Identifying a Small Molecule Blocking Antigen Presentation in Autoimmune Thyroiditis

Abstract

We previously showed that an HLA-DR variant containing arginine at position 74 of the DR␤1 chain (DR␤1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DR␤1-Arg74. We hypothesized that blocking the binding of these peptides to DR␤1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DR␤1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DR␤1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DR␤1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD. Autoimmune thyroid diseases (AITD),2 Graves disease (GD), and Hashimoto thyroiditis (HT), are among the most common autoimmune disorders, afflicting up to 5% of the United States population (1). They are characterized by infiltration of the thyroid by lymphocytes reactive to thyroid antigens and production of thyroid-specific antibodies (2). Complex interaction of genetic susceptibility factors, environmental triggers, and epigenetic alterations leads to the breakdown of tolerance, resulting in AITD (3-5). Currently, there is no satisfactory therapy except for hormone replacement therapy in HT or thyroid suppression or ablation in GD (6 -10). To develop new therapies for AITD a better understanding of their etiology is needed. We have been studying the etiology of AITD using a reverse-genetics approach, i.e. dissecting the mechanisms causing disease through unbiased genetic screening studies. These studies led to the identification of a specific HLA-DR pocket sequence that is strongly associated with AITD (11). The presence of arginine at position 74 of the DR␤ chain renders the individual highly susceptible to AITD, whereas glutamine at position 74 is protective (12). These data were confirmed by other groups (13). The presence of DR␤1-Arg74 (from here on we refer to the HLA-DR3 containing arginine at position 74 as HLA-DR␤1-Arg74) results in a more positively charged P4 pocket. With this structural change in the pocket, the selectivity and binding of pathogenic peptides is affected, conferring higher risk for disease (12).Besides the HLA genes, two thyroid-specific genes, the thyroglobulin (Tg) and thyrotropin receptor (TSHR) genes also contribute to the etiology of AITD (2). Thyroglobulin is the most abundant thyroidal protein (14), and it is the precursor to ...

Flexible peptide recognition by HLA-DR triggers specific autoimmune T-cell responses in autoimmune thyroiditis and diabetes

“…NOD-DR3 mice were used in this experiment because they are susceptible to EAT [28]. Each of the 11 peptides showing positive binding to the APS3v HLA-DR in vitro (2 Tg, 1 Ins, 2 GAD65 and 6 TPO) was used to immunize 3 female NOD-DR3 mice to induce T-cell responses to the peptide.…”

“…Of the 2 thyroglobulin peptides that bound to the APS3v HLA-DR only Tg.1571 stimulated T-cell responses suggesting that it is a major antigenic epitopes for APS3v. Interestingly, Tg.1571 was also identified as an important epitope in AITD [13, 28]. Hence, while some antigenic epitopes (e.g.…”

“…Therefore, an approach to target the HLA pocket predisposing to APS3v for therapeutic purpose would be more advantageous than targeting specific peptides. Non-peptidic small organic molecules targeting susceptible HLA alleles and blocking T-cell responses have recently shown promise as a potential new therapeutic approach in autoimmunity [28, 44–47]. Our lab has identified a small molecule inhibitor, Cepharanthine, that can block T-cell activation by Tg peptides within the HLA-DRβ1-Arg74 pocket [28].…”

“…Non-peptidic small organic molecules targeting susceptible HLA alleles and blocking T-cell responses have recently shown promise as a potential new therapeutic approach in autoimmunity [28, 44–47]. Our lab has identified a small molecule inhibitor, Cepharanthine, that can block T-cell activation by Tg peptides within the HLA-DRβ1-Arg74 pocket [28]. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis.…”

“…Female NOD-DR3 mice, 4–6 weeks old, were injected subcutaneously with the peptides tested in this study in Complete Freund’s Adjuvant (CFA, Sigma) to induce T-cell responses as previously described [27, 28]. Mice were immunized with peptides on day 0 and boosted on day 7, then sacrificed on day 21.…”