Cyclic D-Sulfopeptide Ligands of the Chemokine CCL22 via Mirror-Image mRNA Display Lending Drug Development a Helping Hand
Chemokines are a family of small proteins that signal through G-protein coupled receptors to orchestrate trafficking of leukocytes. The importance of chemokines in immune surveillance, inflammation, and cell homing is evident as dysregulation of the chemokine-receptor signaling axes is associated with a wide range of disease states that include autoimmune disorders, metastatic cancer progression, and chronic inflammation. While considerable therapeutic development in the pharmaceutical industry has been directed toward G-protein coupled receptors, targeting chemokines directly was thought to be impractical because of their small size (<10 kDa). The discovery of a small molecule inhibitor of IL-2 (~15 kDa) in 2003 showed that shallow, solvent exposed clefts on the surface of small proteins can serve as drug discovery sites. Drugging the chemokine ligands directly is an attractive parallel therapeutic strategy as targeting G-protein coupled receptors often comes with unwanted side-effects.
In their recent paper, Zhang and colleagues identified a 24-residue cyclic D–sulfopeptide inhibitor of CCL22, a chemokine associated with allergic inflammation and atopic dermatitis. The use of D–amino acids was selected as small peptide inhibitors composed of L–amino acids are rapidly degraded by endogenous proteases. This is because ribosome-based protein synthesis exclusively uses L–amino acids and endogenous proteases composed of L–amino acids cannot cleave peptides based on D–amino acids because of the inverted chirality.
The authors turned to mirror-image mRNA display to design peptides based on D–amino acids that avoid proteolytic degradation. First, they chemically synthesized both L–CCL22 and D–CCL22, the mirror image (enantiomer) of L–CCL22. To envision how L– and D–CCL22 differ, simply place your hands together palm-to-palm. The plane that separates your palms is a mirror-plane reflecting the fingers from the left hand to the right hand. This renders your left and rights hands unique and trying to superimpose your hands is impossible. Placing the palm of your right hand on the back of your left the reveals that the order of your fingers is reversed. Likewise, L–CCL22 can be represented by the left hand and is not superimposable with D–CCL22 represented by the right hand.
To identify cyclic peptides that bind D–CCL22 Zhang and colleagues generated a library of cyclic L–peptides, with and without tyrosine sulfation, using mirror-image mRNA display to yield a pool of >1012 potential inhibitors. Immobilized D–CCL22 was then used to pan the library of cyclic L–peptides and after several rounds of selection, the top 25 enriched cyclic L–peptide sequences were identified. Chemical synthesis was used to produce their mirror-image peptides using D–amino acids and binding of cyclic D–peptides to L–CCL22 was evaluated using surface plasmon resonance. One cyclic D–sulfopeptide, Dpep24, was shown to have nanomolar affinity for L–CCL22 and exhibited >40-fold selectivity over related CCL chemokines, including CCL17 which also signals through the same G-protein coupled receptor. Dpep24 was also able to inhibit CCL22 recruitment of beta-arrestin 2 to its receptor, CCR4, with an IC50 of 570 nanomolar. These findings demonstrate the utility of mirror-image mRNA display and offer a platform to develop peptide-based inhibitors of the opposite hand.
Biotinylated and fluorescently labeled human and mouse chemokines are available from the Protein Foundry catalog. If your research requires a chemokine with label or functional group not found in our catalog, contact our team and we will work with you to develop a solution.
Zhang BB, Harrison K, Zhong Y, Maxwell JWC, Ford DJ, Calvey LP, So SS, Peterson FC, Volkman BF, Stone MJ, Bhusal RP, Kulkarni SS, Payne RJ. Discovery of Selective Cyclic d- Sulfopeptide Ligands of the Chemokine CCL22 via Mirror-Image mRNA Display with Genetic Reprogramming. J Am Chem Soc. 2024 Dec 18;146(50):34253-34259. doi: 10.1021/jacs.4c12057. Epub 2024 Dec 4. PMID: 39629944.
