Stepan received his Ph.D. from the University of Maastricht, the Netherlands in 2019. During his doctoral trajectory he studied evasins - tick chemokine-binding proteins. After defense, he became a recipient of a two-year Kootstra Fellowship from UMC+ and Maastricht University to continue his work as a postdoctoral fellow. During these two years Stepan was leading a research project focused on screening chemokine-binding cyclic D-peptides from combinatorial phage libraries.

In 2021 the Rubicon Fellowship from the Dutch Research Council was granted to Stepan to fund a two-year postdoctoral position in the lab of prof. Bhattacharya at the University of Oxford, the United Kingdome. In Oxford University he is exploring applicability of directed evolution for modifying chemokine-binding peptides derived from pathogens' and viral proteins.

Chemokines are secreted signaling proteins involved in the development of atherosclerosis, tumor-associated angiogenesis, and neurodegenerative diseases. Therefore, neutralization of chemokines could offer novel beneficial therapeutic strategies.

Although peptides are considered potential chemokine-neutralizing agents, they usually suffer from low chemical and proteolytic stability. Peptide cyclization and incorporation of non-natural amino acids inaccessible to enzymes can prevent peptide proteolysis and immunogenicity, leading to more effective drugs.

Here, we generated cyclic D-peptides from a combinatorial library for interleukin-8, CXCL8, neutralization. First, the D-variant of CXCL8 was synthesized using Boc-SPPS and further used as a target in three rounds of CLIPS phage display selection of the combinatorial library with ten variable residues. Peptide sequences, obtained by NGS of the enriched phage
library, were analyzed and clusterized based on sequence similarity.

Subsequently, 100 sequences from different clusters were selected and synthesized in L-form for screening against D-CXCL8 by SPR biosensor analysis. The best performing CLIPS peptides had KD values of ~170 nM, and eventually four sequences were selected for synthesis in the D-form. Peptides with inverted stereochemistry bound only L-CXCL8 and not D-CXCL8.

Structural features of D-peptides and their complex with L-CXCL8 have been studied using CD and solution NMR spectroscopy, showing that binding caused disruption of CXCL8 dimer, but did not affect the receptor binding site of the chemokine. Nevertheless, all peptides effectively blocked CXCL8-induced chemotaxis of human monocytes at µM concentrations. In the future, selected peptides will be subjected to sequence optimization to increase the affinity for CXCL8