Cepharanthine Blocks the Presentation of Thyroid & Islet Peptides in a Novel Humanized Autoimmune Polyglandular Syndrome Type 3 Variant (APS3v) Mouse Model

Introduction: There is a strong genetic association between autoimmune thyroiditis (AITD) and Type 1 diabetes (T1D), and the co-occurrence of AITD and T1D in the same individual is referred to as Autoimmune Polyglandular Syndrome type 3 variant (APS3v). We previously discovered a unique amino acid signature of the HLA-DR pocket (designated APS3v HLA-DR pocket) that predisposes to APS3v, and we found that both thyroid (Tg.1571, TPO.758) and islet (GAD.492) peptides can bind to this flexible APS3v HLA-DR pocket. The goal of the present study is to identify small molecules that can block the presentation of these thyroid and islet peptides by the APS3v HLA-DR pocket as a potential therapeutic approach for APS3v. Methods: We screened a panel of small molecules using an in vitro screening assay we developed using Baculovirus-generated recombinant APS3v-DR. To validate “hit” compounds discovered by the in vitro assay in a mouse model we established a novel mouse model of APS3v. The APS3v mouse model was established by immunizing humanized NOD-DR3 mice with Tg.1571, TPO.758 and GAD.492 combined, together with adjuvant. We then validated hit small molecules for their effectiveness in blocking stimulation of T-cell responses in the APS3v mouse model. Results: Our screen identified 4 small molecules (S5, S9, S15, S53) that showed more than 50% inhibition in our in vitro assay. We were also able to establish a novel APS3v mouse model. Immunized mice showed an increase in cytokine production in response to all 3 thyroidal and islet peptides. The immunized mice also developed antibody responses against each peptide. When we tested the 4 “hit” compounds, only Cepharanthine (S53) which we previously identified as a small molecule blocking AITD immune responses, could block all 3 thyroidal/islet peptides in the APS3v mouse model. Treating with Cepharanthine prior to inducing APS3v in the mice also reduced T-cell proliferation in the immunized mice. Conclusions: We established a novel humanized APS3v mouse model by co-immunizing 3 thyroidal and islet peptides. We also identified Cepharanthine as a small molecule that can block thyroid and islet specific T-cell responses in this model. Cepharanthine can be further tested in APS3v patients carrying the specific APS3v-DR pocket as a potential therapeutic treatment.