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

“Design, synthesis and anti-AML activity of guanidyl stapled peptide targeting MLAA-34/MST3 interaction. Chao J(1), Shen Y(2), Gao S(3), Shen H(2), Zhang S(1), Xu Y(4), Shi Y(3), Cong W(2), Niu F(5), Li X(6), Chen S(7). Author information: (1)School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China. (2)School of Medicine, Shanghai University, 99 Shangda Road, Shanghai, 200444, China. (3)Institute of Translational Medicine, Shanghai University, 99 Shangda Road, Shanghai, 200444, China. (4)School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China; School of Medicine, Shanghai University, 99 Shangda Road, Shanghai, 200444, China. (5)The department of Hematology, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, 710049, China. Electronic address: niufan@xjtufh.edu.cn. (6)School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China. Electronic address: xiangli@smmu.edu.cn. (7)School of Medicine, Shanghai University, 99 Shangda Road, Shanghai, 200444, China. Electronic address: caroline-sisi-chen@hotmail.com. 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 (KD = 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 (T1/2 = 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. Copyright © 2026 Elsevier Masson SAS. All rights reserved. Conflict of interest statement: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.”