Engineered anti-inflammatory peptides inspired by mapping an evasin–chemokine interaction

Abstract

Chemokines mediate leucocyte migration and homeostasis, and are key targets in inflammatory diseases including atherosclerosis, cytokine storm and chronic auto-immune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines overcoming redundancy, and are effective in several pre-clinical disease models. Their clinical development has not progressed due to concerns regarding potential immunogenicity, parenteral delivery and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple-chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen–deuterium exchange mass spectrometry to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand 8 (CCL8) and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native mass spectrometry approaches showed that BK1.1 binds CCL8, CCL7 and CCL18, and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2 and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2 and CCL3 chemotactic function in vitro. We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo. Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

Structural characterization of anti-CCL5 activity of the tick salivary protein evasin-4

“…Furthermore, the linear peptide Ev4 Glu 14 –Asn 31 , derived from the Evasin-4 sequence, possesses nanomolar affinity to CCL5 on its own. Similar tight affinities have recently been shown for peptides derived from the N terminus of P672 and are potent binders of CCL8 ( 31 ). The N-terminal region of Evasin-4 embodies multiple tyrosines, which are proposed to mimic an N-terminal region of CC-chemokine receptors ( 32 ).…”

“…Development of chemokine-binding peptides and peptidomimetics based on Evasins may provide more perspectives ( 34 ). Recently, several peptides derived from C8-Evasin P672 were shown to effectively block inflammation because of binding to multiple CC-type chemokines ( 31 ). Ev4 Glu 14 –Asn 31 effectively inhibits CCL5-induced monocyte migration.…”

Immunomodulatory Proteins in Tick Saliva From a Structural Perspective

“…Although EVA-1 and EVA-4 have effectively diminished inflammation in the murine model of acute lung injury and myocardial infarction, respectively ( Russo et al., 2011 ; Braunersreuther et al., 2013 ), their application as drugs could be challenging due to the complex structures and difficult production (personal observation). However, it has recently been shown that short linear and cyclic peptides derived from the N -termini of EVA-P672 and EVA-4 retain binding and inhibitory activity comparable to full-length parent proteins ( Darlot et al., 2020 ; Denisov et al., 2020b ). Moreover, these peptides could be further modified and adjusted for neutralization of chemokines of interest.…”

Tick Salivary Compounds for Targeted Immunomodulatory Therapy

“…Due to its broad CC chemokine-binding spectrum, Evasin-4 is considered the most suitable candidate for therapeutic development (86). More recently, Evasin-inspired artificial peptides dramatically reduced inflammation in vivo by targeting multiple chemokines (87). These peptides might therefore provide a route to the development of new anti-inflammatory therapeutics for chronic autoimmune diseases such as rheumatoid arthritis and inflammatory diseases such as atherosclerosis.…”

“…These peptides might therefore provide a route to the development of new anti-inflammatory therapeutics for chronic autoimmune diseases such as rheumatoid arthritis and inflammatory diseases such as atherosclerosis. Moreover, the mechanism of action of these peptides suggests that they might also be useful in acute infectious diseases such as influenza or COVID-19, where exuberant cytokine responses are thought to be at least partially responsible for tissue injury (87).…”