Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases;but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists;for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.

Discovery, structure,and chemical synthesis of disulfide-rich peptide toxins and their analogs

Recently, medicinal peptide molecules are of great interest to many international pharmaceutical companies, mainly because of their relatively lower research costs, shorter research cycles, and the greater likelihood of being drugs, when compared with traditional small molecules. Due to the great variety in molecule structures and the diverse biological functions, disulfide-rich peptide toxins have become a shining molecular library for the development of polypeptide drugs. In view of the increasing amount of related publications, here we summarize the discovery, structural elucidation and chemical synthesis of disulfide-rich peptide toxins and their analogs.

Chemical synthesis and racemic crystallization of rat C5a-desArg

The deletion of the C-terminal arginine of the anaphylatoxin protein C5a reduces it receptor binding affinity.Understanding how C-terminal arginine affects the structure and bioactivity of C5a is important for the development of C5a C-terminal mimics as drug candidates.Herein,we report the total chemical synthesis of rat C5a and its D-enantiomer with its C-terminal arginine deleted,namely L-rC5a-desArg and D-rC5a-desArg.The structure of rC5a-desArg was then determined by racemic crystallography for the first time.The C-terminal residues of rC5a-Arg were found to expand from the fourth helix in a continuous helical confo rmation.This C-terminal conformation is significantly different from that of the previously reported full-length of C5a,indicating that the deletion of C-terminal arginine residue could result in the destruction of a positively charged surface formed by two adjacent Arg residues in C5a.

Design of thiol-containing amino acids for native chemical ligation at non-Cys sites

Protein chemical synthesis usually relies on the use of native chemical ligation that couples peptide thioester with a Cys-peptide. A limitation of this method is the difficulty of finding an appropriate Cys ligation site in many synthetic targets. To overcome this problem, the ligation-desulfurization approach has been developed. This approach involves the use of a thiol-containing amino acid as the ligation partner. After the sequence assembly is completed, the thiol group is removed through a desulfurization reaction to generate the standard amino acids. Currently this strategy has been applied to the ligations at a number of amino acids including Ala, Phe, Val, Lys, Thr, Leu, Pro and Gin. The present article reviews the design and svnthesis of these thiol-containing amino acids for native chemical libation at non-Cys sites.