ABSTRACT
MyD88 is a cytoplasmic adaptor protein that plays a central role in signaling downstream of the TLRs and the IL1R superfamily. We previously demonstrated that MyD88 plays a critical role in EAE, the murine model of multiple sclerosis, and showed that the MyD88 BB-loop decoy peptide RDVLPGT ameliorates EAE. We now designed and screened a library of backbone cyclized peptides based on the linear BB loop peptide, to identify a metabolically stable inhibitor of MyD88 that retains the binding properties of the linear peptide. We identified a novel cyclic peptide protein mimetic that inhibits inflammatory responses to TLR ligands, and NFκB activation in response to IL-1 activation. The inhibitor, c(MyD 4-4), is metabolically stable in comparison to the linear peptide, blocks MyD88 in a specific manner, and inhibits MyD88 function by preventing MyD88 dimerization. Finally, treatment of mice with c(MyD 4-4) reduced the severity of clinical disease in the murine EAE model of multiple sclerosis. Thus, modulation of MyD88-dependent signaling using c(MyD 4-4) is a potential therapeutic strategy to lower innate immune inflammation in autoimmune CNS disease.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Myeloid Differentiation Factor 88/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Binding Sites , Female , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/chemistry , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Peptides, Cyclic/chemistry , Peptides, Cyclic/therapeutic use , Protein Binding , RAW 264.7 Cells , Receptors, Interleukin-1/metabolism , Toll-Like Receptors/metabolismABSTRACT
A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrinâ αvß3 is based on two concepts: a)â screening of systematically designed libraries with spatial diversity and b)â masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1)â initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2)â selection of cyclic peptides with the highest intestinal permeability; 3)â design of sublibraries with the bioactive RGD sequence in all possible positions; 4)â selection of the best ligands for RGD-recognizing integrin subtypes; 5)â fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6)â protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7)â proof of biological effects in mice after oral administration.