Pre-miR-21 Conformational Equilibrium Modulates the Efficacy of the Maturation Inhibitor L50

The cyclic peptide L50 is a promising anticancer candidate that inhibits miR-21 maturation by targeting the Dicer cleavage site of pre-miR-21. A recent study revealed that pre-miR-21 exists in equilibrium between an Ade-29-deprotonated Bulged state and a Dicer-susceptible Ade-29-protonated Paired state. However, the impact of this equilibrium on L50 efficacy remains unclear. Here, we addressed this question by conducting biochemical and structural analyses under various pH conditions. We found that L50 exhibits reduced inhibitory activity and affinity for pre-miR-21 in the Paired state. The analysis of the molecular interface suggested that changes in the L50 binding mode during the conformational transition of pre-miR-21 to the Paired state lead to the loss of key interactions required for high-affinity binding. These findings explain the limited efficacy of L50 in inhibiting miR-21 maturation, provide insights into its optimization, and highlight the importance of considering RNA conformational equilibrium in RNA-targeting drug development.