Supplementary data

Identification of ligand efficient fragment-like hits from an HTS library:

Structure-based virtual screening & docking investigations of 2H- and 3H-pyrazolo

tautomers for aurora kinase A selectivity

Sailu Sarvagalla,†,¶ Vivek Kumar Singh,†,¶ Yi-Yu Ke,,¶ Hui-Yi Shiao, Wen-Hsing Lin,Hsing-

Pang Hsieh , John T. A. Hsu, and Mohane Selvaraj Coumar †,

† Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India ¶ These authors contributed equally to this work  Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan, ROC  To whom correspondence should be addressed: Phone, +91-413-2654950; fax, +91-413- 2655211; e-mail, m [email protected] Contents A. Figures...... S3 B. Tables...... S6

S2 A. Figures

N NH Cl Cl

HN F F H H H N N N N N OMe N N O N O N N O N N S F OMe N OH OH VX-680/MK-0457 (Vertex) MLN8054 (Millenium) MLN8237 (Millenium) CLogP: 3.4, Ml.Wt: 464.2; Pan-selective CLogP: 5.2, Ml.Wt: 476.1; Aurora A selective CLogP: 4.7, Ml.Wt: 518.1; Aurora A selective Withdrawn from Phase II testing Withdrawn from Phase I testing Phase I/II testing

O OH P H OH N H O N N N NH O N H HN N N N HN O NH NH O O O N N NH F O N N N O AZD1152 (AstraZeneca) PHA-739358 (Nerviano) AT-9283 (Astex) CLogP: 3.6, Ml.Wt: 587.2; Aurora B selective N CLogP: 2.5, Ml.Wt: 474.2 CLogP: 2.2, Ml.Wt: 381.2; Phase II testing Pan-selective; Phase II testing Aurora A & B selective; Phase I/IIa testing

O CH3 H3C N CH3 NH2 N N CH3 N N O H C N N O H 3 N N F NH O N HN CF3 N N N N N HN H H GSK1070916 (GSK) R-763/AS-703569 (Rigel) PF3814735 (Pfizer) CLogP: 4.1, Ml.Wt: 507.3; Aurora B & C selective CLogP: 3.1, Ml.Wt: 450.3 CLogP: 3.2, Ml.Wt: 474.2 Phase I testing Phase I testing Aurora A & B selective; Phase I testing

F H2N N Cl O N N COOH S NH O N HN S N NH O N N S S N N H N Cl H N N AMG 900 (Amgen) VX-689/MK-5108 (Vertex) SNS-314 (Sunesis) CLogP: 5.7, Ml.Wt: 503.1 CLogP: 5.2, Ml.Wt: 461.1 CLogP: 5.1, Ml.Wt: 430.0 Phase I testing Phase I testing Pan-selective; Phase I testing

Figure S1. Aurora kinase inhibitors in clinical development and their status.

S3 Figure S2. Superimposition of the original crystal structure conformation (Green colour) of VX- 680 and the docked ligand conformation (Red colour) of VX-680 to 3E5A using Libdock module of Discovery studio v2.5. RMSD = 0.842 Å.

Figure S3. Superimposition of the original crystal structure conformation (Green colour) of VX- 680 and the docked ligand conformation (Red colour) of VX-680 in 3E5A using Glide module of Schrodinger. RMSD = 0.513 Å.

S4

Libdock based VS workflow Glide XP based VS workflow

Protein Ligand Protein Ligand preparation preparation preparation preparation

Defining 337 poses Grid 409 poses binding site generation generation generation

Docking & Subjected Docking & Subjected to Scoring using to docking Scoring using docking Libdock Glide XP 22061 docking 409 docking conformation conformation

Ranking based on Ranking based on Libdock Score Glide XP Score

Top ranked 13 Top ranked 13 compounds selected compounds selected

Combined list

Total of 18 compounds sent for testing

Figure S4. The overall Libdock and Glide screening protocol used to select the 18 compounds for aurora kinase A testing is shown.

S5 B. Tables Table S1: Top 13 Aurora kinase A inhibitors identified through Structure-based virtual screening using libdock module of Discovery Studio.

Aurora Kinase Inhibition

% Inhibition % Inhibition IC50 Sl.No. Compound Structure Libdock Score @ 50 µM @ 20 µM (nM)

H2N 99.1007 89.3 59.1 - a (3) N N N H

b N HN HN H 98.3561 7.3 10.6 - N N N O O H

H2N 94.2136 2.6 0.8 - c N N N H

-

H2N 93.5066 14.1 9.8 d N N N H

O 93.3569 32.0 - - e N

HN N O NH2

f 93.3382 27.1 12.9 -

S6 O

N

HN N O NH2

H2N 92.1884 21.5 5.0 - g HN N

F h 91.7329 73.3 39.9 - HN N N O H

91.4244 13.0 1.3 - i HN N OH O

N 91.1600 14.3 4.1 - j N N

HN N N

F 89.9505 40.4 11.5 -

k HN N N O H

O + N - 89.8993 l HN O 2.5 - N N H m (5)

88.9963 82.5 63.6 S7 -

HN N O O

Table S2: Top 13 Aurora kinase A inhibitors identified through Structure based virtual screening using Glide module of Schrodinger.

Aurora Kinase Inhibition

% Inhibition % Inhibition IC50 Sl.No. Compound Structure Glide Score @ 50 µM @ 20 µM (nM)

H2N a (3) N -12.817 89.3 59.1 - N N H

F

HN b (4) N N O H -12.645 73.3 39.9 -

c - HN N N O -12.524 46.7 24.6 H

F

d - HN -12.432 40.4 11.5 N N O H

e N - N H -12.274 0.0 0.0

S8 O f N HN N 12.204 32.0 - - O NH2

O g (6)

HN N -12.182 98.4 93.5 341

OH HN N h O O -11.781 10.1 8.2 -

O -11.771 27.1 12.9 i N - HN N O NH2

H2N

j N N N H -11.555 14.8 9.8 -

H2N

k HN -11.401 21.5 5.0 - N

O HO l N N -11.262 13.0 1.3 - H

HN N -9.153205933 44.5 29.0 - m O O

Table S3: Aurora kinase A inhibitors identified through sub-structure search of hits identified from Structure based virtual screening

S9 Aurora Kinase Inhibition % % Inhibition IC50 Sl.No. Inhibition Compound Structure @ 50 µM @ 20 µM (nM)

3 H2N N 89.3 59.1 - N N H

7 H2N (Analog N N O of 3) H N 96.5 96.6 852 O 6 98.4 93.5 341 HN N

8 O

(Analog HO 100.0 99.1 3270 of 6) O HN N

O NH O 9 HO 2 S (Analog O 100.0 97.9 1091 of 6) HN N

S10 Table S4. Compound characterization data for hits 3-9 reported in Table 1.

Calculated MF Detected MW HPLC Compd Structure & MW (LCMS)a Purityd

+ a H2N MF: C14H20N4 [M+H] : 245

3 N 100.00% + N N MW: 244.34 H [M+Na] : 267

MF: C13H12FN3O F 4 [M+H]+: 246a 97.52% N MW: 245.25 N N O H H

MF: C14H14N2O2 5 [M+Na]+: 265a 95.58% HN M W: 242.27 N O O

O MF: C14H8N2O 6 [M+H]+: 221b 96.76% MW: 220.23 HN N

H N 2 MF: C14H14N4O [M+H]+: 255a 7 N 99.54% N O + H N MW: 254.29 [M+Na] : 277

O MF: C15H8N2O3 8 [M+H]+: 265b 99.64% HO MW: 264.24 O HN N

O NH O HO 2 MF: C14H9N3O4S S 9 O NDc 93.19% MW: 315.30 HN N aLCMS mass was determined using Agilent MSD-1100 ESI-MS/MS system. bLCMS mass was determined using Agilent MSD-1100 APCI+mode-MS/MS system. cMass not detected by APCI+, APCI- S11 or ESI mode in LCMS. dPurity of the compounds were determined with an Hitachi 2000 series HPLC system using C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm) operating at 25 °C. Elution was carried out using acetonitrile as mobile phase A and water containing 0.1% formic acid +10 mmol NH4OAc as mobile phase B. Elution condition: at 0 min, phase A 10% + phase B 90%; at 45 min, phase A 90% + phase B 10%; at 50 min, phase A 10% + phase B 90%; at 60 min, phase A 10% + phase B 90%. The flow-rate of the mobile phase was 0.5 mL/min, and the injection volume of the sample was 5 μL. Peaks were detected at 254 nm (hit 3 at 235 nm).

Table S5. Kinase selectivity profile of hit 7 at 10 μM.a

Kinase Tested % Inhibition

ABL1 39

CDK2/cyclin A 37

CHEK1 (CHK1) 22

CHEK2 (CHK2) 7

FLT3 85

KDR (VEGFR2) 48

KIT 43

MAP2K1 (MEK1) 28

PLK1 9

aKinase profiling done by Life Technologies Corporation, using Z-LYTE® Screening assay.

S12