Development of a novel RANKL-based peptide, microglial healing peptide1-AcN (MHP1-AcN), for treatment of ischemic stroke

Munehisa Shimamura,Hideyuki Mukai,Shota Yoshida,Nan Ju,Hiroki Hayashi,Hideo Shimizu,Ryosuke Watanabe,Yuka Ikeda,Tomohiro Kawano,Hironori Nakagami,Ryuichi Morishita,Takeshi Okuzono,Hideki Mochizuki

doi:10.1038/s41598-018-35898-z

Abstract

Although the regulation of post-ischemic inflammation is an important strategy to treat ischemic stroke, all clinical trials have failed to show its efficacy. To solve the problem, we previously developed a novel partial peptide of RANKL, microglial healing peptide 1 (MHP1), which could reduce ischemic injury by inhibiting Toll-like receptor (TLR) induced inflammation. However, optimization of the peptide was necessary to increase the stability and efficacies for clinical use. According to information gathered through HPLC/MS in serum, we have newly designed a series of modified MHP1 peptides and have found that N-terminal acetylation and C-terminal amidation in MHP1 (MHP1-AcN), can strengthen its anti-inflammatory effects and increase its stability with anti-osteoclastogenic effects. Anti-TLR activity was reported to be reduced in MHP1 when incubated at 37 °C for 24 hrs, but MHP1-AcN could keep the activity under the same condition. The therapeutic effect of MHP1-AcN was observed in transient ischemic stroke model at lower dose than MHP1. Importantly, MHP1-AcN did not affect thrombolytic effects of tissue plasminogen activator (tPA) and inhibited tPA-induced hemorrhagic transformation. These findings indicated that MHP1-AcN was stable and effective anti-TLR signal peptide and could be a promising agent for treating stroke patients receiving tPA and endovascular therapy.

Highlights

To solve this problem, we developed a novel peptide “microglial healing peptide[1] (MHP1)”, which is a partial peptide of RANKL, to inhibit anti-TLR4-induced inflammation without activating osteoclast[5]
The peptide bonds of microglial healing peptide 1 (MHP1) were cleaved at the N-terminus (RT: 28.1, 28.3, 31.0), C-terminus (RT: 33.1, 33.5), and both termini (RT: 30.5, 31.5, 32.3, 33.4) (Fig. 1B,C), some degradation products, such as “LMVYVVKTSIKIPSSHNLMKGGSTKNWSGNOxygen”, “LMVYVVKTSIKIPSSHNLMKGGS”, or “LMVYVVKTSIKIPSSHNLM”, included the sequence “LMVYVVKTSIKIPSS”, which is a key region for anti-Toll-like receptor (TLR)-signaling[5]
As the N-terminus “LMVYVVKTSIKIPSS” is the key region for the anti-TLR activity in MHP1 and degradation of this region completely loses its activity[5], we designed 3 peptides with an amino acid in this region replaced by D-amino acid

Summary

Introduction

We developed a novel peptide “microglial healing peptide[1] (MHP1)”, which is a partial peptide of RANKL, to inhibit anti-TLR4-induced inflammation without activating osteoclast[5]. MHP1 inhibited TLR2, 7, 8-induced inflammations[5,6], which are other important signals for DAMP in post-ischemic inflammation[7,8]. Since unmodified synthetic peptide loses its activity due to rapid degradation within minutes by enzymes[9], modification of MHP1 was required to increase the stability for clinical use. The effects of the modified MHP1 on tissue plasminogen activator (tPA)-induced thrombolysis and cerebral hemorrhage were examined because tPA is commonly used in the acute stage of ischemic stroke clinically

Methods

Results

Conclusion