Design and functional studies of xylene-based cyclic mimetics of SOCS1 protein

Abstract

Peptidomimetics of Suppressors of cytokine signaling 1 (SOCS1) protein demonstrated valid therapeutic potentials as anti-inflammatory agents. Indeed, SOCS1 has a small kinase inhibitory region (KIR) primarily involved in the inhibition of the JAnus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway Herein, on the basis of previous investigations on a potent mimetic of KIR-SOCS1, named PS5, we designed and evaluated the SAR (Structure Activity Relationship) features of two xylene-based macrocycles analogues of PS5. These novel compounds bear thiol-xylene linkages with mono- and bi-cyclic scaffolds: they were in vitro functionally investigated toward JAK2 catalytic domain, as ligands with microscale thermophoresis (MST) and as inhibitors through LC-MS analyses. To evaluate structural properties Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies were employed along with serum stability assays. Results indicated that a monocycle scaffold is well-tolerated by PS5 sequence enhancing the affinity toward the kinase with a K_D in the low micromolar range and providing consistent inhibitory effects of the catalytic activity, which were evaluated for the first time in the case of SOCS1 mimetics. Conformationally, the presence of xylene scaffold affects the flexibility of the compounds and their stabilities to proteases degradation. This study contributes to the understanding of the factors necessary for accurately mimicking the inhibitory mechanism of SOCS1 protein towards JAK2 and to the translation of proteomimetics into drugs.