Dual Conjugation of Long- and Medium-Chain Fatty Acids to BimBH3 Peptide Yields Ultra Long-Acting Inhibitors of Intracellular PTPN1/2

Fatty acid derivatization is a promising strategy for discovering long-acting peptide therapeutics, but intracellular targeting remains challenging due to insufficient membrane permeability. Here, we report a dual fatty acid conjugation approach to develop ultralong-acting inhibitors of intracellular PTPN1/2. By conjugating a long-chain fatty acid/diacid to the N-terminus and a medium-chain fatty acid/diacid to Lys² of BimBH3 peptide, we achieved efficient cell permeability and uptake for intracellular target inhibition and metabolic stability for long-acting <i>in vivo</i> efficacy. The optimized analogue D6 exhibited potent dual PTPN1/2 inhibition (IC₅₀ = 107.6 nM and 3375 nM), 40-fold improved DPP-IV stability, and prolonged plasma half-life (>200 h) in rats following sc administration. D6 exhibited efficient cell permeability, restored insulin signaling in HepG2 cells and demonstrated once-weekly glycemic control in db/db mice. Molecular docking revealed key interactions with PTPN1/2 active sites. This work demonstrates a strategy for designing cell-permeable, long-acting peptide inhibitors of intracellular targets.