Design, synthesis and anticancer potential of new peptide conjugates via urokinase inhibition: biological and in silico studies

A new series of peptide derivatives was designed, synthesized, and structurally confirmed. The target compounds were evaluated for their anticancer activity against four human cancer cell lines: HepG-2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), HeLa (cervical epithelioid carcinoma), and MDA-MB-231 (triple-negative breast cancer). Eight compounds exhibited exceptional broad-spectrum cytotoxicity, outperforming the standard drug Doxorubicin across most tested cell lines. Consequently, their inhibitory activity against the urokinase-type plasminogen activator (uPA) enzyme was evaluated using Rivaroxaban as a reference standard. Hybrids bearing heterocyclic acid moieties, compounds 13 and 18, emerged as the most potent uPA inhibitors, displaying IC50 of 0.546 ± 0.018 μM and 0.698 ± 0.023 μM, respectively-both superior to Rivaroxaban (IC50 = 0.729 ± 0.024 μM). Mechanistic studies on MCF-7 cells demonstrated that compounds 13 and 18 induced cell cycle arrest at the G2/M phase and triggered apoptosis (31.41% and 23.25%, respectively) with low necrosis (3.81% and 2.94%, respectively, vs. 1.71% in control). RT-qPCR analysis revealed significant upregulation of pro-apoptotic Bax (6.55 and 4.07-fold) and downregulation of anti-apoptotic Bcl-2 (0.45- and 0.22-fold), confirming activation of apoptosis pathways. Molecular docking, binding energy calculations, and surface mapping against uPA highlighted critical interactions with key active site residues (Gly219, Cys220, Arg217, and Gln192), rationalizing their potent enzymatic inhibition. Furthermore, molecular dynamics simulations provided critical atomistic insights into the conformational stability and binding mechanisms, highlighting the therapeutic potential of the identified compounds.