XAN-5

XAN-5 is a mitochondrial DNA G-quadruplex (mtG4) ligand with a Kd value of 3.8 μM. It selectively binds to and stabilizes mtG4 structures, disrupting mitochondrial gene transcription and DNA replication. This compound induces mitochondrial dysfunction, leads to excessive production of reactive oxygen species (ROS), causes G0 phase arrest, and triggers caspase-dependent apoptosis. XAN-5 inhibits autophagy and induces immunogenic cell death. In mouse liver cancer models, XAN-5 inhibits tumor growth and enhances tumor-infiltrating CD4+ and CD8+ T cells. Additionally, XAN-5 targets two tumor resistance mechanisms and is applicable in hepatic cancer research.

XAN-5 stabilizes the mtG4 model mt6363 with a ΔTm of 10 °C when used at 10 μM for 2 hours. It selectively accumulates in the mitochondria of HepG2 cells at 5 μM for 30 minutes. XAN-5 effectively inhibits HepG2 cell proliferation (IC50 = 1.8 μM) with low toxicity to normal liver and breast epithelial cells at concentrations of 0.25-4 μM for 24 hours. It significantly reduces the transcription levels of OXPHOS genes ATP6, CYTB, and COX-1 in HepG2 cells at 1-4 μM for 24 hours. XAN-5, in a dose-dependent manner, inhibits mtDNA replication, reducing D-Loop copy number by 80% at 4 μM for 24 hours. It induces concentration-dependent overproduction of reactive oxygen species, dissipation of mitochondrial membrane potential, and decreased ATP levels in HepG2 cells at 1-4 μM for 24 hours. At 1 μM, XAN-5 causes G0/G1 phase arrest, while at 2 μM and 4 μM, the sub-G0 apoptotic cell population increases progressively. XAN-5 induces dose-dependent apoptosis in HepG2 cells, with 30% Annexin V-positive cells at 4 μM for 24 hours. Additionally, it inhibits autophagy in HepG2 cells, evidenced by decreased LC3B-II conversion, p62 accumulation, suppressed ATG gene expression, and reduced DAPGreen fluorescence intensity, mediated by reactive oxygen species at 1-4 μM for 24 hours. XAN-5 induces immunogenic cell death in HepG2 cells, characterized by increased calreticulin expression, elevated cell surface calreticulin, and higher extracellular ATP levels at 1-4 μM for 24 hours. Furthermore, it significantly inhibits HepG2 cell migration, colony formation, and 3D tumor spheroid growth in a concentration-dependent manner at 0.25-4 μM over 0-7 days.

XAN-5 (10 mg/kg; administered every 48 hours for 6 days) reduces tumor volume by 59% in a BALB/c mouse H22 liver cancer model. It inhibits tumor cell proliferation and increases the frequency of CD4+ and CD8+ T cells in both the tumor and spleen, without notable systemic toxicity at the tested dose.