Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

Abstract

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

“…A monoclonal antibody, CA1974, was identified and shown to have excellent selectivity for the TNF–small molecule complex compared to the apo form of TNF. Consistent with previous observations that small molecules do not ablate engagement with all three receptor subunits 5 , 6 , binding specificity was initially demonstrated using a human TNFR1-capture ELISA in order to present the distorted A/C interface of the trimer. As shown in Fig.…”

“…We solve a crystal structure of the conformation-selective antibody in complex with TNF trimer, small molecule inhibitor, and TNFR1 extracellular domain. The structure confirms other studies suggesting that the small molecule inhibitors stabilise a perturbed conformation of the TNF trimer, resulting in a reduction in receptor stoichiometry, which likely leads to the subsequent inhibition of signalling 5 , 6 . Furthermore, we show that the antibody, in addition to providing insights into the structure and dynamics of TNF and the mode of action of the small molecules, has potential utility as a tool for measuring target occupancy.…”

“…The development of a number of orally active small molecule inhibitors of human TNF that exhibit activity both in vitro and in vivo has recently been described 5 . The small molecule compounds bind in a pocket at the centre of the TNF trimer and stabilise a conformation where a single TNF monomer subunit is perturbed, thereby inducing a molecular asymmetry in the trimer which in turn affects TNFR1 receptor interaction stoichiometry and subsequent signalling.…”

Small Molecule NF-κB Pathway Inhibitors in Clinic

“…TNFR and IL1R mAbs are well-established and approved for various clinical indications, whereas in small molecule inhibitors, there is only one preclinical candidate. UCB-6786 by UCB pharma, a TNFR small molecule inhibitor, has only been tested for in vivo efficacy in collagen antibody induced arthritis mice model [ 37 ].…”

Structure-Based Design, Synthesis and Bioactivity of a New Anti-TNFα Cyclopeptide

“…Recently, a small molecule benzimidazole derivative (UCB-6876) has been discovered, a co-crystal structure of UCB-6876 with TNFα resulted in a change in the spatial arrangement of the TNFα trimer and a loss of its 3-fold symmetry. This compound was also found to occupy the centre of the TNFα trimer filling a hydrophobic binding pocket involving chain A (Y59), chain B (L57, Y119) and chain C (Y59, Y151) [19].…”