Design, synthesis and evaluation of pyrrolobenzodiazepine (PBD)-based PROTAC conjugates for the selective degradation of the NF-κB RelA/p65 subunit.

NF-κB signalling is frequently dysregulated in human cancers making it an attractive therapeutic target. Despite concerted efforts to generate NF-κB inhibitors, direct pharmacological inhibition of the kinases mediating canonical NF-κB has failed due to on-target toxicities in normal tissues. So, alternative strategies, designed to target specific components of the NF-κB signalling machinery, have the potential to selectively inhibit tumour cells whilst reducing the toxicities associated with broad inhibition of NF-κB in non-malignant cells. Here we present evidence that a C8-linked pyrrolobenzodiazepine (PBD) containing proteolysis-targeting chimera (PROTAC) selectively degrades the NF-κB subunit, RelA/p65, in a proteasome-dependent manner. Our lead PROTAC (JP-163-16, 15d) showed cytotoxicity with mean LC(50) values of 2.9 μM in MDA-MB-231 cells, 0.14 μM in MEC-1 cells and 0.23 μM in primary chronic lymphocytic leukaemia cells. In contrast, 15d was two-logs less toxic in primary B- and T-lymphocytes (mean LD(50) 19.1 μM and 36.4 μM, respectively). Importantly, the development of 15d, by conjugating the C8-linked PBD with a cereblon-targeting ligand using a five-carbon linker, abolished the ability of the C8-linked PBD to bind to DNA, whilst demonstrating cytotoxicity in cancer cells associated with the degradation of RelA/p65. Mechanistically, 15d displayed PROTAC credentials through the selective degradation of NF-κB RelA/p65 in a proteasome-dependent manner and showed a five-fold reduction in potency in the cereblon deficient, lenalidomide resistant, myeloma cell line, RPMI-8226. To our knowledge, this work describes the first PROTAC capable of selective degradation of a single NF-κB subunit and highlights the therapeutic potential of our strategy for the treatment of RelA/p65-dependent tumours.