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 102206, China b. Peking University-Tsinghua University-ational Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Tsinghua University, 100084, China # These authors contributed equally. Corresponding Author *(.H.) phone 86-10-80720645, fax 86-10-80720813, e-mail: huangniu@nibs.ac.cn; *(X.Q.) phone 86-10-80726688-8655, fax 86-10-80708048, e-mail: qixiangbing@nibs.ac.cn. S1
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
Figure S1. Structure of representative small-molecule inhibitors of BTK and LCK. S3
Figure S2. Structure and inhibition activity of eight candidates from hierarchical virtual screening against p38α. S4
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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 (http://chemmine.ucr.edu/similarity/). S6
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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
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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
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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
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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
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
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
Vandetanib 0.19 intedanib 0.10 Vemurafenib 0.20 Bosutinib 0.12 Axitinib 0.21 S17
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 2016/0256440 A1 0.34 Thrombin inhibitor 2 US 2016/0251341 A1 0.23 Thrombin inhibitor 3 WO 2016/033635 0.39 Antiprotozo al agent 4 Arch. Pharm. Res. 2014 37, 440 451. 0.24 TRPV1 antagonist 5 WO 2013/049591 A2 0.43 Thrombin and coagulation factor Xa dual inhibitor 6 J. Med. Chem. 2011, 54, 8563 8573 0.62 AR BF3 inhibitor 7 US 2013/0040950 A1 0.39 Thrombin inhibitor 8 WO 2012/073995 A1 0.27 Pesticide 9 Arch. Pharm. Chem. Life Sci. 2007, 340, 17 25 0.27 K-2 receptor inhibitor 10 Bioorg. Med. Chem. Lett. 15 (2005) 5154 5159 0.24 Tubulin polymerizat ion inhibitor S18
11 US 2009/0048301 A1 0.23 Tubulin polymerizat ion inhibitor 12 US 2004/0087798 A1 0.20 5-HT 2c receptor antagonist 13 HETEROCYCLE S, Vol.34, o.1, 1992 0.37 Antimicrobi al:t. viride S19
Table S3. The data collection and refinement statistics of cocrystal structure of p38α in complex with 1. Data collection Space group P212121 Unit cell (a, b, c in Ȧ) 45.15, 86.13, 124.77 Unit cell (α, β, γ in ) 90, 90, 90 Wavelength (Ȧ) 0.979 Resolution range (Ȧ) a 28.83-2.61 (2.75-2.61) Observations 86377 (12095) Unique reflections 15084 (2145) Redundancy 5.7 I/σ 15.4 (5.4) Completeness (%) 97.1 (97.4) R merge b 0.074 (0.275) Structure refinement Resolution range (Ȧ) 2.61 Reflections used 15524 o. heavy atoms 2754 R factor c 0.2155 R free d 0.2604 Rms Deviations Bond length (Ȧ) 0.0107 Bond angles ( ) 1.4908 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
Table S4. The data collection and refinement statistics of cocrystal structure of p38α in complex with 3. Data collection Space group P212121 Unit cell (a, b, c in Ȧ) 44.94, 86.60, 124.68 Unit cell (α, β, γ in ) 90, 90, 90 Wavelength (Ȧ) 0.979 Resolution range (Ȧ) a 33.60-1.70 (1.79-1.70) Observations 396555 (55807) Unique reflections 54433 (7847) Redundancy 7.3 I/σ 15.6 (5.1) Completeness (%) 99.9 (100.0) R merge b 0.080 (0.383) Structure refinement Resolution range (Ȧ) 1.70 Reflections used 54445 o. heavy atoms 2837 R factor c 0.2442 R free d 0.2766 Rms Deviations Bond length (Ȧ) 0.0224 Bond angles ( ) 2.0988 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
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 ± 0.02 0.11 ± 0.01 1.5 ± 0.2 TEC 306.45 ± 75.02 1.86 ± 0.01 7.0 ±2.5 ITK > 1000 D 4.9 ±1.2 ErbB2 > 1000 D 6.4 ± 1.8 JAK3 > 100 D 32 ± 15 S22
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, 101.63 Unit cell (α, β, γ in ) 90.00, 100.04, 90.00 Wavelength (Ȧ) 0.979 Resolution range (Ȧ) a 47.25-2.64 (2.93-2.64) Observations 82813 (10019) Unique reflections 11419 (1422) Redundancy 7.3 I/σ 15.5 (5.4) Completeness (%) 87.7 (77.3) R merge b 0.091 (0.309) Structure refinement Resolution range (Ȧ) 2.64 Reflections used 13056 o. heavy atoms 2839 R factor c 0.2035 0.2606 Rms Deviations Bond length (Ȧ) 0.0123 Bond angles ( ) 1.5842 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
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 65 12 481 Y 11 4 502 175 5 506 160 11 527 Y 111 33 633 260 0 Total 782 65 S24
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 1 0.12 0.25 Ibrutinib 0.22 0.38 2 0.22 0.35 Dasatinib 0.21 0.34 3 0.22 0.34 CGI-1746 0.18 0.28 4 0.21 0.32 R-486 0.18 0.26 5 0.20 0.28 GDC-0834 0.18 0.26 6 0.28 0.32 IAQ 0.27 0.37 S25