Uncompetitive Phospholipase A2 Inhibition by CHEC Sequences Including Oral Treatment of Experimental Autoimmune Myeloencephalitis

Abstract

Catalytic sites of metal dependent phosphatases and phospholipases are characterized by a catalytic histidine (H) and adjacent acidic metal binding residue E (or D). For the secreted phospholipases A2 (sPLA2s), the active sites are stabilized by nearby flanking cystines. CHEC-9 (CHEASAAQC), which displays this organization, is a sPLA2 inhibitor that is an effective treatment for models of traumatic and autoimmune neurodegenerative disorders. We screened modifi- cations of this sequence and found that elimination of the two central alanines followed by cyclization results in another potent sPLA2 inhibitor, CHEC-7 (CHEASQC). CHEC-7, like CHEC-9, had uncompetitive kinetic properties making it ideal for in vivo applications. Subcutaneous injections of CHEC-7 into restrained rats inhibited the transient rise in plasma sPLA2 activity suggesting that this peptide was also effective in vivo. Oral delivery of the peptide attenuated the LPS- induced increases in sPLA2 activity. Thin layer chromatography and mass spectroscopy of representative LPS-treated rats suggested that the peptide also reduced levels of lysophosphatidylcholine, a hydrolytic product of the enzyme reaction. In order to test the efficacy of CHEC-7 for neuroimmune therapies, rats were immunized with spinal cord homogenates to induce experimental autoimmune encephalomyelitis (EAE), followed by daily treatment with oral (1.5 mg/kg) or subcuta- neous (0.1, 0.5, and1.5mg/kg) CHEC-7 and CHEC-9 (n=10 for each different condition). The rats were scored for severity of disease applying a standard EAE scale. Control rats all developed disease over the 23-day course of the experiments. Both CHEC-9 and CHEC-7 produced highly significant reductions in disease severity including via oral delivery. The performance of oral CHEC-7 was notable in that this treatment completely prevented disease in half the rats. The results suggested that continued study of modifications of CHEC sequences would provide stable therapeutic peptides for a variety of inflammatory and autoimmune disorders.