Affinity Capillary Electrophoresis to Probe Drug Interactions with Membrane Proteins in Their Native Lipid Environment

Membrane proteins are major pharmacological targets. Our previous work demonstrated that affinity capillary electrophoresis (ACE) allows robust hit validation and accurate quantification of dissociation constants when applied to purified soluble proteins.1–5 Extending this methodology to membrane proteins, however, introduces a distinct set of challenges. Their intrinsic instability in solution and reliance on native lipid environments continue to limit affinity measurements. Here, we present the first application of ACE to probe molecular interactions directly at the surface of native Golgi membrane fractions isolated from human cancer cells (Figure 1). Using this approach, we quantified the binding affinities of two inhibitors of UDP‑glucose ceramide glucosyltransferase (UGCG), an intracellular membrane enzyme implicated in cancer progression and chemoresistance. ACE enables direct measurement of target engagement for low‑abundance membrane proteins while preserving their native lipid context, providing a robust analytical platform suitable for complex biological samples. This methodology broadens current strategies for membrane‑protein drug discovery and addresses a key gap in evaluating pharmacological interactions under pathologically relevant conditions.