Locally constrained xylene-based cyclic mimetics of SOCS3 protein

The development of SOCS3 peptidomimetics targeting the JAK2 signaling pathway presents a promising strategy for modulating cytokine-driven diseases. In this study, we designed four novel monocyclic analogues of a previously reported linear lead compound (KIRCONG chim PEG), introducing local conformational constraints via regioselective thioether cyclization of either the KIR (thio-monoleft) or CONG (thio-monoright) regions. Each variant was synthesized with one or two PEG moieties (PEG₁ or PEG₂) to modulate solubility and flexibility. Microscale Thermophoresis (MST) revealed that thio-monoright PEG₁ exhibited the highest affinity for the JAK2 catalytic domain, and inhibition assays further confirmed its superior ability to suppress JAK2 mediated phosphorylation. Circular dichroism (CD) and metadynamics simulations indicated enhanced structural organization and intramolecular hydrogen bonding in cyclic analogues, especially thio-monoright PEG₁, as supported by Natural Bond Orbital (NBO) and Second Order Perturbation Theory (SOPT) analyses. Conversely, PEG₂ analogues showed reduced activity and solubility, likely due to increased flexibility and aggregation arising from hydrophobic surface exposure, as demonstrated by fluorescence spectroscopy and solvent-accessibility calculations. Overall, thio-monoright PEG₁ represents a promising conformationally stabilized SOCS3 mimetic with improved bioactivity, providing a rational foundation for further optimization of JAK2 targeting peptide therapeutics.