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.
Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis.
BACKGROUND AND PURPOSEToll-like receptor 7 (TLR7) agonists have potential in the treatment of allergic diseases. However, the therapeutic utility of current low molecular weight TLR7 agonists is limited by their systemic activity, resulting in unwanted side effects. We have developed a series of TLR7-selective 'antedrugs', including SM-324405 and AZ12441970, which contain an ester group rapidly cleaved in plasma to reduce systemic exposure. EXPERIMENTAL APPROACHAgonist activity at TLR7 of the parent ester and acid metabolite was assessed in vitro in reporter cells and primary cells from a number of species. Pharmacokinetics following a dose to the lungs was assessed in mice and efficacy evaluated in vivo with a mouse allergic airway model. KEY RESULTSCompounds were selective agonists for TLR7 with no crossover to TLR8 and were metabolically unstable in plasma with the acid metabolite showing substantially reduced activity in a number of assays. The compounds inhibited IL-5 production and induced IFN-a, which mediated the inhibition of IL-5. When dosed into the lung the compounds were rapidly metabolized and short-term exposure of the 'antedrug' was sufficient to activate the IFN pathway. AZ12441970 showed efficacy in a mouse allergic airway model with minimal induction of systemic IFN-a, consistent with the low plasma levels of compound. CONCLUSIONS AND IMPLICATIONSThe biological and metabolic profiles of these TLR7-selective agonist 'antedrug' compounds are consistent with a new class of compound that could be administered locally for the treatment of allergic diseases, while reducing the risk of systemic side effects.
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