Allosteric Inhibition of Anaplastic Lymphoma Kinase by Nanobody

Dysregulation of the Anaplastic Lymphoma Kinase (ALK) is a key oncogenic driver in several cancers, including non-small cell lung cancer. Although clinically effective, the utility of ATP-competitive ALK inhibitors is frequently undermined by the emergence of acquired drug resistance. This thesis addresses this challenge by exploring allosteric inhibition which targets less conserved regulatory sites to circumvent resistance. This work describes the discovery and characterisation of a novel allosteric inhibitor of the ALK kinase domain. A screen of candidate nanobodies identified B09 as a potent, non-ATP-competitive inhibitor of the hyperactive ALK F1174L mutant. Computational modelling and molecular dynamics simulations predicted that B09 binds to allosteric site, forming a stable parallel β-strand between its CDR3 loop and the ALK activation loop. Furthermore, B09 expression disrupted the formation of EML4-ALK condensates and the binding of adaptor proteins, including SHP2 and IRS1/2. Kinome profiling also revealed that B09 induces a broad downregulation of oncogenic signalling, including the JAK-STAT and PI3K-AKT pathways, with STAT3 identified as a central node of inhibition. In conclusion, this thesis identifies B09 as an allosteric modulator that suppresses ALK activity through a mechanism involving restriction of activation loop dynamics and disruption of critical protein complexes. This research provides mechanistic insights and a novel prospect for the development of next-generation ALK inhibitors designed to overcome drug resistance in ALK-driven cancers.