PFKFB4-IN-1 is a potent and selective ATP-competitive inhibitor of PFKFB4 (IC50 = 4.50 μM) that reduces intracellular PFKFB4 protein levels. It exhibits over 12-fold selectivity for PFKFB1/4 and PFKFB3/4 isoforms. PFKFB4-IN-1 inhibits cancer cell proliferation, induces apoptosis, and suppresses cell migration. It also hinders tumor growth in MDA-MB-231 xenograft mouse models. PFKFB4-IN-1 is applicable for studies related to breast cancer, lung cancer, and liver cancer.

PFKFB4:4.50 μM

PFKFB4-IN-1 (compound 2v) exhibits broad-spectrum anticancer activity with IC50 values of 4.02 μM, 11.07 μM, and 11.2 μM against MCF-7, HepG2, and A549 cells respectively, while demonstrating significantly reduced cytotoxicity towards normal human liver cells (HL7702, IC50 > 50 μM). It inhibits the proliferation of MCF-7 breast cancer cells in a time- and concentration-dependent manner over 0-48 hours. The compound at 6-18 μM over 24 hours impedes migration and colony formation of MCF-7 cells and induces apoptosis through caspase-3 activation and PARP cleavage pathways. PFKFB4-IN-1 suppresses PFKFB4 protein expression in MCF-7 cells, inhibiting glycolytic metabolism and significantly reducing intracellular ATP and NADP+/NADPH levels. It stably binds within the ATP binding pocket of PFKFB4, forming critical hydrogen bonds with Asn168, Gly51, and Tyr428 residues. Additionally, PFKFB4-IN-1 does not significantly increase levels of reactive oxygen species (ROS) or DNA damage in MCF-7 cells.

PFKFB4-IN-1, when administered at a dose of 30 mg/kg through intraperitoneal injection once daily for 14 consecutive days, effectively inhibits tumor growth in an MDA-MB-231 xenograft mouse model. Additionally, PFKFB4-IN-1 shows good acute safety in a zebrafish developmental toxicity model when exposed at 5 μM starting within 24 hours post-fertilization and continuing for 72 hours.