Discovery, synthesis, and structure-activity relationships of 20S-dammar-24-en-2α,3ß,12ß,20-tetrol (GP) derivatives as a new class of AMPKα2ß1γ1 activators.

As a follow-up discovery of AMPK activators from natural products, 20S-dammar-24-en-2α,3ß,12ß,20-tetrol (GP, 1), a dammarane-type triterpenoid, was found to have some favorable metabolic effects on dyslipidemia in Golden Syrian hamsters, and activate AMPKα2ß1γ1 by around 2.4 fold with an EC50 of 5.1µM on molecular level. In order to enhance its potency at AMPK and structure-activity relationship study, GP derivatives were designed, synthesized, and evaluated in pharmacological AMPK activation assays. Structure-activity relationship analysis showed that amine at the 24-position (groups I-IV) effectively and significantly increased the potency and efficacy. GP derivatives 12 and 17-19 exhibited better potency (EC50: 0.3, 0.8, 0.8, and 1.0µM) and efficacy (fold: 3.2, 2.7, 3.0, and 2.8) in the activation of AMPK heterotrimer α2ß1γ1 than positive control (AMP, EC50: 1.6µM, fold: 3.2). Furthermore, the most potent compounds 12 and 17 obviously inhibited glucose output through increasing the phosphorylation of AMPK, without affecting mitochondrial membrane potential or producing cytotoxicity.

Role of activated macrophages in experimental Fusarium solani keratitis.

Macrophages under the conjunctival tissue are the first line defender cells of the corneas. Elimination of these cells would lead to aggravation of fungal keratitis. To determine how the course of fungal keratitis would be altered after the activation of these macrophages, a murine model was achieved by intrastromal instillation of latex beads before the corneas were infected with Fusarium solani. The keratitis was observed and clinically scored daily. Infected corneas were homogenized for colony counts. The levels of the IL-12, IL-4, MPO, MIF and iNOS cytokines were measured in the corneas using real-time polymerase chain reactions and enzyme-linked immunosorbent assays. CD3+, CD4+ and CD8+ lymphocytes in the corneas, submaxillary lymph nodes and peripheral blood were detected using immunohistochemistry and flow cytometry, respectively. The latex bead-treated mice exhibited aggravated keratitis. Substantially increased macrophage and polymorphonuclear leukocyte infiltration was detected in the corneas, although few colonies were observed. There was a marked increase in the IL-12, IL-4, MPO, MIF and iNOS expression in the corneas. The numbers of CD3+, CD4+ and CD8+ lymphocytes and the CD4+/CD8+ ratio were significantly enhanced in the corneas and submaxillary lymph nodes. However, the number of CD4+ lymphocytes was decreased in the peripheral blood, while the number of CD8+ lymphocytes increased. Collectively, our data demonstrate that the activation of macrophages in the cornea may cause an excessive immune response. Macrophages appear to play a critical role in regulating the immune response to corneal infections with F. solani.