Discovery of proteolytically stable monocyte locomotion inhibitory factor peptide through systematic structural optimization

The identification of novel molecular candidates capable of treating osteoarthritis (OA) has significant clinical implications. Monocyte locomotion inhibitory factor peptide (MLIF) is a pentapeptide derived from Entamoeba histolytica. It has been found possesses selective anti-inflammatory effects both in vitro and in vivo. Nonetheless, like many peptide therapeutics, MLIF has relatively poor proteolytic stability and short half-life in vivo, hindering its effective clinical applicability. To overcome these limitations, structural optimizations are needed to enhance the stability of MLIF while preserving or even enhancing its anti-inflammatory activities. Herein, a series of MLIF derivatives were designed and synthesized based on diverse structural modifications including N-terminal modifications, _D-amino acid replacement, N-methylation, sulfhydryl modification, cyclization, and splicing strategy. Among all the MLIF derivatives, MLIF 30 with replacing _L-methionine (Met) with _D-Met and linking the polyethylene glycol (PEG) to cysteine (Cys) of MLIF displayed enhanced in vitro anti-inflammatory activities. Further in vivo experiment demonstrated MLIF 30 could reduce cartilage inflammation and attenuate cartilage damage more effectively in the collagenase induced osteoarthritis (CIOA) mice due to its improved serum stability compared to the linear MLIF. These findings laid foundation for the development of potent and stable anti-inflammatory peptide therapeutics and pushed the frontier of MLIF for clinical OA treatment.