The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice Abdoreza Davoodi-Semiromi1*, Clive H. Wasserfall2, Chang Qing Xia2, Rhonda M. Cooper-DeHoff1, Martin Wabitsch3, Michael Clare-Salzler2, Mark Atkinson 2 * 1 Department of Pharmacotherapy & Translational Research, College of Pharmacy, University of Florida, Gainesville, Florida, United States of America, 2 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, United States of America, 3 Division of Pediatric Endocrinology, Diabetes and Obesity Unit, Department of Pediatrics and Adolescent Medicine, University of Ulm, Eythstr, Ulm, Germany Abstract Background: Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown. Materials and Methods: Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed. Results: AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.Scid mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p,0.004) and lower expression of co- stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system. Conclusion: The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease. Citation: Davoodi-Semiromi A, Wasserfall CH, Xia CQ, Cooper-DeHoff RM, Wabitsch M, et al. (2012) The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice. PLoS ONE 7(5): e36079. doi:10.1371/journal.pone.0036079 Editor: Paolo Fiorina, Children’s Hospital Boston/Harvard Medical School, United States of America Received January 9, 2012; Accepted March 26, 2012; Published May 14, 2012 Copyright: 2012 Davoodi-Semiromi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Juvenile Diabetes Research Foundation International (Innovative grant # 5-2006-937), and National Institutes of Health R21A176394-01 (The American Recovery And Reinvestment Act) awarded to ADS. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dsemiromi@cop.ufl.edu (ADS); atkinson@ufl.edu (MA) Introduction Tyrosine phosphorylation inhibitors, termed tyrphostins, are a family of low molecular weight molecules having the ability to inhibit tyrosine phosphorylation through protein tyrosine kinases (PTKs). The family has at least 31 members, with AG490, also known as tyrphostin B42, being one [1]. AG490 has been widely studied in cancer and autoimmune diseases, but not subject to investigation in the setting of T1D. Tyrosine kinase inhibitors (TKIs) have been broadly used in many cancer clinical trials [2–4], T1D [5,6] in mouse model of multiple sclerosis [7] and in intestinal inflmmation is reported [8]. Oral administration of Sunitinib (Sutent) and Imatinib (Gleevec), two TKIs, have been shown to prevent and reverse T1D in the NOD mouse [5]. In several publications successful remission of T1D has been reported [9–16]; however, when it comes to monotherapy approach long- lasting remission of diabetes is still challlenging [17]. The Jak-Stat signaling pathway plays a critical role in mediating inflammatory immune response to a variety of signals [18–21]. Currently, several Jak-Stat inhibitors have been developed and are under clinical investigation [4,22–25]. AG490 blocks the activa- tion of the Jak and STAT family of molecules [1]. AG490 has been reported to be more potent as a Jak2 inhibitor (4.3-fold) than Jak3 [26]; however, it has no impact on membrane associated kinases such as Lyn, Btk, Syk, Src and Zap70 [27,28] suggesting that it is not a general kinase inhibitor [26,27,29–31]. Additionally, AG490 blocks tumor growth and inhibits/delays the onset of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) [32], inhibits antigen-specific T-cell infiltration in EAE mice [32–34], and significantly extends rat heart allograft survival [35]. AG490 also blocks IL-12 mediated T cell proliferation, inhibits phosphorylation of Stat3, decreases IFN-c production mediated by IL-12 [32], and inhibits differentiation of antigen- PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36079