Anti-inflammatory effects of novel barbituric acid derivatives in T lymphocytes Chenjia Xu a , Arlene R. Wyman a , Manal A. Alaamery a , Shannon A. Argueta a , F. Douglas Ivey a , John A. Meyers b , Adam Lerner b , Tricia H. Burdo a , Timothy Connolly a , Charles S. Hoffman a , Thomas C. Chiles a, ⁎ a Departments of Biology, Boston College, Chestnut Hill, MA 02467, United States b Hematology and Medical Oncology, Boston University Medical Center, Boston, MA 02118, United States abstract article info Article history: Received 30 December 2015 Received in revised form 1 June 2016 Accepted 6 June 2016 Available online xxxx We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phos- phodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a N 95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human periph- eral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PDE7 inhibitory activity (BC12-4) displays similar biological activity, suggesting that BC12 does not act via PDE7 inhibition. To investigate the mechanism of inhibition of IL-2 production by BC12, we performed microarray anal- yses using unstimulated and stimulated Jurkat T cells in the presence or absence of BC12 or BC12-4. Our studies show these compounds affect the transcriptional response to stimulation and act via one or more shared targets to produce both anti-inflammatory and pro-stress effects. These results demonstrate potent immunomodulatory activity for BC12 and BC12-4 in T lymphocytes and suggest a potential clinical use as an immunotherapeutic to treat T lymphocyte-mediated diseases. © 2016 Elsevier B.V. All rights reserved. Keywords: T lymphocyte Interleukin 2 Cyclic nucleotide phosphodiesterase Pro-inflammatory cytokines Transcription factor 1. Introduction T cells contribute multiple functions to adaptive immunity, including the activation of B-lymphocytes, cell-mediated immunity and in atten- uation of normal immune responses [1]. In response to activation, CD4 + T lymphocytes can differentiate into a variety of distinct effector subtypes, including Th1, Th2, Tregs, and Th17 cells [1]. In addition to their role in adaptive immunity, CD4 + T lymphocytes may also contrib- ute to inflammatory responses, autoimmunity, and hypersensitive reac- tions [2]. An early event in the differentiation of naïve T cells is stimulation of the T-cell antigen receptor (TCR) together with antigen- bound MHC II complex by antigen presenting cells [3]. Engagement of the TCR together with co-stimulatory signals delivered via CD3 receptors activate signaling pathways that lead to interleukin 2 (IL-2) expression, which in turn, promotes T cell proliferation [4]. Activation of the p21ras pathway and the downstream MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 contribute via AP-1 to the activation of the IL-2 gene promoter [5]. TCR signaling also regulates IL-2 gene expression by promoting translocation of NF-κB and the nuclear factor of activated T cells (NFAT) into the nucleus [6]. Subsequent lineage-specific differentiation is dependent on numerous extrinsic factors, including the cytokine mi- lieu and the nature of the antigen presenting cells [1]. Understanding how extrinsic signals modulate endogenous T cell signaling pathways is essential to understanding normal as well as aberrant T cell growth and effector responses. Cyclic nucleotide phosphodiesterases (PDEs) are a family of related phosphohydrolases that catalyze the hydrolysis of the 3′ cyclic phos- phate bonds of adenosine and/or guanosine 3′,5′ cyclic monophosphate to produce 5′AMP and 5′GMP, respectively [7–9]. PDE families are cate- gorized based on their substrate specificity, their sensitivity to pharma- cological inhibitors, and the presence of conserved domains outside of the catalytic domains [7–9]. Differences in tissue-specific expression International Immunopharmacology 38 (2016) 223–232 Abbreviations: IL-2, interleukin 2; PHA, phytohaemagglutinin; PMA, phorbol-12- myristate-13-acetate; PDE, phosphodiesterase; TNFα, tumor necrosis factor-α; TCR, T- cell antigen receptor; IFNγ, interferon-γ; ELISA, enzyme-linked immunosorbent assay. ⁎ Corresponding author at: Department of Biology, Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, United States. E-mail address: chilest@bc.edu (T.C. Chiles). http://dx.doi.org/10.1016/j.intimp.2016.06.004 1567-5769/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp