Identification and validation of a small molecule targeting ROR1 for the treatment of triple negative breast cancer

Shradheya R R Gupta; Tram M Ta; Maryam Khan; Archana Singh; Indrakant K Singh; Bela Peethambaran

doi:10.3389/fcell.2023.1243763

Identification and validation of a small molecule targeting ROR1 for the treatment of triple negative breast cancer

Front Cell Dev Biol. 2023 Sep 13:11:1243763. doi: 10.3389/fcell.2023.1243763. eCollection 2023.

Authors

Shradheya R R Gupta¹, Tram M Ta², Maryam Khan², Archana Singh³, Indrakant K Singh^{1

4

5}, Bela Peethambaran²

Affiliations

¹ Molecular Biology Research Laboratory, Department of Zoology, Deshbandhu College, University of Delhi, New Delhi, India.
² Department of Biology, Saint Joseph's University, Philadelphia, PA, United States.
³ Department of Botany, Hans Raj College, University of Delhi, New Delhi, India.
⁴ Delhi School of Public Health, Institute of Eminence, University of Delhi, New Delhi, India.
⁵ Norris Comprehensive Cancer Center, Division of Medical Oncology, University of Southern California, Los Angeles, CA, United States.

Abstract

Introduction: Breast cancer is the most common cancer in women, with roughly 10-15% of new cases classified as triple-negative breast cancer (TNBC). Traditional chemotherapies are often toxic to normal cells. Therefore, it is important to discover new anticancer compounds that target TNBC while causing minimal damage to normal cells. Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is an oncofetal protein overexpressed in numerous human malignancies, including TNBC. This study investigated potential small molecules targeting ROR1. Methodology: Using AutoDock Vina and Glide, we screened 70,000 chemicals for our investigation. We obtained 10 representative compounds via consensus voting, deleting structural alerts, and clustering. After manual assessment, compounds 2 and 4 were chosen for MD simulation and cell viability experiment. Compound 4 showed promising results in the viability assay, which led us to move further with the apoptosis assay and immunoblotting. Results: Compound 4 (CID1261330) had docking scores of -6.635 and -10.8. It fits into the pocket and shows interactions with GLU64, ASP174, and PHE93. Its RMSD fluctuates around 0.20 nm and forms two stable H-bonds indicating compound 4 stability. It inhibits cell proliferation in MDA-MB-231, HCC1937, and HCC1395 cell lines, with IC₅₀ values of approximately 2 μM to 10 μM, respectively. Compound 4 did not kill non-malignant epithelial breast cells MCF-10A (IC₅₀ > 27 μM). These results were confirmed by the significant number of apoptotic cells in MDA-MB-231 cells (47.6%) but not in MCF-10A cells (7.3%). Immunoblot analysis provided additional support in the same direction. Discussion: These findings collectively suggest that compound 4 has the potential to effectively eliminate TNBC cells while causing minimal harm to normal breast cells. The promising outcomes of this study lay the groundwork for further testing of compound 4 in other malignancies characterized by ROR1 upregulation, serving as a proof-of-concept for its broader applicability.

Keywords: MCF-10A; MDA-MB-231; Ror1; TNBC; breast cancer; kinase.

Grants and funding

The research in BP’s laboratory was supported by the Abraham Edythe Endowed Funds in Natural Products awarded to BP by St. Joseph’s University.