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Herein we investigated the structural and cellular effects ensuing from the cyclization of a potent inhibitor of JAK2 as mimetic of SOCS1 protein, named PS5. The introduction of un-natural residues and a lactam internal bridge, within SOCS1-KIR motif, produced candidates that showed high affinity toward JAK2 catalytic domain. By combining CD, NMR and computational studies, we obtained valuable models of the interactions of two peptidomimetics of SOCS1 to deepen their functional behaviors. Notably, when assayed for their biological cell responses mimicking SOCS1 activity, the internal cyclic PS5 analogues demonstrated able to inhibit JAK-mediated tyrosine phosphorylation of STAT1 and to reduce cytokine-induced proinflammatory gene expression, oxidative stress generation and cell migration. The present study well inserts in the field of low-molecular-weight proteomimetics with improved longtime cellular effects and adds a new piece to the puzzled way for the conversion of bioactive peptides into drugs.

[Image: see text] The immunomodulatory effects of Suppressor of Cytokine Signaling (SOCS) proteins, that control the JAK/STAT pathway, indicate them as attractive candidates for immunotherapies. Recombinant SOCS3 protein suppresses the effects of inflammation, and its deletion in neurons or in immune cells increases pathological blood vessels growth. Recently, on the basis of the structure of the ternary complex among SOCS3, JAK2, and gp130, we focused on SOCS3 interfacing regions and designed several interfering peptides (IPs) that were able to mimic SOCS3 biological role in triple negative breast cancer (TNBC) models. Herein, to explore other protein regions involved in JAK2 recognition, several new chimeric peptides connecting noncontiguous SOCS3 regions and including a strongly aromatic fragment were investigated. Their ability to recognize the catalytic domain of JAK2 was evaluated through MST (microscale thermophoresis), and the most promising compound, named KIRCONG chim, exhibited a low micromolar value for dissociation constant. The conformational features of chimeric peptides were analyzed through circular dichroism and NMR spectroscopies, and their anti-inflammatory effects were assessed in cell cultures. Overall data suggest the importance of aromatic contribution in the recognition of JAK2 and that SOCS3 peptidomimetics could be endowed with a therapeutic potential in diseases with activated inflammatory cytokines.

Suppressors of Cytokine Signaling (SOCS) proteins are negative regulators of JAK proteins that are receptor-associated tyrosine kinases, which play key roles in the phosphorylation and subsequent activation of several transcription factors named STATs. Unlike the other SOCS proteins, SOCS1 and 3 show, in the N-terminal portion, a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Drug discovery processes of compounds based on KIR sequence demonstrated promising in functional in vitro and in inflammatory animal models and we recently developed a peptidomimetic called PS5, as lead compound. Here, we investigated the cellular ability of PS5 to mimic SOCS1 biological functions in vascular smooth muscle cells and simultaneously we set up a new binding assay for the screening and identification of JAK2 binders based on a SPR experiment that revealed more robust with respect to previous ELISAs. On this basis, we designed several peptidomimetics bearing new structural constraints that were analyzed in both affinities toward JAK2 and conformational features through Circular Dichroism and NMR spectroscopies. Introduced chemical modifications provided an enhancement of serum stabilities of new sequences that could aid the design of future mimetic molecules of SOCS1 as novel anti-inflammatory compounds.