TLR7 stimulation of APCs results in inhibition of IL-5 through type I IFN and Notch signaling pathways in human peripheral blood mononuclear cells.

TLR7 agonists modulate Th2 immune responses through mechanisms that have not been fully elucidated. Suppression of IL-5 production from Ag- or phytohemagglutinin-stimulated human PBMCs by the TLR7 antedrug AZ12441970 was mediated via type I IFN-dependent and type I IFN-independent mechanisms through TLR7 activation of plasmacytoid dendritic cells, B cells, and monocytes. The type I IFN-dependent inhibition of T cell-derived IL-5 was mediated by IFN-α acting directly on activated T cells. IL-10 was shown not to be involved in the type I IFN-independent inhibition of IL-5 and the mechanism of inhibition required cell-cell interaction. Notch signaling was implicated in the inhibition of IL-5, because addition of a γ-secretase inhibitor blocked the type I IFN-independent suppression of IL-5. Accordingly, AZ12441970 induced high levels of the notch ligands Dll1 and Dll4 mRNA, whereas immobilized DLL4 resulted in the suppression of IL-5 production. Therefore, we have elucidated two mechanisms whereby TLR7 agonists can modulate IL-5 production in human T cells. The suppression of Th2 cytokines, including IL-5, would be of benefit in diseases such as atopic asthma, so we assessed TLR7 function in PBMC from asthmatics and showed equivalent activity compared with healthy volunteers. Demonstrating this function is intact in asthmatics and knowing it links to suppression of Th2 cytokines support the case for developing such compounds for the treatment of allergic disease.

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT-1.

We have recently described the immunosuppressive properties of AR-C117977 and AR-C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT-1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR-C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft-versus-host (GvH) response in the rat, and in high- and low-responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT-1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT-1.

Mechanism of action of inhibition of allergic immune responses by a novel antedrug TLR7 agonist.

Triggering innate immune responses through TLRs is expected to be a novel therapeutic strategy for the treatment of allergic diseases. TLR agonists are able to modulate Th2 immune responses through undefined mechanisms. We investigated the mechanism of action of the suppression of Th2 immune responses with a novel antedrug TLR7 agonist. The antedrug is rapidly metabolized by plasma esterases to an acid with reduced activity to limit systemic responses. Topical administration of this compound inhibited features of the allergic airway inflammatory response in rat and murine allergic airways model. Type I IFN played a role in the suppression of Th2 cytokines produced from murine splenocytes. Inhibition of Th2 immune responses with the antedrug TLR7 agonist was shown to be via a type I IFN-dependent mechanism following short-term exposure to the compound, although there might be type I IFN-independent mechanisms following long-term exposure. We have demonstrated that local type I IFN signaling and plasmacytoid dendritic cells, but not Th1 immune responses, are required for in vivo efficacy against murine airway Th2-driven eosinophilia. Furthermore, migration of dendritic cell subsets into the lung was related to efficacy and is dependent on type I IFN signaling. Thus, the mechanism of action at the cytokine and cellular level involved in the suppression of Th2 allergic responses has been characterized, providing a potential new approach to the treatment of allergic disease.

CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.

Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.

AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.

In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3+/-1.4 nM, 1.29+/-0.09 nmol per ml of packed cells and 12.2+/-1.1 s-1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7-10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011-20021].

The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donor-specific suppression, reducing acute and chronic allograft rejection in the rat.

BACKGROUND: In a search for immunosuppressive drugs having novel mechanisms, monocarboxylate transporter (MCT-1) inhibitors were identified that markedly inhibited immune responses. Here, we report the effects of AR-C117977, a potent MCT-1 inhibitor, on alloimmune responses in the rat. METHODS: In vitro activity was determined in a rat mixed lymphocyte response (MLR). In vivo activity was tested in a graft versus host response (GVHR) and in both high (DA to PVG) and low (PVG to DA) responder cardiac allograft models. To assess induction of donor-specific suppression recipients of allogeneic hearts surviving longer than 100 days received a second transplant either of the same donor strain or a third-party donor strain. Effects on chronic graft rejection were assessed histologically by evaluating vasculopathy in long-term surviving grafts and in an obliterative bronchiolitis (OB) model. RESULTS: AR-C117977 inhibited the rat MLR and was more potent than cyclosporin A (CsA). In the rat GVHR model, AR-C117977 gave a dose-related inhibition. In the high responder cardiac allograft model, graft survival in excess of 100 days was achieved with AR-C117977 compared with 20 days with CsA and all the long-term survivors exhibited donor-specific suppression on retransplantation. In the low responder model, both AR-C117977 and CsA induced survival in excess of 100 days. Histology of the long-term surviving grafts suggested reduced vasculopathy associated with chronic rejection. Furthermore, AR-C117977 inhibited the occlusion of transplanted trachea in a OB model. CONCLUSION: This report describes a MCT-1 specific inhibitor having immunosuppressive activity on alloimmune responses and inducing donor-specific suppression.

Monocarboxylate transporter MCT1 is a target for immunosuppression.

Current immunosuppressive therapies act on T lymphocytes by modulation of cytokine production, modulation of signaling pathways or by inhibition of the enzymes of nucleotide biosynthesis. We have identified a previously unknown series of immunomodulatory compounds that potently inhibit human and rat T lymphocyte proliferation in vitro and in vivo in immune-mediated animal models of disease, acting by a novel mechanism. Here we identify the target of these compounds, the monocarboxylate transporter MCT1 (SLC16A1), using a strategy of photoaffinity labeling and proteomic characterization. We show that inhibition of MCT1 during T lymphocyte activation results in selective and profound inhibition of the extremely rapid phase of T cell division essential for an effective immune response. MCT1 activity, however, is not required for many stages of lymphocyte activation, such as cytokine production, or for most normal physiological functions. By pursuing a chemistry-led target identification strategy, we have discovered that MCT1 is a previously unknown target for immunosuppressive therapy and have uncovered an unsuspected role for MCT1 in immune biology.