Yongchun Shen

Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)–containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti–double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Discovery of anti-inflammatory clinical candidate E6201, inspired from resorcylic lactone LL-Z1640-2, III.

Inspired by natural product, LL-Z1640-2, clinical candidate, E6201 (22) was discovered in a medicinal chemistry effort through total synthesis. The modification on C14-position to N-alkyl substitution showed to be potent in vitro and orally active in vivo in anti-inflammatory assays.

Antiproliferative triterpenoid saponins of Dodonaea viscosa from the Madagascar dry forest.

Bioassay-guided fractionation of an EtOH extract obtained from the roots of the Madagascan plant Dodonaea viscosa led to the isolation of two new antiproliferative oleanane-type triterpenoid saponins, dodoneasides A and B (1 and 2). The structures of these two new compounds were elucidated using 1D and 2D NMR experiments and mass spectrometry. Compounds 1 and 2 showed antiproliferative activity against the A2780 human ovarian cancer cell line with IC(50) values of 0.79 and 0.70 muM, respectively.

Antiproliferative acetogenins from a Uvaria sp. from the Madagascar dry forest.

Investigation of the endemic Madagascan plant Uvaria sp. for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of two new acetogenins. The structures of these two compounds were elucidated on the basis of analysis of their 1D and 2D NMR spectra, circular dichroism, and mass spectrometric data, together with chemical modification. The two acetogenins display weak antiproliferative activity against the A2780 ovarian cancer, the A2058 melanoma, and the H522 lung cancer cell lines.

Isolation and synthesis of antiproliferative eupolauridine alkaloids of Ambavia gerrardii from the Madagascar Dry Forest.

Investigation of the Madagascan endemic plant Ambavia gerrardii for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the three new alkaloids 8-hydroxyeupolauridine (1), 9-methoxyeupolauridine 1-oxide (2), and 11-methoxysampangine (3) and the three known alkaloids 4-6. The structures of 1 and 2 were confirmed by synthesis. Compounds 3, 4, and 6 showed moderate to good antiproliferative activities, with IC50 values of 10.3, 3.5, and 0.60 μM, respectively, against the A2780 human ovarian cancer cell line and with IC50 values of 0.57, 1.77, and 0.58 μM, respectively, against the H460 human lung cancer cell line.

Discovery of an in vitro and in vivo potent resorcylic lactone analog of LL-Z1640-2 as anti-inflammatory lead, II

The potent in vitro lead compound, ER-803064 (2), a MEK1 and MEKK1 inhibitor inspired from natural product LL-Z1640-2 (f152A1), was further optimized to improve in vitro and in vivo potency. The modifications on C14 position led to discovery of the lead compounds 28 and 29, which regained full in vitro potency of f152A1 and showed higher in vivo potency by iv administration.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

Antiproliferative Compounds from Cleistanthus boivinianus from the Madagascar Dry Forest1

The two new lignans 3α-O-(β-d-glucopyranosyl)desoxypodophyllotoxin (1) and 4-O-(β-d-glucopyranosyl)dehydropodophyllotoxin (2) were isolated from Cleistanthus boivinianus, together with the known lignans deoxypicropodophyllotoxin (3), (±)-β-apopicropodophyllin (4), (−)-desoxypodophyllotoxin (5), (−)-yatein (6), and β-peltatin-5-O-β-d-glucopyranoside (7). The structures of all compounds were characterized by spectroscopic techniques. Compounds 1, 4, and 5 showed potent antiproliferative activities against the A2780 ovarian cancer cell line, with IC50 values of 33.0 ± 3.6, 63.1 ± 6.7, and 230 ± 1 nM, respectively. Compounds 2 and 7 showed only modest A2780 activities, with IC50 values of 2.1 ± 0.3 and 4.9 ± 0.1 μM, respectively, while compounds 3 and 6 had IC50 values of >10 μM. Compound 1 also had potent antiproliferative activity against the HCT-116 human colon carcinoma cell line, with an IC50 value of 20.5 nM, and compound 4 exhibited modest antiproliferative activity against the A2058 human caucasian metastatic melanoma and MES-SA human uterine sarcoma cell lines, with IC50 values of 4.6 and 4.0 μM, respectively.

Antiproliferative and antiplasmodial compounds from selected Streptomyces species.

In continuation of our ongoing search for bioactive compounds from microbial extracts, we performed antiproliferative and/or antimalarial assays on extracts of 806 microbial species isolated from Madagascan marine organisms, on 1317 species isolated from Madagascan soil samples and on a Streptomyces species (S.4) from a marine sponge collected from the Florida Keys. This work identified active extracts from four Streptomyces isolates (S.1, S.2, S.3 and S.4). The extracts of Streptomyces S.1 and S.2 showed antiproliferative activity against the A2780 ovarian cancer cell line, while those of S.3 and S.4 displayed both antiproliferative and antimalarial activity. Bioassay-guided fractionation coupled with dereplication of the active extracts led to the identification and isolation of nonactin (1), monactin (2), dinactin (3), ±-nonactic acid (4), toyocamycin (5), piperafizine A (6) and a new dipeptide named xestostreptin (7). The structures of all isolated compounds 1-7 were elucidated by analyses of their NMR spectroscopic and mass spectrometric data, and were confirmed by comparison with the data reported in the literature. Compound 6 was crystallized and subjected to X-ray diffraction analysis to confirm its structure as piperafizine A (6). Compounds 1-3 displayed strong antiproliferative activity against A2780 ovarian cancer cells (IC50 values of 0.1, 0.13 and 0.2 μM, respectively), A2058 melanoma cells (IC50 values of 0.2, 0.02 and 0.02 μM, respectively), and H522-T1 non small-cell cancer lung cells (IC50 values of 0.1, 0.01 and 0.01 μM, respectively), while compounds 4 and 7 exhibited weak antiplasmodial activity against the Dd2 strain of Plasmodium falciparum, with IC50 values of 6.5 and 50 μM, respectively.