Structure-Guided SOCS3 Peptidomimetics: Design and Functional Characterization

Suppressor of Cytokine Signaling 3 (SOCS3) is a pivotal negative regulator of the JAK/STAT pathway, and its loss or silencing is frequently associated with hyperactivated STAT3 signaling in aggressive cancers, including Triple-Negative Breast Cancer (TNBC). In this study, we present the rational design, biophysical characterization, and cellular evaluation of novel SOCS3-derived peptidomimetics that incorporate a hitherto unexploited structural determinant of the SOCS3/JAK2/Gp130 interface: the BC loop. The synthesis of individual and chimeric peptides was guided by structural analysis of the ternary complex, which combined the KIR/ESS regions with a stabilized BC loop. The results of the study revealed that the chimeric construct, KIRESS BC loop-chim, exhibited markedly improved affinity for JAK2 (KD ∼ 10 μM) in comparison to the affinity of the isolated regions. This was determined by means of MicroScale Thermophoresis (MST). Circular dichroism (CD) and fluorescence spectroscopy demonstrated that turn-inducing motifs stabilize native-like conformations, correlating with enhanced serum stability. To preliminarily evaluate potential cellular effects, we assessed their serum stabilities and their cytotoxicity in MDA-MB-231 and MDA-MB-468 cells once conjugated to a small Cell-Penetreting Peptide (CPP). In both cases, the good biocompatibility of the designed mimetics appeared promising for evaluating signaling-dependent effects. These findings validate a multiregion, structure-guided design strategy and identify an improved SOCS3 proteomimetic scaffold with potential for targeting dysregulated JAK/STAT signaling in cancer.