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1 In Silico Identification of a ovel Hinge-Binding Scaffold for Kinase Inhibitor Discovery Yanli Wang a#, Yuze Sun b,a#, Ran Cao a#, Dan Liu a, Yuting Xie a, Li Li a, Xiangbing Qi a*, and iu Huang a* a. ational Institute of Biological Sciences, Beijing, o. 7 Science Park Road, Zhongguancun Life Science Park, Beijing , China b. Peking University-Tsinghua University-ational Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Tsinghua University, , China # These authors contributed equally. Corresponding Author *(.H.) phone , fax , huangniu@nibs.ac.cn; *(X.Q.) phone , fax , qixiangbing@nibs.ac.cn. S1

2 Table of contents Figure S1. Structure of representative inhibitors of BTK and LCK. Figure S2. Structure and inhibition activity of virtual screening candidates. Figure S3. Comparison of MD and PDB structures for p38α MAP kinase. S3 S4 S5 Figure S4. Representative binding modes with Thr(GK) targeting characteristics. S6 Figure S5. Comparison of computational results with crystal structure for lead. Figure S6. 1 H MR and HPLC of 1. Figure S7. 1 H MR and HPLC of 3. Figure S8. 1 H MR and HPLC of 4. Figure S9. 1 H MR, HPLC and HRMS of 7. Figure S10. 1 H MR, HPLC and HRMS of 8. Table S1. Structural comparison of 1 and representative kinase drugs. S7 S8 S9 S10 S12 S14 S15 Table S2. Comparison of 1 with similar chemical structures in existing patents and literatures searched in SciFinder. Table S3. The statistics of cocrystal structure of p38α and 1. Table S4. The statistics of cocrystal structure of p38α and 3. Table S5. Kinase inhibition of 8 examined by Reaction biology. Table S6. The statistics of cocrystal structure of BTK and 8. Table S7. LC-MS/MS analysis of covalent interaction of 8. S18 S20 S21 S22 S23 S24 Table S8. Structural comparison of 7 and 8 with representative LCK and BTK inhibitors. S25 S2

3 Figure S1. Structure of representative small-molecule inhibitors of BTK and LCK. S3

4 Figure S2. Structure and inhibition activity of eight candidates from hierarchical virtual screening against p38α. S4

5 S5

6 Figure S4. Representative binding modes for small-molecule kinase inhibitors with gatekeeper(thr) targeting characteristics. Tc values are determined by comparing both structures of 1 and selected inhibitor with online protocol ( S6

7 S7

8 Figure S6. 1 H MR and HPLC of lead compound -(2-chloro-6-fluorobenzyl) -3-(furan-2-yl)-1H-1,2,4-triazol-5-amine (1). S8

9 S9

10 Figure S8. 1 H MR and HPLC of compound 3-(3-aminophenyl)--(2-chloro-6 -fluorobenzyl)-1h-1,2,4-triazol-5-amine (4). S10

11 S11

12 Figure S9. 1 H MR, HPLC and HRMS of compound -(2-chloro-6-fluoroben zyl)-3-(3,4-dimethoxyphenyl)-1h-1,2,4-triazol-5-amine (7). S12

13 S13

14 Figure S10. 1 H MR, HPLC and HRMS of compound -(3-(5-((2-chloro-6-fl uorobenzyl)amino)-1h-1,2,4-triazol-3-yl)phenyl)acrylamide (8). S14

15 Table S1. The structural comparison of 1 and representative small molecular drugs of kinase. Representative kinase inhibitor Structure Similarity (TC) Representative kinase inhibitor Structure Similarity (TC) Imatinib 0.15 Cabozantinib 0.13 Gefitinib 0.20 Pazopanib 0.15 Erlotinib 0.18 Ponatinib 0.12 Sorafenib 0.18 Regorafenib 0.18 Dasatinib Cl O H S H OH 0.15 Tofacitinib 0.10 S15

16 Sunitinib 0.13 Afatinib 0.19 Lapatinib 0.16 Dabrafenib 0.14 ilotinib H O H 0.13 Ibrutinib 0.16 CF 3 Crizotinib 0.20 Trametinib 0.12 Ruxolitinib 0.13 Ceritinib 0.15 S16

17 Vandetanib 0.19 intedanib 0.10 Vemurafenib 0.20 Bosutinib 0.12 Axitinib 0.21 S17

18 Table S2. Comparison of compound 1 with similar chemical structures in existing patents and literatures searched in SciFinder database. ID. Representative Structure Reference Tc Target/ Activity 1 US 2016/ A Thrombin inhibitor 2 US 2016/ A Thrombin inhibitor 3 WO 2016/ Antiprotozo al agent 4 Arch. Pharm. Res , TRPV1 antagonist 5 WO 2013/ A Thrombin and coagulation factor Xa dual inhibitor 6 J. Med. Chem. 2011, 54, AR BF3 inhibitor 7 US 2013/ A Thrombin inhibitor 8 WO 2012/ A Pesticide 9 Arch. Pharm. Chem. Life Sci. 2007, 340, K-2 receptor inhibitor 10 Bioorg. Med. Chem. Lett. 15 (2005) Tubulin polymerizat ion inhibitor S18

19 11 US 2009/ A Tubulin polymerizat ion inhibitor 12 US 2004/ A HT 2c receptor antagonist 13 HETEROCYCLE S, Vol.34, o.1, Antimicrobi al:t. viride S19

20 Table S3. The data collection and refinement statistics of cocrystal structure of p38α in complex with 1. Data collection Space group P Unit cell (a, b, c in Ȧ) 45.15, 86.13, Unit cell (α, β, γ in ) 90, 90, 90 Wavelength (Ȧ) Resolution range (Ȧ) a ( ) Observations (12095) Unique reflections (2145) Redundancy 5.7 I/σ 15.4 (5.4) Completeness (%) 97.1 (97.4) R merge b (0.275) Structure refinement Resolution range (Ȧ) 2.61 Reflections used o. heavy atoms 2754 R factor c R free d Rms Deviations Bond length (Ȧ) Bond angles ( ) a Values in parentheses are for the data in the outer shell. b R merge = Σ I i I m /ΣI i, where I i is the intensity of the measured reflection and I m is the mean intensity of all symmetry related reflections. c R factor =Σ F o F c /ΣF o, where F o and F c are the observed and calculated structure factor amplitudes. d R free is the same as R work, but calculated on random 5% reflections not used in refinement. S20

21 Table S4. The data collection and refinement statistics of cocrystal structure of p38α in complex with 3. Data collection Space group P Unit cell (a, b, c in Ȧ) 44.94, 86.60, Unit cell (α, β, γ in ) 90, 90, 90 Wavelength (Ȧ) Resolution range (Ȧ) a ( ) Observations (55807) Unique reflections (7847) Redundancy 7.3 I/σ 15.6 (5.1) Completeness (%) 99.9 (100.0) R merge b (0.383) Structure refinement Resolution range (Ȧ) 1.70 Reflections used o. heavy atoms 2837 R factor c R free d Rms Deviations Bond length (Ȧ) Bond angles ( ) a Values in parentheses are for the data in the outer shell. b R merge = Σ I i I m /ΣI i, where I i is the intensity of the measured reflection and I m is the mean intensity of all symmetry related reflections. c R factor =Σ F o F c /ΣF o, where F o and F c are the observed and calculated structure factor amplitudes. d R free is the same as R work, but calculated on random 5% reflections not used in refinement. S21

22 Table S5. IC 50 (nm) values of 8 against selected kinases examined by reaction biology corporation. Kinase IC 50 (nm) 8 Ibrutinib Ibrutinib (Data from Ref. 1 ) BTK 1.91 ± ± ± 0.2 TEC ± ± ±2.5 ITK > 1000 D 4.9 ±1.2 ErbB2 > 1000 D 6.4 ± 1.8 JAK3 > 100 D 32 ± 15 S22

23 Table S6. The data collection and refinement statistics of cocrystal structure of BTK in complex with 8. Data collection Space group P21 Unit cell (a, b, c in Ȧ) 59.59, 53.60, Unit cell (α, β, γ in ) 90.00, , Wavelength (Ȧ) Resolution range (Ȧ) a ( ) Observations (10019) Unique reflections (1422) Redundancy 7.3 I/σ 15.5 (5.4) Completeness (%) 87.7 (77.3) R merge b (0.309) Structure refinement Resolution range (Ȧ) 2.64 Reflections used o. heavy atoms 2839 R factor c Rms Deviations Bond length (Ȧ) Bond angles ( ) a Values in parentheses are for the data in the outer shell. b R merge = Σ I i I m /ΣI i, where I i is the intensity of the measured reflection and I m is the mean intensity of all symmetry related reflections. c R factor =Σ F o F c /ΣF o, where F o and F c are the observed and calculated structure factor R free d amplitudes. d R free is the same as R work, but calculated on random 5% reflections not used in refinement. S23

24 Table S7. LC-MS/MS analysis of covalent interaction between BTK and 8. Cysteine ID in BTK On surface or not Identified times in total Identified times for 8 labeling 464 Y Y Y Total S24

25 Table S8. Structural comparison of 7 and 8 with representative LCK and BTK inhibitors (LE = pic 50 /umber of heavy atoms). 7 (LE: 0.42) 8 (LE: 0.44) LCK Structure Tc LE BTK Structure Tc LE Ibrutinib Dasatinib CGI R GDC IAQ S25