Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

Abstract

Highlights d NanoBRET assays were created to monitor the interaction of IL-23 with its receptor d The affinities of IL-23 for IL23R, IL12Rb1 and heteromer complexes were measured d Receptor monomers were found to associate in complexes in the absence of IL-23 d IL-23 binding induced a rearrangement of the N-terminal domains of the receptor

Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

“…In this study we validate the use of a previously reported TAMRA labelled version of IL-23 (IL23-TMR 24 ) for use in the characterisation of reported receptor antagonists at both IL23R and IL12Rβ1 and design and characterise a novel fluorescent cyclic peptide probe (P630-TMR) specific to the IL23R receptor subunit only. We then use P630-TMR to measure IL23R specific inhibitors, demonstrating how the probes can be used together to classify IL-23 receptor antagonists.…”

“…A possible explanation for the observed modulation in BRET efficiency with IL12Rβ1 is a modification of the position of the IL23R conjugated NanoLuc-tag caused by the association of the subunits in an inactive complex. The further reduction in BRET efficiency observed when the NanoLuc tag is placed on the N-terminus of IL12Rβ1 is most likely due to the increased distance between the N-terminus of IL12Rβ1 and the P630-TMR binding epitope, however the successful measurement of a BRET signal is consistent with the receptor subunits forming a complex in the absence of IL-23, as has been previously suggested 24,36 .…”

“…The mean value generated for P630 Ki was 38.0 ± 9.0 nM (± SEM, n=25). The Cheng-Prusoff equation was then used as previously described 24 to generate an IL23-TMR Kd value of 32.0 pM from the gradient of the linear regression of the data plotted in Figure 4D and the measured P630 Ki value. The experiment was also repeated with different concentrations of P630-TMR being competed by IL-23.…”

“…Phosphorylation of STAT3 was measured using an AlphaLISA Surefire Ultra assay as previously described 24 .…”

“…Instead, these peptides have been validated using either, functional read-outs which do not allow detailed pharmacological characterisation of the peptide's binding mechanism or immobilised purified protein techniques such as ELISA and SPR which do not allow the measurement of the interactions with the full-length dimeric receptor expressed in the cell membrane [19][20][21][22][23] . This second point is important as it has previously been demonstrated that when the subunits of the IL-23 receptor are purified as truncates their affinity for IL-23 is markedly reduced 24,25 .…”