Structure-Activity Relationship Investigations of Novel Constrained Chimeric Peptidomimetics of SOCS3 Protein Targeting JAK2

Abstract

SOCS3 (suppressor of cytokine signaling 3) protein suppresses cytokine-induced inflammation and its deletion in neurons or immune cells increases the pathological growth of blood vessels. Recently, we designed several SOCS3 peptidomimetics by assuming as template structures the interfacing regions of the ternary complex constituted by SOCS3, JAK2 (Janus Kinase 2) and gp130 (glycoprotein 130) proteins. A chimeric peptide named KIRCONG chim, including non-contiguous regions demonstrated able to bind to JAK2 and anti-inflammatory and antioxidant properties in VSMCs (vascular smooth muscle cells). With the aim to improve drug-like features of KIRCONG, herein we reported novel cyclic analogues bearing different linkages. In detail, in two of them hydrocarbon cycles of different lengths were inserted at positions i/i+5 and i/i+7 to improve helical conformations of mimetics. Structural features of cyclic compounds were investigated by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopies while their ability to bind to catalytic domain of JAK2 was assessed through MST (MicroScale Thermophoresis) assay as well as their stability in biological serum. Overall data indicate a crucial role exerted by the length and the position of the cycle within the chimeric structure and could pave the way to the miniaturization of SOCS3 protein for therapeutic aims.

Keywords: JAK/STAT; SOCS3; cytokine signaling; mimetic peptides; stapled peptides.

Figure 1

Chemical structures of KIRCONG analogues with residues involved in cycle formation colored: (A) KIRCONG amide (blue: Lys, purple: Asp), (B) KIRCONG disulfide (green: Cys), (C) KIRCONG i/i+5 (orange: (S)-N- 2-(4′-pentenyl) alanine) and (D) KIRCONG i/i+7 (orange: (S)-N-2-(4′-pentenyl) alanine, red: (R)-2-(7′-octenyl) alanine).

Figure 2

CD spectra of: (A) KIRCONG amide; (B) KIRCONG disulfide; (C) KIRCONG i/i+5; (D) KIRCONG i/i+7 in TFE/buffer 15-65% v/v. KIRCONG i/i+7 resulted not soluble in 65/25 v/v, TFE/buffer and the related spectrum is absent.

Figure 3

NMR analysis of KIRCONG i/i+7 in H₂O/TFE 60/40 v/v. (A) Chemical shift deviations of Hα protons from random coil values (∆δ_Hα). Standard amino acids are reported with the one letter code whereas, B stands for β-Alanine, Z stands for (R)-N-2-(7′-octenyl) alanine and X stands for (S)-2-(4′-pentenyl) alanine. ∆δ_Hα for Z and X is set equal to 0 as the reference random coil value is missing. For ∆δ_Hα evaluation β-Alanine was assimilated to Glycine. (B) NOEs pattern. (C) Ribbon representation of KIRCONG i/i+7 NMR structures: 20 conformers are superimposed on the backbone atoms of residues 3–17, and (D) ribbon representation of the first NMR conformer. The aliphatic linker between (R)-N-2-(7′-octenyl) alanine (residue n.8) and (S)-2-(4′-pentenyl) alanine (residue n.15) is shown in black. The NMR structure was generated from 261 upper distance limits (170 intraresidue, 58 short-range, 27 medium-range and 6 long-range) and 73 angular constraints.

Figure 4

Serum stability assay of KIRCONG chim derivatives. The serum stability was evaluated by incubation in 25% FBS for 42 h. Residual peptide amount is expressed as the percentage of the initial amount versus time.

Figure 5

Left column: binding isotherms for MST signals versus peptide concentrations. Right column: thermophoretic traces of MST assays for the binding to JAK2 of (A) KIRCONG amide; (B) KIRCONG disulfide; (C) KIRCONG i/i+5; (D) KIRCONG i/i+7.