The observation that Cys111 could act as reactive nucleophilic residue on SOCS2 prompted us to design covalent binder 15 (hence afterward referred to as MN551), by replacing the allyl group in 14 with a more electrophilic chloroacetamido group. We chose 14 as scaffold ligand instead of 13 (hydrogen instead of fluorine at the para position) due to convenient availability of 3-(Boc-amino) benzylamine as reagent for the incorporation of the chloroacetamido group during the synthesis of MN551 (Supplementary Scheme 3). The chloroacetamide moiety newly introduced in MN551 covalently modified Cys111, as confirmed by protein X-ray crystallography and mass spectrometry experiments (Figure ). A co-crystal structure of SBC preincubated with MN551 for 2 h prior to crystallisation revealed the formation of a covalent bond between the chloroacetamide group of MN551 and Cys111. The binding mode for the remaining portion of the MN551 molecule remained unchanged compared to the parental analogues. Only a 1:1 covalent SOCS2:MN551 adduct was observed using intact mass analysis by electrospray protein mass spectrometry (Figure , 2B), and no reactivity was observed with a C111S mutant (Figure ), consistent with high specificity of binding and modification site. Time-dependent reaction monitored by mass spectrometry showed that MN551 covalently modified recombinant SOCS2 protein to stoichiometric occupancy within 2 h at Cys111 (Figure ). Binding of MN551 to SBC was further validated with DSF and ITC (Figure ). MN551 enhanced the thermal stability of SBC as demonstrated by a 6 o C shift in the melting temperature (Tm). The reversible binding affinity of MN551 to SBC was determined by ITC to be Ki = 2.2 µM (Figure ). To confirm that MN551 blocks binding of the natural substrates of SOCS2, we performed competition ITC experiments of GHR_pY595 peptide against SBC either unmodified or preincubated with equimolar amount of MN551 for 2 hours at room temperature. In the presence of MN551, GHR was no longer able to bind to SBC, nor to compete out the inhibitor, demonstrating covalent saturation of the SOCS2 binding site by MN551 (Figure ). With the specific covalent modification of Cys111 established, we decided to fully characterise MN551 as a covalent SOCS2 inhibitor. To this end, we developed a covalent fluorescence polarisation assay to monitor displacement of a non-covalent fluorescent probe 26 (KD = 77.56 nM, as measured with FP, Supplementary Figure ) by MN551, to ultimately determine the covalent efficiency kinact/KI that is a critical parameter to characterize covalent inhibitors (Figure , 2H, Supplementary Figure ). Due to the covalent nature of the competing inhibitor, a time-dependent reduction in the fraction of protein-bound probe is observed. Plotting the rate (kobs) of time-dependent reduction in probe FP signal with MN551 concentrations, yields the parameters kinact=2.1×10 -4 s -1 (maximum potential rate of inactivation) and KI=3.6 µM (concentration of MN551 at which kobs=kinact/2) (details in methods and Supplementary Figure ) . We also calculated Ki = 1.1 µM at time = 0 and unlike the Ki determined by ITC measurement, calculated Ki from the FP represents the initial reversible binding affinity of MN551. Figure captures the initial rates to give an accurate estimation of the efficiency of covalent bond formation, kinact/KI=58 M -1 s -1 . This value illustrates reasonable specificity for a covalent inhibitor, with nonetheless scope for future optimization. In addition to characterising the kinetic covalency parameters, we have also assessed the stability of the MN551 by monitoring its reactivity (half-life >70 min) with reduced glutathione (GSH) (Supplementary Figure ) .