Design, synthesis and anti-AML activity of guanidyl stapled peptide targeting MLAA-34/MST3 interaction

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy, where drug resistance and systemic toxicities remain major clinical challenges. Recently, MLAA-34 has emerged as a promising anti-AML target that interacts with the MST3 protein. Herein, based on the binding epitope of the MST3 protein, we established a computer-aided rational design workflow to engineer an optimized 18-residue linear baseline peptide (MST3-0), and subsequently developed a series of all-hydrocarbon and guanidyl-stapled analogues. Among these, the optimal guanidyl-stapled peptide, MST3-9-3d, synthesized via our solid-phase side-chain guanidyl-construction strategy, exhibited a markedly increased α-helical content and enhanced proteolytic stability against chymotrypsin. Crucially, isothermal titration calorimetry (ITC) confirmed that MST3-9-3d possessed the highest binding affinity (K_D = 4.69 ± 0.69 μM) to MLAA-34. Benefiting from the introduced guanidyl groups, MST3-9-3d displayed dramatically improved cellular uptake and potent anti-proliferative activity against OCI-AML3 cells (IC₅₀ = 22.37 μM), outperforming both its linear and hydrocarbon-stapled counterparts. Furthermore, MST3-9-3d demonstrated favorable biocompatibility with negligible hemolytic toxicity and high selectivity over normal bone marrow-derived macrophages. In vivo pharmacokinetic evaluation in mice revealed a prolonged half-life (T_1/2 = 3.52 h) and sustained systemic exposure. Molecular dynamics simulations unveiled that the guanidyl staple not only rigidified the peptide conformation but also directly engaged in target recognition by forming extra intermolecular hydrogen bonds. Together, these findings highlight MST3-9-3d as a highly promising lead inhibitor targeting the MLAA-34/MST3 interaction for AML therapy.