A small shared epitope-mimetic compound potently accelerates osteoclast-mediated bone damage in autoimmune arthritis.

We have recently proposed that the shared epitope (SE) may contribute to rheumatoid arthritis pathogenesis by acting as a ligand that activates proarthritogenic signal transduction events. To examine this hypothesis, in this study we characterized a novel small SE-mimetic compound, c(HS4-4), containing the SE primary sequence motif QKRAA, which was synthesized using a backbone cyclization method. The SE-mimetic c(HS4-4) compound interacted strongly with the SE receptor calreticulin, potently activated NO and reactive oxygen species production, and markedly facilitated osteoclast differentiation and function in vitro. The pro-osteoclastogenic potency of c(HS4-4) was 100,000- to 1,000,000-fold higher than the potency of a recently described linear SE peptidic ligand. When administered in vivo at nanogram doses, c(HS4-4) enhanced Th17 expansion, and in mice with collagen-induced arthritis it facilitated disease onset, increased disease incidence and severity, enhanced osteoclast abundance in synovial tissues and osteoclastogenic propensities of bone marrow-derived cells, and augmented bone destruction. In conclusion, c(HS4-4), a highly potent small SE-mimetic compound enhances bone damage and disease severity in inflammatory arthritis. These findings support the hypothesis that the SE acts as a signal transduction ligand that activates a CRT-mediated proarthritogenic pathway.

Studying protein-peptide interactions using benzophenone units: a case study of protein kinase B/Akt and its inhibitor PTR6154.

Protein-protein interactions (PPIs) govern nearly all processes in living cells. Peptides play an important role in studying PPIs. Peptides carrying photoaffinity labels that covalently bind the interacting protein can be used to obtain more accurate information regarding PPIs. Benzophenone (BP) is a useful photoaffinity label that is widely used to study PPIs. We developed a one-pot two-step synthesis for the preparation of novel BP units. To map the binding site more thoroughly, linkers of various lengths were attached to the BP moiety. These units can be incorporated into peptide sequences using well-established solid phase peptide synthesis (SPPS) protocols. As a proof of concept, we studied the interaction between protein kinase B (PKB/Akt) and its synthetic peptide inhibitor, PTR6154. The methodology is general and can be implemented to study PPIs in a variety of biological systems.

Developing potent backbone cyclic peptides bearing the shared epitope sequence as rheumatoid arthritis drug-leads.

Rheumatoid arthritis (RA) is a common human leukocyte antigen-associated disease. Most RA patients have a five-residue sequence motif called the shared epitope (SE) in the DRß-chain of the HLA-DRB1 protein. The SE was found to activate nitric oxide (NO) production, suggesting a possible mechanism for RA development. The native conformation of the SE is presumed to be an α-helix, thus using cyclic peptides to stabilize this conformation may produce a potent SE mimetic which will have drug-like properties. We present the development of a backbone cyclic SE mimetic that activates NO production in the low nM range. Circular dichroism analysis revealed a conformational change from for the parent linear peptides to the cyclic analogs. The most active cyclic analog is completely stable towards trypsin/chymotrypsin degradation while the linear 15-mer analogs completely degraded within 30 min. The outcome of this study is a potent cyclic peptide with drug-like properties that can be used as a template for drug development.