Shared epitope-antagonistic ligands: a new therapeutic strategy in mice with erosive arthritis

Abstract

Objective: The mechanisms underlying bone damage in rheumatoid arthritis (RA) are incompletely understood. We recently identified the shared epitope (SE), an HLA-DRB1-coded 5-amino acid sequence motif carried by the majority of RA patients as a signal transduction ligand that interacts with cell surface calreticulin and accelerates osteoclast (OC)-mediated bone damage in collagen-induced arthritis (CIA). Given the role of the SE/calreticulin pathway in arthritis-associated bone damage, we sought to determine the therapeutic targetability of calreticulin.

Methods: A library of backbone-cyclized peptidomimetic compounds, all carrying an identical core DKCLA sequence, was synthesized. The ability of these compounds to inhibit SE-activated signaling and OC differentiation was tested in vitro. The effect on disease severity and OC-mediated bone damage was studied by weekly intraperitoneal administration of the compounds to DBA/1 mice with CIA.

Results: Two members of the peptidomimetics library were found to have SE-antagonistic effects and antiosteoclast differentiation effects at picomolar concentrations in vitro. The lead mimetic compound, designated HS(4-4)c Trp, potently ameliorated arthritis and bone damage in vivo when administered in picogram doses to mice with CIA. Another mimetic analog, designated HS(3-4)c Trp, was found to lack activity, both in vitro and in vivo. The differential activity of the 2 analogs depended on minor differences in their respective ring sizes and correlated with distinctive geometry when computationally docked to the SE binding site on calreticulin.

Conclusion: These findings identify calreticulin as a novel therapeutic target in erosive arthritis and provide sound rationale and early structure/activity relationships for future drug design.

Figure 1. SEAL activity in vitro

A. Interaction between linear DKCLA and recombinant CRT; B. Linear DKCLA (508 μM) specifically inhibits the interaction between SE ligand 65–79*0401 (171 μM) and CRT; C. Linear DKCLA specifically inhibits the interaction between SE ligand 65–79*0404; D. Dose-response inhibition of the interaction between SE ligand 65–79*0401 (171 μM) and CRT by Linear DKCLA; E. Dose-response inhibition of the interaction between SE ligand 65–79*0404 (169 μM) and CRT by Linear DKCLA; F. Linear DKCLA (170 μM) specifically inhibits SE ligand 65–79*0401(57μM)-activated NO signaling; G. Structural formula of the cDKCLA compound library. H. Dose-response curve of the inhibitory effect of HS(4–4)c Trp on SE ligand 65–79*0401 (57 μM)-activated signaling. I. Competitive inhibition of SE ligand 65–79*0401 (229 μM) binding to CRT by HS(4–4)c Trp; J. HS(4–4)c Trp inhibits unstimulated (left) and SE ligand 65–79*0401 (29 μM)-activated (right) osteoclastogenesis in primary mouse BMCs. Asterisks denote statistical significance compared to the respective control culture: PBS, or 65–79*0401; K. Compound HS(3–4)c Trp does not inhibit osteoclastogenesis in mouse BMC; L. HS(4–4)c Trp and HS(3–3)c Trp inhibit SE ligand-activated osteoclastogenesis in human PBMC. #, p < 0.05 compared to PBS cultures; * p < 0.05 compared to cultures with 65–79*0401.

Figure 2. HS(4–4)c Trp ameliorates CIA

A. HS(4–4)c Trp, 36 pg/gm (blue) or 360 pg/gm (red), or PBS (black) was administered ip weekly and day of arthritis onset was recorded. N=10 per group. B. CIA mice were treated as above and arthritis incidence was recorded over time; C. CIA mice were treated as above and joint swelling was recorded over time. P values were calculated using a paired t-test compared to PBS treated mice; D. Synovial OCs were counted in the joints of CIA mice treated as above. N=5 per group. E. Wrist histology of CIA mice treated with PBS (left column), or 360 pg/gr of HS(4–4)c Trp (right column). Sections were stained with either TRAP (upper row, 4x magnification), or H&E (lower row, 100x magnification). The region identified by a white arrow is magnified in the boxed image in the lower right corner of the image. F. Micro-CT images of front (left) and rear (right) paws of CIA mice treated with PBS (upper row), 36 pg/gr (middle row), or 360 pg/gr (bottom row) of HS(4–4)c Trp. G. Bone erosion scores of CIA mice treated as above.

Figure 3. HS(3–4)c Trp is inactive

A. Arthritis day of onset in DBA/1 CIA mice treated with HS(3–4)c Trp (administered ip weekly at a dose of 360 pg/gr- blue) or with PBS (black). N=10 per group. B. Arthritis incidence curves of CIA mice treated with 360 pg/gr of HS(3–4)c Trp (blue) or PBS (black). N=10 per group. C. Arthritis scores of CIA mice treated with 360 pg/gr of HS(3–4)c Trp (blue) or with PBS (black). N=10 per group; D. Comparative micro-CT images of paws of CIA mice treated with PBS (upper panel) or HS(3–4)c Trp, 360 pg/gr (lower panel); E. OCs were counted in the joints of CIA mice treated with 360 pg/gr HS(3–4)c Trp (blue) or PBS (black) as above. N=5 per group.

Figure 4. Docking models

Ligands (cyan) were docked onto the CRT surface (light brown). Side chains of interacting CRT residues are highlighted in orange. Dotted lines represent chemical bonds. Red areas represent surface of key CRT residues that interact with ligands. Inserted boxes depict chemical interactions. The CRT residues are identified in a three-letter format. Ligand residues are shown in a single-letter format.