OPEN-ACCESS PEER-REVIEWED
Dr. Ningaraj Belagalla1*, Dr. Jaya Philip2, Dr. Sagar Ashok Jadhav3, Dr. Meenu Mangal4, Debojit Samajdar5, Dr. Praveen Katiyar6
1*Assistant Professor, Department of Entomology, School of Agriculture, SR University, Warangal-506371, Telangana, India
2Assistant professor, Department of Microbiology, Patna Women’s College Autonomous Patna University
3Assistant Professor, Dr. Shivajirao Kadam College of Pharmacy, Kasabe Digraj, Sangli, Maharashtra
4Professor, Department of Chemistry, Poddar International College, Mansarovar, Jaipur-302020, Rajasthan, India
5M. Pharm in Pharmaceutical Chemistry, Department of Pharmaceutical Chemistry, BCDA College of Pharmacy & Technology, Hridaypur, Kolkata-700127, West Bengal, India
6Associate Professor, School of Health Sciences, CSJM University, Kanpur
Abstract
This study investigates protein-ligand interactions using Surface Plasmon Resonance (SPR) and Mass Spectrometry (MS) to analyze the binding properties of four ligand-protein pairs: Warfarin with human serum albumin (HSA), Erlotinib with receptor tyrosine kinases (RTK), Methotrexate with an enzyme inhibitor, and Trastuzumab with an antibody. SPR analysis revealed varied binding kinetics for these interactions. Warfarin-HSA demonstrated a high affinity with a dissociation constant (KD) of 4.6 µM, while Erlotinib-RTK showed a KD of 13.3 µM, indicating moderate affinity. Methotrexate and Trastuzumab interactions also exhibited distinct kinetics with KDs of 53.7 µM and 30.4 µM, respectively. The binding kinetics were further analyzed by calculating the association rate constant (ka) and dissociation rate constant (kd). For Warfarin-HSA, ka was 1.2 × 105 M-1 s-1 and kd was 5.5 × 10-4 s-1, while Erlotinib-RTK had ka of 7.5 × 104 M-1 s-1 and kd of 1.0 × 10-3 s-1. MS analysis corroborated these results, providing mass shifts corresponding to the formation of protein-ligand complexes, with observed shifts of 12 Da for Warfarin-HSA and 5 Da for Erlotinib-RTK. The study confirms the reliability of SPR and MS for accurately measuring the binding affinity and interactions of protein-ligand complexes in drug discovery, with high reproducibility and statistical significance (p-value < 0.05).
Keywords: Protein-ligand interactions, Surface Plasmon Resonance (SPR), Mass Spectrometry (MS), Human serum albumin (HSA), Receptor tyrosine kinases (RTK).
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