Modulation of SERCA1a by Sesquiterpene Lactones: Insights into Inhibition Mechanisms

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a) is a key regulator of intracellular calcium homeostasis and an emerging therapeutic target in cancer, cardiac, and metabolic diseases. Sesquiterpene lactones such as thapsigargin (TG) and trilobolide (TB) inhibit SERCA1a via transmembrane binding, yet systematic comparisons of their inhibitory potency and biophysical mechanisms remain limited. Here, we investigated the inhibitory effects of TG, TB, and two TB derivatives (TB-44, TB-46(2)) using kinetic, thermodynamic, and docking analyses. SERCA1a activity in isolated sarcoplasmic reticulum vesicles was quantified by an NADH-coupled ATPase assay, with Ca²⁺- and ATP-dependence modeled by Michaelis-Menten and Hill fits. All compounds inhibited SERCA1a in a dose-dependent manner, with TB most potent (IC₅₀ = 0.36 µM vs. TG 0.60 µM) and TB-44 the most effective derivative (IC₅₀ = 1.47 µM). Each reduced V_max while largely preserving Ca²⁺ sensitivity, consistent with stabilization of a low-turnover SERCA1a conformation and impaired ATP-driven catalysis. Notably, TB's enhanced potency, despite intermediate calculated binding energy (ΔG = - 9.1 kcal/mol), correlated with moderate lipophilicity (logP = 2.43) and favorable electrostatic contacts with Gln259. These data highlight that effective SERCA1a inhibition by sesquiterpene lactones reflects an interplay between lipophilicity, membrane partitioning, and electrostatic-hydrophobic complementarity, rather than binding affinity alone.