Signal Transduction of Hyaluronic Acid-Peptide Conjugate for Formyl Peptide Receptor Like 1 Receptor

Agonistic and antagonistic peptides for formyl peptide receptor like 1 (FPRL1) receptor have been investigated as novel drug candidates for inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis. In this work, a novel protocol for the synthesis of hyaluronic acid (HA)-peptide (CWRYMVm) conjugate for FPRL1 receptor was successfully developed for further clinical applications of peptide drugs. Aminoethyl methacrylated HA (HA-AEMA) was synthesized by the coupling reaction of tetrabutyl ammonium salt of HA (HA-TBA) and AEMA using benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP) in dimethyl sulfoxide (DMSO). Then, HA-AEMA was conjugated with CWRYMVm in water via Michael addition reaction between methacrylate group of HA-AEMA and thiol group in cysteine. The formation of HA-peptide conjugate was confirmed by H-1 NMR and gel permeation chromatography (GPC). The average number of conjugated peptide molecules could be controlled from 5 to 23 per single HA chain. The HA-peptide conjugate showed serum stability longer than four days. In vitro signal transduction activity of the HA-peptide conjugate for FPRL1 receptor was confirmed from the elevated levels of phospho-extracellular signal-regulated kinase (pERK) and calcium ion in FPRL1 overexpressing RBL-2H3 cells. The partially decreased biological activity of HA-peptide conjugates by the steric hindrance of HA was recovered after its degradation by hyaluronidase treatment.