Targeted Penetrating Motif Engineering of BH3 Mimetic: Harnessing Non‐Canonical Amino Acids for Coinhibition of MCL‐1 and BCL‐xL in Acute Myeloid Leukemia

Abstract Acute Myeloid Leukemia (AML) remains a formidable clinical challenge, predominantly due to the emergence of resistance to existing therapeutic regimens, including BCL‐2 inhibitors like Venetoclax. Here, a novel approach is introduced by engineering BH3 mimetics utilizing non‐canonical amino acids (ncAAs) to achieve dual inhibition of MCL‐1 and BCL‐xL. Through site saturation mutagenesis scanning, the I58(Chg) mutation is identified, significantly enhancing binding affinity with IC ₅₀ values of 2.77 n m for MCL‐1 and 10.69 n m for BCL‐xL, reflecting an increase of fourfold or more. The developed vMIP‐II‐TAT‐I peptide, incorporating a CXCR4‐targeted penetrating motif, demonstrated superior cellular uptake, with mean fluorescence intensity (MFI) 7.2‐fold higher in CXCR4‐positive AML cells and exhibited a high selectivity index (SI) for AML cells, with minimal impact on normal human hematopoietic stem cells (HSCs). When combined with Venetoclax, this peptide induced synergistic apoptosis, reducing tumor burden and prolonging survival in an AML mouse model, with median survival extended to 53 days from 37 days with Venetoclax alone. These findings reveal the therapeutic potential of dual inhibition in overcoming Venetoclax resistance and selectively targeting leukemic cells with reduced off‐target effects, while laying the foundation for developing advanced BH3 mimetics with enhanced targeting, binding affinity, and efficacy for AML treatment.