Hazards of using “masking protocols” when performing ligand binding assays: lessons to be learned from the sigma-1 and sigma-2 receptors

H. Abbas¹, D. Ferry², S. T. Safrany³. ¹Pharmacy, University of Wolverhampton, Wolverhampton, United Kingdom, ²Eli Lilly, Indianapolis, ³RCSI-Bahrain, Adliya, Bahrain.

Introduction: Sigma-2 receptors are emerging cancer therapeutic targets [1]. Unfortunately, quantitation of sigma-2 receptors frequently employs the use of the non-selective pan sigma radioligand, di-O-tolyl guanidine (DTG) in the presence of sigma-1 selective agents ((+) pentazocine or dextrallorphan) to “mask” this receptor [2, 3]. Here, we explain why this approach should not be used.

Methods: We have calculated saturation and competition binding data using previously published affinities of sigma ligands [4, 5, 6] using standard rectangular hyperbolic and sigmoidal parameters. We have determined how many sigma-1 receptors remain masked and how many sigma-2 receptors are undiscovered using these protocols. Data analysis has been performed using GraphPad Prism.

Results: Modelling the two concentrations of (+) pentazocine widely used in “masking” protocols (100 nM [2], 1 μM [3]), we calculate that 85-99% of sigma-1 receptors are bound; additionally, 1-58% of sigma-2 receptors are also bound. The addition of the non-selective radioligand [³H] DTG at concentrations frequently used for saturation binding of sigma-2 receptors (up 256 nM) can compete out up to 51% of (+) pentazocine from sigma-1 receptors. In contrast, up to 27% of sigma-2 receptors may remain bound by (+) pentazocine. Modelling performed using dextrallorphan (1 μM) identifies that it would bind 86-98% of sigma-1 receptors and 2.2-6.3% of sigma-2 receptors. DTG would displace up to 16% of dextrallorphan from sigma-1 receptors. Up to 15% of the dextrallorphan would also remain bound to sigma-2 receptors.

Conclusions: The protocols currently used are misleading, and will label a significant proportion of sigma-1 receptors when quantifying sigma-2 receptors. These results may explain why all systems tested appear to have sigma-2 receptors. We advise researchers to stop using such masking protocols.

References:

[1] van Waarde A et al., (2015) Biochim Biophys Acta 1848: 2703-2714;

[2] Chu UB and Ruoho AE (2015) Curr Protoc Pharmacol71: 1.34.1-1.34.21;

[3] Abate C et al., Eur J Med Chem 69: 920-930;

[4] Xu R et al., (2015) Bioorg Med Chem 23: 222-230;

[5] Vilner BJ and Bowen WD (2000) J Pharmacol Exp Ther 292: 900-911;

[6] Lever JR et al., (2006) Synapse 59: 350-358.