The University of Toledo Translation Journal of Medical Sciences 
Nephrology Abstract, Department of Medicine Research Symposium UTJMS 2023 May 5; 11(1):e1-e1 

Computational and Experimental Analysis 
Reveals the Arachidonic Acid Metabolite 
20-Hydroxyeicosatetraenoic Acid is a Novel
Ligand of the Na/K-ATPase
Dhilhani Faleel1*, Shungang Zhang1, Jacob A. Connolly1, Deepak Malhotra1, Steven T. Haller2, John R. Falck1, 
David J. Kennedy2 
1Division of Nephrology, Department of Medicine, The University of Toledo, 
Toledo, OH 43614 
2Division of Cardiovascular Medicine, Department of Medicine, The University of 
Toledo, Toledo, OH 43614 

*Corresponding author: Dhilhani.Faleel@rockets.utoledo.edu

Published: 05 May 2023 

Objective: We sought to determine the ability of 20-HETE to bind with the NKA relative to other 
known NKA ligands using a computational molecular modeling approach. We further sought to test the 
ability of 20-HETE to stimulate NKA mediated signaling in renal proximal tubule cells.  

Methods: Computational molecular modeling to investigate the interaction of 20-HETE and NKA was 
performed using Maestro software analysis (Schrodinger 2021-2).  In vitro experiments of NKA 
signaling were performed with both 20-HETE and its stable analog, 5,14-20-HEDE, in renal LLC-PK1 
proximal tubule cells. 

Results: First, we performed induced fit docking to predict the binding free energy of both 20-HETE 
and its stable analog, 5,14-20-HEDE, in comparison with the well-established cardiotonic steroid NKA 
ligand telocinobufagin. This docking analysis predicted that 20-HETE and 5,14-20-HEDE interact with 
the NKA with similar binding free energy as cardiotonic steroids (Predicted binding free energies: 
telocinobufagin =-9.2; 20-HETE= -8.5 and 5,14-20-HEDE = -8.18). Further this computational 
modeling demonstrated that all of these molecules interact in the same binding pockets of the NKA. 
Next, our in-vitro experiments showed that 20-HETE and its analog 5,14-20-HEDE increased MAPK 
activation in a dose dependent manner from 10 nM to 10 uM in LLC-PK1 cell lines. This MAPK 
activation was significantly reduced after pretreatment with pNaKtide, a specific inhibitor of the NKA-
Src signaling complex (1uM pNaKtide, 30 minutes).   

Conclusion: The result of these study suggests that 20-HETE interacts with NKA in similar manner as 
cardiotonic steroids and is capable of inducing NKA signaling in renal proximal tubules. 

https://dx.doi.org/10.46570/utjms.vol11-2023-767 

https://dx.doi.org/10.46570/utjms.vol11-2023-767
mailto:Dhilhani.Faleel@rockets.utoledo.edu