Directly capturing native fatty acid synthase from cancer cells by affinity-based probe for precise inhibitor discovery

Target-based screening is vital for drug lead discovery, yet it often involves isolating, purifying or immobilizing specific targets. However, limitations arise, such as the use of cell-free systems or non-diseased tissue, potentially leading to screening inaccuracies. Moreover, protein purification and immobilization may impact the target's structural integrity and active sites. To address these limitations, we have developed a precise inhibitor discovery method that involves directly capturing native fatty acid synthase (FASN) from cancer cells based on affinity-based protein profiling (AfBPP) technology, with FASN as the designated target. Firstly, we initiated the design and synthesis of a FASN affinity-based probe (FAP), which comprised of a FASN-specific inhibitor-based warhead linked through an ester bond to a multifunctional small molecule incorporating both a photoreactive group and an alkyne handle. The warhead and photoreactive group enable the probe to precisely recognize and covalently capture of FASN from cancer cell lysates. The warhead, originally bound to FASN, was then disassociated through acid treatment, followed by hydrolysis to remove it according to the prodrug principle, ultimately yielding FASN with an alkyne handle. Subsequently, the captured FASN was immobilized onto the azide-functionalized silica gel (PFASG) materials through a click reaction, leading to the formation of FASN immobilized PFASG (FASN/PFASG) affinity materials. FASN/PFASG affinity materials were successfully confirmed by material surface characterization and enzyme activity testing. Moreover, FASN/PFASG affinity materials demonstrated exceptional binding capacity, selectivity and durability. Finally, we employed FASN/PFASG affinity materials in the screening of FASN inhibitors from Ginkgo biloba leaves extracts. This study presents a novel approach for directly capturing specific targets from human cancer cells, eliminating the necessity for traditional protein purification and facilitating inhibitor screening.