脂肪酸合成酶酰基载体蛋白结构域纳米抗体的筛选及鉴定

Screening and characterization of nanobodies against the acyl carrier protein domain of fatty acid synthase

摘要:

脂肪酸合成酶(fatty acid synthase, FASN)是催化脂肪酸从头合成的关键酶，其在多种肿瘤中的异常高表达与肿瘤侵袭性及不良预后密切相关，是抗肿瘤药物开发的重要靶点。然而，现有针对FASN催化结构域的小分子抑制剂常面临靶点同源性高、潜在脱靶风险大等挑战。为寻找更具特异性的新型调控位点，本研究以FASN高度动态的酰基载体蛋白(acyl carrier protein, ACP)结构域为靶标开展纳米抗体筛选，旨在评估并验证其作为潜在调控靶点的可行性。首先，利用纳米抗体酵母表面展示库(库容量1×10⁸)进行2轮磁珠分选和1轮流式筛选，筛选并获得了18种靶向ACP的纳米抗体。其次，使用表面等离子共振实验验证其结合能力。最后，使用细胞内外活性检测方法并通过与穿膜肽TAT融合，进一步表征纳米抗体对FASN活性的抑制效果。结果表明，多株纳米抗体与靶蛋白具有明确的结合活性，亲和力达纳摩尔级别。其中，NbB-11对FASN抑制活性表现最佳，在3 μmol/L浓度下对FASN体外活性的抑制率为28.2%。此外，通过与穿膜肽TAT融合构建的TAT-NbB-11蛋白成功实现了跨膜递送，在4 μmol/L浓度下表现出22.0%的细胞增殖抑制率。本研究成功淘选出靶向FASN的高亲和力纳米抗体，初步验证了ACP结构域作为FASN活性调控靶点的可行性，为后续的纳米抗体工程化改造及体内验证提供了基础先导分子。

Abstract:

Fatty acid synthase (FASN) is a key enzyme catalyzing the de novo biosynthesis of fatty acids. Its aberrant overexpression in multiple tumor types is closely correlated with tumor invasiveness and poor prognosis, making it a critical target for anti-cancer drug development. However, existing small-molecule inhibitors targeting the catalytic domains of FASN frequently encounter challenges such as high target homology and considerable off-target risk. To explore novel regulatory sites with higher specificity, this study targeted the highly dynamic acyl carrier protein (ACP) domain of FASN for nanobody selection, aiming to evaluate and validate its feasibility as a potential therapeutic target. Initially, utilizing a yeast surface-display nanobody library (with a capacity of 1×10⁸), two rounds of magnetic-activated cell sorting (MACS) and one round of fluorescence-activated cell sorting (FACS) were performed, and 18 nanobodies targeting the ACP domain were obtained. Subsequently, their binding affinities were verified using surface plasmon resonance (SPR) assays. Finally, the inhibitory effects of these nanobodies on FASN activity were further characterized using both cell-free and cell-based activity assays, combined with fusion to the cell-penetrating peptide TAT. The results demonstrated that several nanobodies exhibited distinct binding activity to the target protein, with affinities reaching the nanomolar range. Among them, NbB-11 exhibited the most potent inhibitory activity toward FASN, achieving a 28.2% inhibition rate of FASN in vitro activity at a concentration of 3 μmol/L. Furthermore, the TAT-NbB-11 fusion protein, constructed by linking NbB-11 with the cell-penetrating peptide TAT, was successfully delivered across the membrane and demonstrated a 22.0% cell proliferation inhibition rate at a concentration of 4 μmol/L. In conclusion, this study successfully selected high-affinity nanobodies targeting FASN and preliminarily validated the feasibility of the ACP domain as a regulatory target for FASN activity. These findings provide fundamental lead molecules for subsequent nanobody engineering and in vivo validation.