The development of specific inhibitors that can block nuclear factor-B (NF-B) activation is an approach for the treatment of cancer, autoimmune, and inflammatory diseases. Several diterpenoids, oridonin, ponicidin, xindongnin A, and xindongnin B were isolated from the herb Isodon rubescens. These compounds were found to be potent inhibitors of NF-B transcription activity and the expression of its downstream targets, cyclooxygenase-2 and inducible nitric-oxide synthase. The mechanisms of action of the diterpenoids against NF-B are similar, but significant differences were also identified. All of the diterpenoids directly interfere with the DNA-binding activity of NF-B to its response DNA sequence. Oridonin and ponicidin have an additional impact on the translocation of NF-B from the cytoplasm to nuclei without affecting IB-␣ phosphorylation and degradation. The effect of these compounds on the interaction of NF-B with consensus DNA sequences is unique. Different inhibitory effects were observed when NF-B bound to various DNA sequences. Both p65/p65 and p50/p50 homodimers, as well as p65/p50 heterodimer association with their responsive DNA, were inhibited. Kinetic studies on NF-B-DNA interaction indicate that the diterpenoids decrease the B max app but have no effect on K d app . This suggests that this class of compounds interacts with both p65 and p50 subunits at a site other than the DNA binding site and subsequently modulates the binding affinity of the transcription factor toward DNA with different NF-B binding sequences. The diterpenoid structure could therefore serve as a scaffold for the development of more potent and selective NF-B inhibitors that target regulated gene transcription.

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Phenanthroindolizidine-based tylophora alkaloids have been reported to have potential antitumor, anti-immuno and anti-inflammatory activity. The structure-activity relationships of a series of tylophora alkaloids were studied to guide future drug design. Our results indicate that although these compounds are structural analogs, their potency of cytotoxicity, selectivity against NF-κB signaling pathway, and their inhibitory effects against protein and nucleic acid synthesis are different. Because they do not have an identical spectrum of targets, the studied compounds are structural, but may not be functional analogs. KeywordsTylophora alkaloids; Structure; activity relationship; Protein; DNA and RNA synthesis Tylophora alkaloids originate from various plants of the Asclepiadaceae family, such as Tylophora, that are native of India and Southeast Asia. 1 They have antitumor, 2-6 antiinflammatory, 7 anti-arthritis, 8 and anti-lupus activity in vivo. 9 Due to their diverse and potent pharmacological actions, they continue to be targets for synthesis, modification, structureactivity relationship (SAR) studies since their first isolation in 1935. 10 Their molecular mechanisms of action of antitumor and anti-inflammatory activity include: inhibitory effect on protein synthesis 2 and nucleic acid synthesis, 2,11 inhibitory effect on RNA transcription which are controlled by cyclic AMP response elements (CREs), activator protein-1 (AP-1) sites, and NF-κB binding sites, 6 and ability to suppress the expression of a subset of proteins, such as cyclin D1, cyclin B1 and CDK4. 12 Tylocrebrine, a positional isomer of tylophorine, was found in clinical trials to have intolerable central nervous system (CNS) side effects. To *Corresponding author. Yung-Chi Cheng: tel.: +1-203-785-7118; fax: +1-203-785-7129; e-mail: E-mail: yccheng@yale.edu. Note: Yung-Chi Cheng is a fellow of the National Foundation for Cancer Research. † These authors contributed equally to this work.Supplementary data Supplementary data associated with this article (Synthesis of phenanthrene-based tylophorine derivatives described in Scheme 1, synthesis of tylophora alkaloids exemplified in Scheme 2, IC 50s of the inhibitory effect of tylophora alkaloids and PBTs against endogenous NF-κB, CRE, AP-1 and GRE mediated transcription in HepG2 cells listed in Table S1) can be found, in the online version, at doi: xxxxxxx.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. offset such CNS side effects, a series of phenanthrene-based tylophorine derivatives (PBTs) with increased polarity was synthesized to limit the crossin...

Nuclear factor-B (NF-B) has been recognized to play a critical role in cell survival and inflammatory processes. It has become a target for intense drug development for the treatment of cancer, inflammatory, and autoimmune diseases. Here, we describe a potent NF-B inhibitor, eriocalyxin B (Eri-B), an ent-kauranoid isolated from Isodon eriocalyx, an anti-inflammatory remedy. The presence of two ␣,␤-unsaturated ketones give this compound the uniqueness among the ent-kauranoids tested. Eri-B inhibited the NF-B transcriptional activity but not that of cAMP response element-binding protein. It suppressed the transcription of NF-B downstream gene products including cyclooxygenase-2 and inducible nitric-oxide synthase induced by tumor necrosis factor-␣ or lipopolysaccharide in macrophages and hepatocarcinoma cells. Chromatin immunoprecipitation assay indicated that Eri-B selectively blocked the binding between NF-B and the response elements in vivo without affecting the nuclear translocation of the transcription factor. Down-regulation of the endogenous p65 protein sensitized the cells toward the action of the compound. Furthermore, in vitro binding assays suggested that Eri-B reversibly interfered with the binding of p65 and p50 subunits to the DNA in a noncompetitive manner. In summary, this study reveals the novel action of a potent NF-B inhibitor that could be potentially used for the treatment of a variety of NF-B-associated diseases. Modification of the structure of this class of compounds becomes the key to the control of the behavior of the compound against different cellular signaling pathways.NF-B is the transcription factor that controls inflammatory and immune responses and cell growth and survival. Since the discovery of NF-B in 1986 (Sen andBaltimore, 1986), both the cytoplasmic and nuclear regulations of NF-B have been well-characterized. In the classic activation pathway that can be stimulated by TNF-␣ and LPS, the NF-B heterodimers of p65:p50 mainly localize in the cytoplasm through their interaction with IB␣. Activation of the cells leads to the phosphorylation of IB-␣ by the IKK complex (which consists of IKK␣, IKK␤, and NF-B essential modulator/IKK␥), rapid ubiquitination, and subsequent degradation of IB␣ through the 26S proteasome pathway. This promotes the translocation of the p65:p50 complexes into the nucleus where they bind to the NF-B response elements and regulate the expression of their downstream genes (Chen and Greene, 2004;Hayden and Ghosh, 2004). Many of these steps have become targets for drug development

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