Biological Characterisation of the Cellular Target Engagement and Function of SOCS2 and CISH using novel covalent inhibitor, MN714

Abstract

E3 handles are powerful tools that can be leveraged to uncover more about the biology of a particular E3 ligase. SOCS2, suppressor of cytokine signalling 2, is a key negative regulator of cytokine signalling pathways. Dysregulation of SOCS2 has been found in many diseases such as cancer, inflammatory disease, and metabolic disorders. Recently, a first of its kind SOCS2 inhibitor was published. This inhibitor was built to mimic the phosphotyrosine binding site and block SOCS2 via its SH2 domain. Whilst recombinant studies proved covalent modification and native substrate displacement, it was yet to be proven in a cellular environment. As part of this research, we aimed to investigate the effects of SOCS2 inhibition by MN551 as well as establish protocols to later study its effect at an entire proteome level. The growth hormone receptor system was utilised as this is a well-known SOCS2 target.

We achieved native substrate displacement as a result of MN551- SOCS2 inhibition as well as demonstrating this on live cells using the pro-drug, MN714. This indirect target engagement assay underwent multiple rounds of optimisation to reach the end result in two biologically different cell lines. MS proteomics experiments were carefully designed in order to better control for the unwanted oxidative stress caused by POM group cleaving and formaldehyde formation. New MS data revealed potential on-target proteins up regulated as a result of covalent SOCS2 inhibition.

Designing and establishing the protocols presented in this research serves as a basis for further investigation into the therapeutic potential of SOCS2 inhibition. Modulating SOCS2 activity to increase cytokine signalling or inhibiting it could provide benefit in systems where its activity is undesirable.

There is scope to harness SOCS2 E3 ligase function to make PROTACs and uncover potential protein targets to degrade in signalling pathways where SOCS2 plays a crucial role.

Date of Award	2024
Original language	English
Awarding Institution	University of Dundee
Supervisor	Alessio Ciulli (Supervisor), Doreen Cantrell (Supervisor) & Ignacio Moraga Gonzalez (Supervisor)