There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

人们对开发抗体作为信号通路的调节剂有相当大的兴趣。高等真核生物中最重要的信号通路之一是磷酸肌醇 3 激酶 (PI3K) 通路，它在生长、代谢和免疫中起基本作用。IB 类 PI3K，PI3Kγ，是由与 p101 或 p84 调节亚基结合的催化 p110γ 亚基组成的异二聚体复合物。PI3Kγ 是多种免疫信号传导过程中的关键成分，并且依赖于 Ras 和 G 蛋白偶联受体 (GPCR) 的激活来介导其细胞作用。在这里，我们描述了使用氢-氘交换 (HDX) 质谱 (MS) 快速有效地表征多种 PI3Kγ 结合单链骆驼纳米抗体，用于结构和生化研究。我们确定了刺激脂质激酶活性、阻断 Ras 激活和特异性抑制 p101 介导的 GPCR 激活的纳米抗体。总体而言，我们的工作揭示了对 PI3Kγ 调控的深入了解，并确定了可用于治疗开发的位点。