PHARMACOPHORE DEVELOPMENT AND SCREENING FOR DISCOVERY OF POTENTIAL INHIBITORS OF ADAMTS-4 FOR OSTEOARTHRITIS THERAPY

PriyankaVerma, [b] Krishna Dalal, [b]and Madhu Chopra,*[a]

Corresponding author: Dr.Madhu Chopra

[a] Dr.Madhu Chopra

Laboratory of Molecular Modelling& Drug Design,

Dr. B. R. AmbedkarCenter for Biomedical Research,

University of Delhi, Delhi 110007, India

Tel.: +91-11-27666272; fax: +91-11-27666248;

E-mail: ,

[b] Dr.PriyankaVerma,

Department of Biophysics

All India Institute of Medical Sciences

Ansari Nagar, New Delhi-110029, India

E-mail:

Dr. Krishna Dalal

Department of Biophysics

All India Institute of Medical Sciences

Ansari Nagar, New Delhi-110029, India

E-mail:

Supplementary Material

Contents

Purity of Maybridge compounds / S2
Controls used in in-vitro study / S2
Selectivity of Compounds / S2
Table ST1: Scores of 10 common feature hypotheses (HipHop) / S2
Fingerprint based similarity analysis / S3
Table ST2: Tanimoto coefficients (Tc)of all 13 compounds / S3

Purity of Maybridge compounds:

MaybridgeHitFinder™ compounds have been analyzed as having a purity of ≥90%, with the majority having a purity of ≥95%.

Controls used in in-vitro study:

We have used SensoLyte® 520 Aggrecanase-1 Assay Kit *Fluorimetric* having:

•Positive control contains the aggrecanase enzyme without test compound.

•Inhibitor control contains aggrecanase and TAPI-O inhibitor (supplied in assay kit).

Selectivity of Compounds:

We have performed an assay which was specific for ADAMTS-4 only. The SensoLyte®520 Aggrecanase-1 Assay Kit is optimized to detect activity of ADAMTS- 4 and for screening of inhibitors. This kit contains a novel internally quenched 5-FAM/TAMRAFRET substrate for aggrecanase-1 (km~5M). Active ADAMTS-4 cleaves the FRET substrate into two separate fragments resulting in an increase of 5-FAM fluorescence which can be monitored atexcitation/emission= 490 nm/520 nm. The cleavage of the FRET ADAMTS-4 substrate by other members of aggrecanase family, such as ADAMTS-1 and ADAMTS-5, is negligible. The assay can detect as low as 0.1 ng/mL active aggrecanase-1.

Table ST1: Scores of 10 common feature hypotheses (HipHop)

S.no / Features / Rank / Direct Hit / Partial Hit / Max Fit
1 / RNZZHA / 127.472 / 111111 / 0 / 6
2 / RNZZHA / 127.472 / 111111 / 0 / 6
3 / RRNZHA / 127.453 / 111111 / 0 / 6
4 / RRNZHA / 127.453 / 111111 / 0 / 6
5 / RRNZHA / 127.453 / 111111 / 0 / 6
6 / RRNZHA / 127.453 / 111111 / 0 / 6
7 / RNZZHA / 127.357 / 111111 / 0 / 6
8 / RNZZHA / 127.357 / 111111 / 0 / 6
9 / RRNZHA / 127.334 / 111111 / 0 / 6
10 / RRNZHA / 127.334 / 111111 / 0 / 6

Fingerprint based similarity analysis

The chemical similarity search was conducted with DS using the standard protocol. The pairwise similarity was assessed by calculating Tanimoto coefficients (Tc)between ordered 13 compounds and training compounds set and each active compound in the test set. The values of the Tanimoto coefficients was obtained in the range of 0.08 – 0.33. These low value of Tanimoto similarity index suggest the novelty of the compounds screened in this work.

Table ST2:Tanimoto coefficients (Tc)of all 13 compounds

Test&Training set compound / 257 (Tc) / 433 (Tc) / 409 (Tc) / 168 (Tc) / 298 (Tc) / 301 (Tc) / 411 (Tc) / 180 (Tc) / 452 (Tc) / 421 (Tc) / 295 (Tc) / 414 (Tc) / 430 (Tc)
T1 / 0.15 / 0.24 / 0.22 / 0.18 / 0.17 / 0.08 / 0.27 / 0.14 / 0.11 / 0.23 / 0.25 / 0.20 / 0.21
T2 / 0.20 / 0.25 / 0.25 / 0.21 / 0.23 / 0.09 / 0.32 / 0.15 / 0.12 / 0.24 / 0.29 / 0.21 / 0.21
T3 / 0.22 / 0.26 / 0.25 / 0.21 / 0.23 / 0.10 / 0.31 / 0.14 / 0.12 / 0.25 / 0.31 / 0.22 / 0.22
T4 / 0.18 / 0.24 / 0.23 / 0.2 / 0.22 / 0.10 / 0.33 / 0.15 / 0.12 / 0.22 / 0.27 / 0.20 / 0.20
T5/Pdb / 0.19 / 0.25 / 0.24 / 0.20 / 0.22 / 0.10 / 0.33 / 0.14 / 0.11 / 0.23 / 0.28 / 0.20 / 0.21
T6 / 0.20 / 0.25 / 0.25 / 0.20 / 0.22 / 0.10 / 0.33 / 0.14 / 0.11 / 0.24 / 0.28 / 0.21 / 0.21
TS1 / 0.16 / 0.24 / 0.23 / 0.18 / 0.16 / 0.09 / 0.28 / 0.13 / 0.11 / 0.23 / 0.26 / 0.20 / 0.21
TS2 / 0.21 / 0.27 / 0.26 / 0.19 / 0.19 / 0.11 / 0.32 / 0.14 / 0.11 / 0.25 / 0.30 / 0.22 / 0.22
TS3 / 0.12 / 0.20 / 0.22 / 0.14 / 0.10 / 0.09 / 0.23 / 0.13 / 0.12 / 0.23 / 0.25 / 0.20 / 0.20
TS4 / 0.21 / 0.29 / 0.27 / 0.25 / 0.23 / 0.09 / 0.33 / 0.19 / 0.17 / 0.28 / 0.30 / 0.25 / 0.25
TS5 / 0.22 / 0.28 / 0.27 / 0.25 / 0.26 / 0.08 / 0.31 / 0.17 / 0.18 / 0.27 / 0.31 / 0.24 / 0.24
TS6 / 0.21 / 0.27 / 0.26 / 0.19 / 0.19 / 0.11 / 0.32 / 0.14 / 0.11 / 0.25 / 0.30 / 0.22 / 0.22
TS7 / 0.21 / 0.27 / 0.26 / 0.19 / 0.2 / 0.11 / 0.32 / 0.14 / 0.11 / 0.25 / 0.30 / 0.22 / 0.22
TS8 / 0.26 / 0.27 / 0.27 / 0.17 / 0.18 / 0.13 / 0.33 / 0.15 / 0.12 / 0.25 / 0.30 / 0.23 / 0.23
TS9 / 0.19 / 0.26 / 0.26 / 0.18 / 0.18 / 0.10 / 0.31 / 0.14 / 0.11 / 0.25 / 0.29 / 0.22 / 0.22
TS10 / 0.24 / 0.24 / 0.24 / 0.18 / 0.22 / 0.10 / 0.33 / 0.15 / 0.12 / 0.23 / 0.28 / 0.20 / 0.20
TS11 / 0.17 / 0.24 / 0.26 / 0.2 / 0.16 / 0.11 / 0.27 / 0.16 / 0.20 / 0.28 / 0.29 / 0.25 / 0.24
TS12 / 0.22 / 0.26 / 0.25 / 0.17 / 0.17 / 0.15 / 0.31 / 0.15 / 0.11 / 0.24 / 0.30 / 0.22 / 0.22

S1