resuspended in lysis buffer (50 mm Tris ph 7.4, 500 mm NaCl, 10 mm Imidazole, 1 mm TCEP, 5% glycerol) supplemented with protease inhibitors (Roche). The resuspended cells were lysed by sonication and the lysate was clarified by centrifugation (36,000xg, 45 min, 4 ). The clarified lysate was applied to Ni-affinity resin previously equilibrated with 25 mm HEPES ph 7.5, 500 mm NaCl, 5 mm TCEP, 5% glycerol. Bound protein was washed with 10 CV of 25 mm HEPES ph 7.5, 500 mm NaCl, 5 mm TCEP, 5% glycerol, 10 mm imidazole and then eluted with the same wash buffer supplemented with 300 mm imidazole. The eluted protein was desalted into 25mM HEPES ph 7.5, 500 mm NaCl, 5 mm TCEP using a PD10 desalting column (GE Life Sciences). The N-terminal 6xHis tag was removed by overnight digestion with Tev protease (His-tagged) at 4 C. Uncleaved protein and Tev protease were removed by flowing the digestion mixture over Ni-affinity resin equilibrated with 25 mm HEPES ph 7.5, 500 mm NaCl, 5 mm TCEP and the flow through collected. The cleaved protein was further purified using size exclusion chromatography using a Superdex S200 (GE Life Sciences) column. Fraction containing protein were combined and concentrated to a 15 mg/ml using a 10 kda MWCO spin concentrator (Amicon). Crystallization and data collection Crystallization experiments were carried out using the sitting-drop vapor diffusion setup with 0.4 μl drops (0.2 μl protein + 0.2 μl reservoir solution). Prior to crystallization, the inhibitor was added to the purified DYRK1A kinase domain to a final concentration of ~0.5 mm. Crystallization experiments were carried out at both 4 and 20. Crystals for the DYRK1A-Inhibitor complex appeared in 2-3 weeks from 0.1 M Bicine ph 8.5, 15% PEG 20000, 3% Dextran sulfate at 4. Crystals were cryopreserved in reservoir solution supplemented with 20% v/v ethylene glycol and flash frozen in liquid nitrogen prior to data collection. X-ray diffraction data were collected from frozen crystals on ALS beamline 5.0.3. Structure determination and refinement Diffraction data were processed using HKL2000 S1 and the structure was solved by molecular replacement using PHASER S2 as implemented in the CCP4 software package. S3 Structure refinement was carried out in PHENIX S4 alternated with manual fitting in Coot. S5 Restraints for the inhibitor were generated with GRADE as part of the autobuster software package. S6,S7 Due to the relatively low resolution of the structure group B-factor refinement (one per residue) and a single TLS group per chain were employed in the refinement protocol using PHENIX. S48
Resolution of X-ray co-crystal structure Despite the moderately low resolution of the structure (3.7 Å) the electron density in the active site and for the inhibitor is very well defined and supports the depicted binding mode. This is likely due to the low mosaicity of the crystal (0.325 ), the low Wilson b-factor (24.87 Å 2 ) and the presence of 3 copies of DYRK1A in the crystallographic asymmetric unit which allowed for the use of NCS restraints during refinement. Also, the average b-factors for the protein and across all three copies of the inhibitor are quite low (48.12 Å 2 and 44.92 Å 2 respectively) relative to the overall resolution. More specifically, the average b-factor of the inhibitor bound to chain A is 24.89 Å 2, the inhibitor bound to chain B is 41.79 Å 2 and the inhibitor bound to chain C is 68.01 Å 2. The structure and interactions depicted in Figure 5 are the inhibitor bound to chain A (the binding mode in the other chain is the same). Figure S73 depicts the 2FoFc electron density map for the inhibitor bound to chain A contoured to 1.5σ. The quality and shape of the electron density allowed for unambiguous placement of the inhibitor molecule. The observed electron density does not support a flipped binding mode. Figure S73. 2FoFc electron density map for GNF2133 bound to chain A. S49
Table S3. Crystallographic data and refinement statistics DYRK1A-GNF2133 PDB Code Wavelength 0.9765 Resolution Range (Å) 48.27 3.703 (3.835 3.703) Space Group P3112 Unit Cell (a, b, c, α, β, γ) 112.884, 112.884, 304.651, 90, 90, 120 Unique Reflections 23849 (2168) Multiplicity 8.4 (8.4) Completeness (%) 98.48 (86.14) Mean I/sigma(I) 24.9 (1.4) Wilson B-factor 24.87 Rmeas 0.122 (0.00) Rpim 0.048 (0.580) Reflections used for R-free 1155 (133) Rwork 27.24 (36.30) Rfree 29.04 (38.33) Number of non-hydrogen atoms 8203 macromolecules 8107 ligands 96 solvent 0 Protein residues 989 RMS (bonds) 0.003 RMS (angles) 0.64 Ramachandran favored (%) 91.7 Ramachandran allowed (%) 7.46 Ramachandran outliers (%) 0.84 Clashscore 8.18 Average B-factor 48.08 macromolecules 48.12 ligands 44.92 Statistics for the highest-resolution shell are shown in parentheses. S50
Part V. Two-week in vivo safety study of GNF2133 In order to evaluate potential off-target (non-islet) cellular proliferation, GNF2133 was administered to 8-week old male HanWistar rats by oral gavage for two weeks, at doses of 0 (vehicle control) and 100 mg/kg/day with 5 animals in each group. Samples of the liver, heart, kidney and pancreas were taken at necropsy, fixed in 10% neutral buffered formalin for 48 h, embedded in paraffin and then processed to slide. Immunohistochemical staining (Ki67) was performed using a Leica Bond RX autostainer using Leica BOND Polymer Refine Detection kit (Leica, Buffalo Grove, IL) except the deparaffinization and heat antigen retrieval steps. Slides were deparaffinized using xylene, 100% ethanol and 95% ethanol (2 changes of 3 minutes each), then rehydrated in deionized water. Slides were submitted to heat-induced antigen retrieval by covering them with Biocare Reveal Decloaker solution in the Biocare Medical Decloaking Chamber (Biocare Medical, Concord, CA) for 1 minute at 125. Slides were washed with deionized water then Leica Bond Wash solution at room temperature. Slides were incubated with Leica Bond 3% hydrogen peroxide in methanol for 5 minutes to quench endogenous peroxidase activity then washed 3 times with Leica Bond wash solution. Slides were then incubated with the primary antibody, a rabbit monoclonal anti-human Ki-67 (Biocare Medical, clone SP6, Concord, CA; 1/25 for 1 hour) or a non-immune isotype-matched control (isotype negative control rabbit IgG Biocare, Concord, CA). Staining visualization was obtained by incubation with Leica Bond Polymer for 8 minutes followed by Leica Bond DAB Refine for 10 minutes. Counterstaining was done using Leica Bond Hematoxylin for 10 minutes then dehydrated, cleared and cover slipped with a synthetic mounting medium. The stained slides were scanned at 40x using Leica s Aperio eslide Manager on an Aperio AT Turbo scanner and evaluated using HALO image analysis software (Indica Labs, Albuquerque, NM) to determine any differences in the percentage of cells staining positive for Ki67 in rats given GNF2133. The number of proliferating cells (Ki67 positive cells) divided by the total tissue area (the result multiplied by 100) were reported as the percent of proliferating cells per micrometer square of tissue for each animal and the fold (multiple) difference between the values from control and treated animals was determined as an index of GNF2133-induced proliferation within the organs (Figure 6). S51
Part VI. Kinase inhibition profiling and Ba/F3 cellular profiling of GNF2133 Table S4. GNF2133 in enzymatic cell free Caliper microfluidic kinase assay a target % inhibition concentration (µm) target % inhibition concentration (µm) Abl 18.45 2 MapK13 2.555 0.2 AKT1 3.085 0.2 MapK14 5.535 0.2 AKT2 3.185 0.2 MAPK3 0.565 0.2 AKT3 3.385 0.2 MAPKAPK2 2.745 0.2 ALK 9.69 0.2 MAPKAPK3 3.745 0.2 AMP-a1b1g1 8.065 2 MARK1 2.79 0.2 AMP-a2b1g1 0.075 2 MARK3 2.725 0.2 ARG 4.005 2 MARK4 0.455 0.2 ARK5 3.205 2 MEK1 9.185 2 AURA 1.63 0.2 MEK2 11.1 2 AURB 0.38 0.2 MELK 15.46 2 AURC 5.11 0.2 MER 3.1 2 Axl 5.705 2 Met 4.645 0.2 BLK 6.225 0.2 MINK 39.145 2 BMX 6.855 0.2 MKNK1 75.65 2 braf 3.255 2 MRCKα 0.705 0.2 BRK 18.185 2 MRCKβ 0.08 2 BRSK1 5 0.2 MSK1 4.685 0.2 BRSK2 2.255 0.2 MSK2 3.245 0.2 BTK 5.74 0.2 MSSK1 2.455 0.2 CAMK1A 4.09 0.2 MST1 3.61 0.2 CAMK1D 2.86 0.2 MST2 5.445 0.2 CAMK2A 3.115 0.2 MST3 2.385 2 CAMK2B 0.785 2 MST4 0 0.2 CAMK2D 0 0.2 mtor 12.385 2 CAMK2G 0 0.2 MUSK 3.255 0.2 CAMK4 0 0.2 NDR1 3.49 0.2 CDK1 11.26 2 NDR2 4.195 0.2 CDK2 30.215 2 NEK1 5.61 0.2 CDK2-cycE 37.69 2 Nek2 3.02 0.2 CDK3-cycE 29.66 2 NEK6 1.49 2 CDK4-cycD 3.065 2 NEK7 0 0.2 CDK5 21.625 2 NEK9 0.12 0.2 CDK5-p25 26.48 2 P38b 1.045 0.2 CDK6-cycD3 4.715 0.2 P38g 2.345 0.2 CDK7 20.25 2 p70s6k 6.95 2 CDK9-cycT1 61.45 2 p70s6k2 2.2 2 S52 (continued on next page)
Table S4. (continued) target % inhibition concentration (µm) target % inhibition concentration (µm) CHEK1 2.35 0.2 PAK1 1.21 0.2 CHEK2 8.145 0.2 PAK2 2.77 2 CK1a 29.34 2 PAK3 3.815 0.2 CK1-epsilon 35.165 2 PAK4 2.195 2 CK1-γ1 11.785 2 PAK5 4.03 2 CK1- γ2 2.115 2 PAK6 2.07 2 CK1- γ3 3.885 2 PAR-1Bα 3.755 2 ckit 10.12 2 PASK 0.145 0.2 CLK1 94.545 2 PDGFRα 74.48 2 CLK2 95.835 2 PDGFRβ 65.95 2 CLK3 15.36 2 PDK1 2.095 2 CLK4 102.415 2 PERK 4.91 2 craf 3.655 2 PHKγ1 13.295 2 CSK 7.115 0.2 PHKγ2 11.515 2 DAPK1 1.49 0.2 PI3Kα 15.275 2 DAPK3 3.395 2 PI4Kβ 33.565 2 DCAMKL2 3.1 2 Pim1 4.89 2 DDR1 13.73 2 Pim2 0 0.2 DDR2 1.22 2 Pim3 0.325 2 DYRK1A 97.695 2 PKA 6.615 0.2 DYRK1B 97.185 2 PKACB 0 0.2 DYRK2 95.055 2 PKCa 2.015 0.2 DYRK3 70.425 2 PKC-β1 5.45 2 DYRK4 2.205 0.2 PKC-β2 3.82 0.2 EGFR 4.695 0.2 PKC-ε 13.15 0.2 EphA1 9.43 2 PKC-η 2.32 2 EphA2 5.385 2 PKC-γ 2.66 0.2 EphA3 6.495 2 PKC-ι 0 0.2 EphA4 10.745 2 PKC-θ 1.44 0.2 EphA5 9.68 2 PKC-ϛ 13.4 0.2 EphA8 8.995 2 PKN1 0 0.2 EphB1 11.875 2 PKN2 1.785 0.2 EphB2 18.985 2 PLK1 1.33 2 EphB3 5.635 2 PLK3 0 0.2 EphB4 5.595 2 PLK4 0 0.2 ErbB2 0 0.2 PRAK 2.915 0.2 ErbB4 0.92 0.2 PRKACA 0.885 0.2 FAK 2.93 0.2 PRKD1 3.485 0.2 (continued on next page) S53
Table S4. (continued) target % inhibition concentration (µm) target % inhibition concentration (µm) FER 4.99 0.2 PRKD2 0.825 0.2 FES 2.58 2 PRKD3 4.785 0.2 FGFR1 18.795 2 PRKG1 3.33 0.2 FGFR2 29.615 2 PRKG2 23.84 2 FGFR3 4.71 0.2 PRKX 0.425 0.2 FGFR4 1.895 0.2 PTK5 2.085 0.2 FGR 3.125 0.2 PYK2 4.24 0.2 Flt1 58.84 2 Ret 5.42 0.2 Flt3 86.9 2 RIPK2 91.89 2 Flt4 90.49 2 ROCK1 5.57 2 FMS 18.555 2 ROCK2 11.7 2 Fyn 7.67 0.2 RON 1.53 2 GRK6 0 0.2 ROS 14.64 2 GRK7 5.885 0.2 RSK1 10.53 2 GSK3α 66.99 2 RSK2 16 2 GSK3β 49.64 2 RSK3 9.955 2 HASPIN 65 2 RSK4 13.15 2 HCK 3.905 2 SGK1 5.3 2 HIPK1 20.73 2 SGK2 5.83 0.2 HIPK2 13.615 2 SGK3 4.185 0.2 HIPK3 8.93 2 SLK 7.45 0.2 HIPK4 79.74 2 SNF1LK 1.44 2 IGF1R 1.22 0.2 SNF1LK2 15.56 2 IKKα 0 0.2 SPHK1 0 0.2 IKKβ 7.87 0.2 SPHK2 0 0.2 IKKe 13.28 2 SRC 4.05 0.2 InsR 4.21 2 SRMS 4.86 0.2 Irak1 13.51 2 SRPK1 1.12 0.2 Irak4 0 0.2 SRPK2 0.665 0.2 IRR 0.04 0.2 STK16 0 0.2 ITK 8.215 0.2 SYK 5.23 0.2 JAK1 2.72 2 TAK1-TAB1 17.185 2 JAK2 0 0.2 TAOK2 1.225 0.2 JAK3 5.49 0.2 TAOK3 37.54 2 Jnk1 38.14 2 TBK1 12.38 2 Jnk2 48.075 2 Tec 5.745 0.2 Jnk3 20.37 2 TIE2 1.325 0.2 (continued on next page) S54
Table S4. (continued) target % inhibition concentration (µm) target % inhibition concentration (µm) KDR 86.31 2 TNIK 64.24 2 LATS1 3.21 0.2 TNK2 3.815 2 LATS2 2.97 0.2 TrkA 10.745 0.2 Lck 6.09 0.2 TrkB 3.995 0.2 LOK 3.98 2 TrkC 6.165 0.2 LRRK2(G2019S) 76.6 2 TSSK1 11.85 2 LTK 11.74 2 TSSK2 2.8 0.2 Lyn 6.64 0.2 TTK 25.3 2 LynB 10 2 TXK 18.37 2 MAP4K2 28.865 2 TYK2 34.695 2 MAP4K4 46.92 2 TYRO3 2.78 0.2 MAP4K5 0 0.2 YES 6 0.2 MapK1 4.685 0.2 ZAP70 1.73 2 a GNF2133 was screened at 0.2 or 2.0 µm against 250 kinases. Results were reported as %inhibition, where larger values indicate stronger inhibition. S55
Table S5. GNF2133 in Ba/F3 Cellular Kinase Profiling Ba/F3 IC 50 (μm) Ba/F3 IC 50 (μm) BaF3 Parental 3.136 KDR 4.569 BCRABL >10.7 KITQ 6.027 BRAF-V600E >10.7 LCK2.1 2.093 Jak2_clone1_CTG 3.019 LYN1.2 4.735 Jak2_V617F_EpoR_CTG 9.011 MER3.2 6.14 KIF5B-RETV804M >10 MET-3.1 2.502 KRasG12V_CTG 7.872 PDGFRa-Q2 9.331 NPM-ALK 6.128 PDGFRb 7.731 Blk4.1 0.903 RETQ1 >10 BMX1.1 6.117 RETQ1 4.075 EPHB4 4.977 RON4.1 4.465 FGFR1 4.01 ROS-1.1 0.906 FGFR2 5.216 SRC3.3 3.979 FGFR3Q2 7.401 SRC3.3 4.575 FGFR4Q4 6.667 SYK-IS 3.381 FGR2.1 6.237 TIE1-Q2 1.608 FLT1Q1 6.386 TRKA-Q3 >10.7 FLT31.2 3.671 TRKB-Q2 7.304 FMS-Q.3 5.905 TRKC-Q2 6.856 IGF1R-N-FL 3.509 TYRO3-2.2 6.575 INSR_1.2 2.903 ZAP70S 3.761 JAK1S-1.2 4.795 Tpr_Met_CTG 4.075 JAK3-S1.2/sACP 1.42 BAF3/WT >8.722 JAK2 3.506 S56
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