Probing Functional Heteromeric Chemokine Protein–Protein Interactions through Conformation‐Assisted Oxime Ligation

Abstract

Protein–protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES‐PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function.

Chemokines from a Structural Perspective

“…In this regard, chemokine heterodimerization can modulate the overall signaling response of GPCRs, thereby providing a general mechanism for regulating chemokine function. The recent synthesis and in vitro / in vivo testing of a covalently-linked CXCL4/CCL5 heterodimer has validated the functional relevance of chemokine heterodimers in GPCR-mediated signal transduction [ 42 , 89 ].…”

“…Based on these studies, other chemokine-derived peptides were designed and shown to be effective chemokine antagonists [ 42 ]. However, it was the design and synthesis of a covalently-linked CXCL4/CCL5 heterodimer [ 89 ] that has provided the most compelling evidence in vitro and in vivo it validated the biological relevance of chemokine heterodimers [ 42 ]. These studies may contribute to the development and promise of novel chemokine antagonists for use in the clinic.…”

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…[47] Tilman Hackeng (CardiovascularR esearch Institute Maastricht/Maastricht University,T he Netherlands) discussed recent applicationso fo xime ligation reactions to probe functional heteromeric chemokinep rotein-protein interactions. [48] He furthermorer eporteda no xime ligationr eaction that can be accelerated under aqueous conditions, at low concentrationsa nd at neutral pH by freezing. [49] Phil Dawson (The Scripps Research Institute,U SA) described how as mall combinatorial peptidelibrary can be built through sequential click reactions starting from as ingle parent peptide.…”

“…To explore different medical and biotechnological applications, biomolecules (such as peptides, DNA, and antibodies) as well as small molecules (dyes, radioisotopes, MRI contrast agents, drugs) were coupled in a precise manner to proteins of interest . Tilman Hackeng (Cardiovascular Research Institute Maastricht/Maastricht University, The Netherlands) discussed recent applications of oxime ligation reactions to probe functional heteromeric chemokine protein–protein interactions . He furthermore reported an oxime ligation reaction that can be accelerated under aqueous conditions, at low concentrations and at neutral pH by freezing .…”

Catalysis of Hydrazone and Oxime Peptide Ligation by Arginine

“…1,2 Hydrazone and oxime formation show useful reaction rates at mildly acidic pH (4-5) but are very slow processes in water at neutral pH, which is the preferred media for manipulating complex biomolecules such as proteins. As a consequence, many studies focused on accelerating these reactions by thermal activation, 3 by freezing, 4 by protein-protein complex formation, 5 by addition of organic co-solvents 3 or catalysts. 1,[6][7][8][9] Nucleophilic catalysis by amine compounds such as aniline 6,8 (or derivatives thereof 10,11 ) or N,N-dimethylaminoethylamine 7,12 has proven especially powerful in this regard (Figure 1).…”