Preliminary Anti-Melanoma Activity of a Chlorogenic Acid-Based PROTAC Targeting MDM4, a Candidate Protein Identified by Proteomics

Chlorogenic acid (CGA), which is ubiquitous in diverse botanical sources, demonstrates considerable anticancer potential through modulation of multiple targets or signaling pathways, thereby posing substantial challenges for mechanistic elucidation and target identification. Based on the proteolysis targeting chimera (PROTAC) technology's ability to induce targeted protein degradation via ubiquitin-proteasome pathway recruitment, we synthesized a panel of CGA-PROTACs. These compounds incorporated the natural product CGA as the target-binding ligand, conjugated to pomalidomide (an E3 ligase-recruiting moiety) via various synthetic linkers. The findings indicated that compound <b>A7</b>, linked with an alkane linker, exhibited a notable anti-proliferative effect on 4T1 and A375 cells in vitro. The IC₅₀ value of <b>A7</b> on A375 cells reached 69.70 μM, which is 2.2 times better than the effect of the precursor compound CGA (IC₅₀ = 148.80 μM). Mouse double minute 4 (MDM4) was confirmed as a potential target of compound <b>A7</b> through a combination of proteomics, Western blot analysis and molecular dynamics simulation. CGA-PROTAC <b>A7</b> treatment led to a dose-dependent reduction in MDM4 protein levels while significantly upregulating p53 and p21 protein expression, and thus inhibited proliferation, induced G2/M phase cell cycle arrest, and markedly enhanced apoptosis in melanoma A375 cells. This study successfully applied an effective strategy for target identification and medication discovery of natural compounds. In addition, CGA-PROTAC <b>A7</b> was synthesized in one step with an overall yield of 45.96%, providing a feasible route for synthesis and establishing a basis for the combination of natural product polyphenols with PROTAC technology.