Comparative Saturation Binding Analysis of 64Cu-Labeled Somatostatin Analogues Using Cell Homogenates and Intact Cells

Abstract

The development of novel ligands for G-protein-coupled receptors (GPCRs) typically entails the characterization of their binding affinity, which is often performed with radioligands in a competition or saturation binding assay format. Since GPCRs are transmembrane proteins, receptor samples for binding assays are prepared from tissue sections, cell membranes, cell homogenates, or intact cells. As part of our investigations on modulating the pharmacokinetics of radiolabeled peptides for improved theranostic targeting of neuroendocrine tumors with a high abundance of the somatostatin receptor sub-type 2 (SST₂), we characterized a series of ⁶⁴Cu-labeled [Tyr³]octreotate (TATE) derivatives in vitro in saturation binding assays. Herein, we report on the SST₂ binding parameters measured toward intact mouse pheochromocytoma cells and corresponding cell homogenates and discuss the observed differences taking the physiology of SST₂ and GPCRs in general into account. Furthermore, we point out method-specific advantages and limitations.

Figure 1

Comparison of saturation binding data using cell homogenates (CH) and intact cells (IC) (A/B) saturation binding of [⁶⁴Cu]Cu-NODAGA-NES5-TATE toward MPC cell homogenates (A) and intact cells (B) with data for total and nonspecific binding (in the presence of 1 μM DOTA-TATE) shown as filled and open circles, respectively. Plot in B was published recently by us (Adapted with permission from Brandt et al. Copyright 2023, American Chemical Society). Regression analysis was performed by using the model of “one site—total and nonspecific binding” as implemented in GraphPad Prism (Version 9.4.1). (C,D) Correlation plots for the ratio paired t tests of K_d (C) and B_max (D) data with results of the tests given in the boxes.