Structure‐activity studies of peptidomimetics based on kinase‐inhibitory region of suppressors of cytokine signaling 1

Abstract

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.

Peptides as Therapeutic Agents for Inflammatory-Related Diseases

“…Ongoing cellular experiments in mouse VSMCs demonstrated that pretreatment of cells with new analogues reduced STAT1 and STAT3 phosphorylation induced by a combination of pro-inflammatory cytokines (IFNγ plus IL-6). Internalized peptides were also able to inhibit STAT1 nuclear translocation [ 16 ].…”

Proteomimetics of Natural Regulators of JAK–STAT Pathway: Novel Therapeutic Perspectives

“…Subsequently, by carrying out a screening of “combinatorial focused libraries” in positional scanning (PS) format ( Marasco et al, 2008 ; Lonardo et al, 2010 ; Ponticelli et al, 2008 ) of new KIR sequence, a new lead compound, named PS5, carrying the mutations His 54 /Cys(Acm), Phe 55 /Arg, and Arg 56 /Gln was investigated ( Figure 2 ). It demonstrated the ability to bind to the JAK2 catalytic site more efficiently than KIR, as evident by the comparison of K D values reported in Table 2 , and to drastically reduce STAT1,3 activation induced by IFN-γ and IL-6 and downstream genes ( Doti et al, 2012 ; Madonna et al, 2013 ; La Manna et al, 2017b ). In detail, PS5 effects were analyzed in keratinocytes, and explants of human skin were activated by IFN-γ that have a crucial role in type-1 skin disorders.…”

Antioxidant Effects of PS5, a Peptidomimetic of Suppressor of Cytokine Signaling 1, in Experimental Atherosclerosis

“…PS5 was revealed to be able to bind to JAK2 with K D values in the nanomolar range [45,46] and was tested in cultures of human keratinocytes and murine VSMCs. In these cell types, we demonstrated a direct effect of PS5 on JAK/STAT since PS5 was revealed to be able to reduce the phosphorylation of STAT1 and STAT3 and the expression levels of interferon regulatory factor-1 (IRF-1) and caused a strong reduction in the expression of inflammatory genes such as intercellular adhesion molecule-1 (ICAM-1), human leukocyte antigen-DR isotype (HLA-DR), C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine ligand 2 (CCL2) and CCL5 [46,47]. Here, to evaluate the possibility to extend the therapeutic potential of PS5 to cardiovascular diseases, we investigated its antioxidant and atheroprotective effects both in vitro, in VSMCs and macrophages, and in vivo, in a mouse model of atherosclerosis.…”

“…The related screening in positional scanning format provided PS5 as a lead compound that bears an un-natural residue Cys (-Acm), at position 54, reported in blue along with the other non-native residue Gln at position 56 ( Figure 1 b). PS5 was revealed to be able to bind to JAK2 with K D values in the nanomolar range [ 45 , 46 ] and was tested in cultures of human keratinocytes and murine VSMCs. In these cell types, we demonstrated a direct effect of PS5 on JAK/STAT since PS5 was revealed to be able to reduce the phosphorylation of STAT1 and STAT3 and the expression levels of interferon regulatory factor-1 (IRF-1) and caused a strong reduction in the expression of inflammatory genes such as intercellular adhesion molecule-1 (ICAM-1), human leukocyte antigen-DR isotype (HLA-DR), C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine ligand 2 (CCL2) and CCL5 [ 46 , 47 ].…”