Type 2 diabetes (T2D) poses a global burden, with insulin resistance driven by c-Jun N-terminal kinase 1 (JNK1) hyperactivation. Here, we have reported the rational design and synthesis of five novel peptide inhibitors (JKPs 1-5) targeting JNK1. By using a pharmacophore model and virtual screening, we identified JKPs with sub-micromolar binding to JNK1, with JKP-5 showing the highest affinity (Kd = 40.16 ± 2.11 nM) and high selectivity. Additionally, JKP-5 was confirmed to possess excellent serum stability and cell permeability. Functional validation experiments demonstrated that, in insulin-resistant HepG2 cells, JKP-5 remarkably enhanced glucose uptake and restored glycogen content without cytotoxicity. Importantly, in diabetic db/db mice, JKP-5 lowered non-fasting blood glucose, improved glucose tolerance, and decreased serum insulin levels, indicating improved insulin sensitivity. Notably, it showed no hypoglycemic effects in normal mice. These findings validate JKP-5 as a potent, safe lead compound targeting JNK1-mediated insulin resistance, highlighting structure-based design and peptide synthesis as effective strategies for T2D therapy.
