(T1230-03-15) In Silico Screening Study of Potential Flavonoids as ADAMTS-5 Exosite Inhibitors for the Treatment of Knee Osteoarthritis

Purpose: Osteoarthritis is one of the chronic joint disorders that results in the degradation and damage of cartilage tissue. ADAMTS-5 is a potential pharmacological target in the treatment of knee osteoarthritis as it is the enzyme responsible for the aggrecan degradation in cartilage. The earliest aggrecanase inhibitors chelated the catalytic zinc ion to target the active site of ADAMTS-5. Owing to the lack of selectivity of the chelated zinc active site, exploring the Exosite domain is promising. The purpose of the study was to obtain a potential inhibitor for ADAMTS-5 Disintegrin and Spacer domains by in silico screening of flavonoids, due to their aggrecanase inhibitory properties. This would pave the pathway for the development of novel therapeutics for knee osteoarthritis.

Methods: The Disintegrin domain (Dis) of the ADMATS-5 structure (PDB: 2RJQ) was explored, and the 3D model of ADAMTS-5 Spacer domain (Sp) was predicted by Modeler in BIOVIA Discovery Studio. The high throughput screening study of the library of 847 flavonoids belonging to families of Flavonol, Anthocyanin, Flavanone, Flavanol, Isoflavanone and Flavone was performed to identify the potential flavonoids which had favorable ADMET and drug likeness properties by applying Lipinski Rule of five, Ghose filter, Veber filter, Egan Filter, Muegge filter, and PAINS score. Furthermore, the selected flavonoids were subjected to mutagenicity study based on TOPKAT_Ames_Mutagenecity Scoring. Following that, the lead compounds were further analyzed by molecular docking study with CDOCKER algorithm to understand the binding affinity of the flavonoid with ADAMTS-5 Exosite domain. Molecular Dynamic Simulation for the Dis Domain were performed to assess the complex stability over time points of 0ps to 50000ps using BIOVIA Discovery Studio 2024.

Results: The predicted 3D protein structure for the Sp domain was verified with Modeler and demonstrated a score of 140.03, which was in the acceptable range. The filtered library resulted in 16 flavonoids which are Eriodictyol, Biochanin A, Epigallocatechin, Cyanidin, Hesperetin, Naringenin, Kaempferol, Isohamnetin, Myriecetin, Quercetin, Calycosin, Fomononetin, Genistein, Chrysin, Jaceosidin and Luteolin. Molecular Docking provided insights into the interaction residues of ADAMTS-5 Dis and Sp domains. The residues in the Dis domain includes GLU538, VAL537, ALA536, PRO535. The residues in the Sp domain includes ILE738, HIS537, LYS579.  Among the 16 flavonoids docked at the Dis and the Sp domains, the highest binding affinity was shown by Eriodictyol with -23.102 kcal/mol and -14.74 kcal/mol respectively. The Cdocker Interaction Energy at both domains for this lead compound was determined to be -29.06 kcal/mol and -23.49 kcal/mol. The Molecular Dynamic Simulations were conducted for the targeted Dis domain of ADAMTS-5 empty protein structure and protein ligand complex with Eriodictyol. The results illustrated a stability of the empty protein and the protein-ligand complex from 0ps to 50000ps, with an average RMSD of 2.13Å and 1.97Å and RMSF values of 1.18Å and 1.05Å respectively.

Conclusion: A study was conducted to assess the ability of flavonoids to interact with the potential inhibitory effects on ADAMTS-5 at the Exosite domain which includes the Disintegrin and Spacer domains. The rigorous in silico screening revealed lead flavonoids that demonstrated key interactions at the Exosite domain which provides an avenue towards precision targeting. Similar RMSD values from MD simulation of the empty protein and the protein-ligand complex indicates the stable nature of the flavonoid with ADAMTS-5. The RMSD curve suggests a strong binding of Eriodictyol with ADAMTS-5 Exosite domain. Our research will open the door for more investigation of the inhibitory ability of Eriodictyol in the treatment of knee osteoarthritis.

References: 1. Santamaria S, Yamamoto K, Botkjaer K, Tape C, Dyson MR, McCafferty J, Murphy G, Nagase H. Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2). Biochem J. 2015 Nov 1;471(3):391-401. doi: 10.1042/BJ20150758. Epub 2015 Aug 24. PMID: 26303525; PMCID: PMC4613496
2. Cuffaro D, Ciccone L, Rossello A, Nuti E, Santamaria S. Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition. J Med Chem. 2022 Oct 27;65(20):13505-13532. doi: 10.1021/acs.jmedchem.2c01177. Epub 2022 Oct 17. PMID: 36250680; PMCID: PMC9620172
3. Santamaria, S., Cuffaro, D., Nuti, E. et al. Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide. Sci Rep 11, 949 (2021). https://doi.org/10.1038/s41598-020-80294-1

Acknowledgements: We would like to thank the Department of Computer Science, Digital Engineering and Artificial Intelligence, and the Department of Pharmaceutical Sciences, at Long Island University for all their support throughout the project. This work was supported by the Dassault Systems, BIOVIA platform. There are no conflicts to disclose regarding the work of this project.

Figure 1: 3D representation of ADAMTS-5 and Eriodictyol interaction at Disintegrin Domain.

Figure 2: 2D representation of ADAMTS-5 and Eriodictyol at Spacer Domain.

Figure 3: RMSD of ADAMTS-5 at Disintegrin Domain with and without Eriodictyol.