Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 M, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Adiponectin plays a pivotal role in the pathogenesis of diabetes and is considered to be involved in various pathological conditions associated with diabetes; however, its role in wound repair is unknown. In this study, we elucidated the involvement of adiponectin in cutaneous wound healing in vitro and in vivo. Normal human keratinocytes expressed adiponectin receptors, and adiponectin enhanced proliferation and migration of keratinocytes in vitro. This proliferative and migratory effect of adiponectin was mediated via AdipoR1/AdipoR2 and the ERK signaling pathway. Consistent with in vitro results, wound closure was significantly delayed in adiponectin-deficient mice compared with wild-type mice, and more importantly, keratinocyte proliferation and migration during wound repair were also impaired in adiponectin-deficient mice. Furthermore, both systemic and topical administration of adiponectin ameliorated impaired wound healing in adiponectin-deficient and diabetic db/db mice, respectively. Collectively, these results indicate that adiponectin is a potent mediator in the regulation of cutaneous wound healing. We propose that upregulation of systemic and/or local adiponectin levels is a potential and very promising therapeutic approach for dealing with diabetic wounds.

Accumulating epidemiologic evidence has revealed that metabolic syndrome is an independent risk factor for psoriasis development and is associated with more severe psoriasis. Adiponectin, primarily recognized as a metabolic mediator of insulin sensitivity, has been newly drawing attention as a mediator of immune responses. Here we demonstrate that adiponectin regulates skin inflammation, especially IL-17-related psoriasiform dermatitis. Mice with adiponectin deficiency show severe psoriasiform skin inflammation with enhanced infiltration of IL-17-producing dermal Vg4 þ gd-T cells. Adiponectin directly acts on murine dermal gd-T cells to suppress IL-17 synthesis via AdipoR1. We furthermore demonstrate here that the adiponectin level of skin tissue as well as subcutaneous fat is decreased in psoriasis patients. IL-17 production from human CD4-or CD8-positive T cells is also suppressed by adiponectin. Our data provide a regulatory role of adiponectin in skin inflammation, which would imply a mechanism underlying the relationship between psoriasis and metabolic disorders.

Summary To guide development of new drugs targeting methionyl-tRNA synthetase (MetRS) for treatment of human African trypanosomiasis, crystal structure determinations of Trypanosoma brucei MetRS in complex with its substrate methionine and its intermediate product methionyl-adenylate were followed by those of the enzyme in complex with high-affinity aminoquinolone inhibitors via soaking experiments. Drastic changes in conformation of one of the two enzymes in the asymmetric unit allowed these inhibitors to occupy an enlarged methionine pocket and a new so-called auxiliary pocket. Interestingly, a small low-affinity compound caused the same conformational changes, removed the methionine without occupying the methionine pocket, and occupied the previously not existing auxiliary pocket. Analysis of these structures indicates that the binding of the inhibitors is the result of conformational selection, not induced fit.

Urea-based methionyl-tRNA synthetase inhibitors were designed, synthesized and evaluated for their potential towards treating human African trypanosomiasis (HAT). With the aid of a homology model and a structure-activity-relationship approach, low nM inhibitors were discovered that show high selectivity towards the parasite enzyme over the closest human homolog. These compounds inhibit parasite growth with EC50 values as low as 0.15 μM while having low toxicity to mammalian cells. Two compounds (2 and 26) showed excellent membrane permeation in the MDR1-MDCKII model, and encouraging oral pharmacokinetic properties in mice. Compound 2 was confirmed to enter the CNS in mice. Compound 26 had modest suppressive activity against T. brucei rhodesiense in the mouse model, suggesting that more potent analogs or compounds with higher exposures need to be developed. The urea-based inhibitors are thus a promising starting point for further optimization towards the discovery of orally available and CNS active drugs to treat HAT.

Psoriasis is an inflammatory cutaneous disorder characterized by marked epidermal thickening and Th1 and Th17 cell infiltration. At present, the contribution of B cells to the pathogenesis of psoriasis is unclear. In mice, topical application of imiquimod induces inflamed skin lesions and serves as an experimental animal model for human psoriasis. In this study, we showed that imiquimod-induced skin inflammation was more severe in CD19(-/-) than WT mice. These inflammatory responses were negatively regulated by a unique IL-10-producing CD1d(hi)CD5(+) regulatory B cell subset (B10 cells) that was absent in CD19(-/-) mice and represented only 1-2% of splenic B220(+) cells in WT mice. Splenic B10 cells entered the circulation and migrated to draining LNs during imiquimod-induced skin inflammation, thereby suppressing IFN-γ and IL-17 production. Furthermore, adoptive transfer of these B10 cells from WT mice reduced inflammation in CD19(-/-) mice. The present findings provide direct evidence that B10 cells regulate imiquimod-induced skin inflammation and offer insights into regulatory B cell-based therapies for the treatment of psoriasis.

The immunological significance of IL-27 has been reported and discussed in various Th1/Th17-mediated inflammatory diseases. However, its importance in psoriasis is unknown. We investigated pathophysiological roles of IL-27 in psoriasis in this study. Serum IL-27 levels in psoriatic patients were significantly higher than those in healthy controls, and correlated with disease severity and serum IFN-gamma levels. An immunohistochemical analysis revealed the infiltration of IL-27-secreting cells in the papillary dermis of psoriatic skin lesions but not in skin lesions with atopic dermatitis or normal skin. Furthermore, IL-27 alone greatly induced in vitro CXCL9, CXCL10, and CXCL11 production and tyrosine phosphorylation of signal transducer and activator of transcription 1 in normal human keratinocytes, while it suppressed the tumor necrosis factor-alpha-induced production of IL-1alpha and CCL20. These results indicate that IL-27 may promote the onset of psoriasis, while it may simultaneously attenuate the expanded inflammation in this disease. Our results implicate potential therapeutic effects of IL-27 for psoriasis.

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