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Table 1. K D values (expressed in µM) of SOCS-KIR mimetic peptides in complex with JAK2and pJAK2KIR New-KIR PS-5 PS-12 PS-11JAK2 87±9 27±5 5.2±1.0 5.2±1.6 5.8±1.1p-JAK2 81±8 16±4 0.8±0.1 4.7±0.5 NCTo further improve binding affinity of new-KIR sequence, following a combinatorialapproach, we screened a “focused simplified peptide library“, in postional scanning format,PS-SSPL [5]. This library was designed randomizing the three residues in positions 55-57of New-KIR, that appeared not directly involved in the interaction with JAK2 catalyticregion. The combination of identified amino acids in each randomized position led to thedesign of 12 new peptides. Their binding capacities to JAK2 peptides were analyzedthrough direct ELISA and we selected two peptides from the experiment involving JAK2(PS-11 and PS-12), and one peptide (PS-5) from that vs pJAK2. Single peptides selectedfrom PS-SSPL screening, were further analyzed in dose-response direct binding ELISAtoward JAK2 peptides. Data fitting provided K D values in the low micromolar rangeshowing major affinities toward JAK2 region in respect to natural sequences (Table 1).Then competition experiments were carried out employing PS-5 and PS-12 ascompetitors of the KIR-JAK2 peptide complexes and both inhibited the binding of JAK2peptides to immobilized KIR in a dose-dependent fashion and data fitting provided IC50values in the low micromolar range, fully consistent with estimated K D values by directbinding, while new-KIR peptide resulted less able to compete with KIR, in the exploredconcentration range.In order to perform cell-based experiments we conjuugated KIR sequences to Tat-CPPas carrier. To assess whether PS-5 peptide could inhibit JAK2/STAT1 signaling, weinvestigated the effect of these conjugated peptides on the phosphorylation of STAT1 andrelated genes expressions in human keratinocites. Cells pretreated with peptides andsubsequently stimulated with INF-γ showed a sensible decrease of phosphorylation level ofINF- -receptor, consequently, in IFN- -induced STAT1 tyrosine phosphorylation while theexpression of ICAM1 was highly reduced in the presence of PS5.In conclusion, here we show that SOCS-1-KIR binds to JAK2 catalytic site with about80 µM affinity then, following an Ala-scanning approach, that the deletion of last sixresidues of SOCS-1 KIR (62SDYRRI67) improved the affinity of this domain to JAK2peptides, providing lower K D value of about 4-fold. Then we have opportunely designed afocused peptide library in which residues directly involved in complex with JAK2, (52-53,57-61), remained unchanged, while positions 54-56 were randomized following oursimplified approach. From the screening of PS-SSPL we have selected several newsequences with K D values in the very low micromolar range (6-fold improvement incomparison to new-KIR). The ability to mime SOCS1 of these new peptide sequences werealso evaluated in preliminary cellular assays confirming their capacity to interfere withIFN- -induced STAT1 phosphorylation and related genes expression, suggesting theirpotential application as modulators of disorders involving SOCSs over-expression. Weselected new and more potent ligands as antagonists of SOCS-1 (K D values are in the highnanomolar range that are 15-fold lower respect to w-t KIR) representing good candidate astherapuetic agents.References1. Alexander, W.S., et al. Annu Rev Immunol. 22, 503-529 (2004).2. Yoshimura, A., et al. Nature Rev. Imm. 7, 454 (2007).3. Giordanetto, F., et al. Protein Engineering 16(2), 115 (2003).4. Albanesi, C., et al. Curr. Drug Targets Inflamm. Allergy 4(3), 329 (2005).5. Marasco, D., et al. Curr. Protein Pept. Sci. 9(5), 447 (2008).539