Pharmacology of Natural Products

We are seeking for distinct natural compounds, usually plant-derived secondary metabolites that target pharmacologically relevant pathways. In that regard, chronic inflammatory diseases and so far incurable types of cancer are of primary interest. The ultimate goal is the development of novel drugs and the identification of novel therapeutic targets.
To achieve these tasks, we isolate and purify compounds of interest to chemical homogeneity. Further, we characterize these compounds including their structural identification. Depending on molecular pharmacological data, molecular modeling, and docking experiments, we may also synthesize semi-synthetic derivatives. The respective compounds will then be extensively investigated for their pharmacological properties at the molecular, cellular and systemic level. Subsequently, some examples are given.

Tirucallic Acids Are Novel Pleckstrin Homology Domain-Dependent Akt Inhibitors Inducing Apoptosis in Prostate Cancer Cells

Activation of the serine/threonine kinase Akt is associated with aggressive clinical behavior of prostate cancer. Our studies have shown that the human prostate cancer cell lines LNCaP and PC-3 express predominantly Akt1 and Akt2. Selective down-regulation of Akt1, but not Akt2 by shRNA reduced the viability of prostate cancer cells. In addition, structurally different Akt inhibitors were cytotoxic for the prostate cancer cells confirming that the Akt pathway is indispensable for their viability. We have purified the tetracyclic triterpenoids 3-oxo-tirucallic acid, 3-a-acetoxy-tirucallic acid, and 3-b-acetoxy-tirucallic acid from the oleogum resin of Boswellia carterii to chemical homogeneity. Specifically the acetoxy-derivatives potently inhibited the activities of human recombinant Akt1 and Akt2. In addition, they inhibited constitutively active Akt immunoprecipitated from PC-3 cells, whereas IkB kinases remained unaffected. Docking data indicated that these tetracyclic triterpenoids form hydrogen bonds within the phosphatidylinositol binding pocket of the Akt pleckstrin homology domain. Accordingly, 3-b-acetoxy-tirucallic acid did not inhibit the activity of Akt1 lacking the PH domain. In the prostate cancer cell lines investigated, these compounds inhibited the phosphorylation of cellular Akt and the Akt signaling pathways including GSK-3b and BAD phosphorylation, nuclear accumulation of p65, the androgen receptor, b-catenin, and c-myc. These events culminated in the induction of apoptosis in prostate cancer, but not in nontumorigenic cells. The tirucallic acid derivatives inhibited proliferation and induced apoptosis in tumors xenografted onto chick chorioallantoic membranes and decreased the growth of pre-established prostate tumors in nude mice without overt systemic toxicity. Thus, tirucallic acid derivatives represent a new class of Akt inhibitors with antitumor properties.

Targeting NF-kB with a Natural Triterpenoid Alleviates Skin Inflammation in a Mouse Model of Psoriasis

Psoriasis vulgaris is a common chronic inflammatory skin disease involving cytokines and an activated cellular immune system. At variance to skin from patients with atopic dermatitis or from healthy subjects, human psoriatic skin lesions exhibit strong activation of transcription factor NF-kB that is mainly confined to dermal macrophages, whereas only a few dendritic cells but no CD3+ lymphocytes show activated NF-kB. Because NF-kB signaling is required for the induction and/or function of many cytokines and aberrant cytokine expression has been proposed as an underlying cause of psoriasis, we investigated whether NF-kB targeting would affect the course of the disease in the CD18 hypomorphic (CD18hypo) mouse model of psoriasis. When mice with severe psoriasiform lesions were treated systemically or locally with the IkB kinase inhibitor acetyl-11-keto-b-boswellic acid (AKbBA), NF-kB signaling and the subsequent NF-kB-dependent cytokine production as shown by the TNF-a production of macrophages were profoundly suppressed. In addition, application of the compound i) counteracted the intradermal MCP-1, IL-12 and IL-23 expression in previously lesional skin areas, ii) led to resolution of the abundant immune cell infiltrates, and iii) significantly reduced the increased proliferation of the keratinocytes. Overall, the AKbBA treatment was accompanied by a profound improvement of the psoriasis disease activity score in CD18hypo mice with reconstitution of a nearly normal phenotype within the chosen observation period. Our data demonstrate that NF-kB signaling is pivotal for the pathogenesis in the CD18hypo mouse model of psoriasis. Therefore, targeting NF-kB might provide an effective strategy for the treatment of psoriasis.

Inhibition of IκB Kinase Activity by Acetyl-Boswellic Acids Promotes Apoptosis in Androgen-Independent Prostate Cancer Cells

Signaling through NF-κB has been implicated in the malignant phenotype as well as chemoresistance of various cancers. Here we used the natural NF-κB-inhibiting compounds acetyl-β-boswellic acid (AβBA) and acetyl-11-keto-β-boswellic acid (AKβBA) to inhibit proliferation and to elicit cell death in chemoresistant androgen-independent PC-3 prostate cancer cells in vitro and in vivo. Induction of apoptosis was demonstrated in cultured PC-3 cells by several parameters including mitochondrial cytochrome c release and DNA fragmentation. At the molecular level, the isolated compounds inhibit constitutively activated NF-κB signaling by intercepting the IκB kinase (IKK) activity. By contrast, signaling through the interferon-stimulated response element remained unaffected suggesting specificity for IKK inhibition. The impaired phosphorylation of p65 and the reduced nuclear translocation of NF-κB proteins were associated with down-regulation of the constitutively overexpressed and NF-κB-dependent antiapoptotic proteins Bcl-2 and Bcl-xL. In addition, expression of cyclin D1, a crucial cell cycle regulator, was reduced as well. Down-regulation of IKK by antisense oligodeoxynucleotides confirmed the essential role of IKK inhibition for the proliferation of the PC-3 cells. Both compounds tested were active in vivo, yet AKβBA proved to be far superior. Indeed, topical application of a specifically designed water-soluble AKβBA-γ-cyclodextrin complex on PC-3 tumors xenografted onto chick chorioallantoic membranes concentration-dependently inhibited the cancer cell proliferation and elicited apoptosis. Similarly, in nude mice carrying PC-3 tumors, systemic application of AKβBA-γ-cyclodextrin complexes inhibited tumor growth and triggered apoptosis in the absence of detectable systemic toxicity. Thus, AKβBA and related compounds acting on IKK might provide a novel approach for the treatment of chemoresistant human tumors such as androgen-independent human prostate cancers.

Beta  

Methods

  • Protein expression
  • Western blotting and immunostaining
  • In vitro/ex vivo kinase assay
  • Co-immunoprecipitation
  • Electrophoretic mobility shift assays
  • RT-PCR
  • ELISA
  • Flow cytometric analysis of cell surface proteins
  • Tumor xenografts on the chick chorioallantoic membrane (CAM assay)
  • Tumor xenografts in athymic NMRI nu/nu mice in vivo 
  • Immunoh
  • istochemistry
  • In vitro topoisomerase assays
  • Molecular modeling
  • Laser scanning microscopy
  • Fluorescence microscopy

 

Publications (selected) 

Büchele B, Zugmaier W, Lunov O, Syrovets T, Merfort I, Simmet Th. Surface plasmon resonance analysis of NF-kB protein interactions with the sesquiterpene lactone helenalin. Anal Biochem 2010; 401:30-7

Estrada A, Syrovets T, Büchele B, Schimana-Pfeifer J, Schmidt T, Pitterle K, Lunov O and Simmet Th. Tetracyclic triterpenoids inhibit Akt and induce apoptosis in prostate cancer cells. Mol Pharmacol. 2010; 77:378-87

Wang H, Syrovets T, Kess D, Büchele B, Hainzl H, Lunov O, Weiss JM, Scharffetter-Kochanek K, Simmet Th. Targeting NF-kB with a natural triterpenoid alleviates skin inflammation in a mouse model of psoriasis. J Immunol. 2009; 183:4755-63

Cuaz-Pérolin C, Billiet L, Baugé E, Copin C, Scott-Algara D, Genze F, Büchele B, Syrovets T, Simmet Th, Rouis M. Anti-inflammatory and anti-atherogenic effects of the NF-kB inhibitor acetyl-11-keto-b-boswellic acid in LPS-challenged apoE-/- mice. Arterioscler Thromb Vasc Biol. 2008; 28:272-7

Büchele B, Zugmaier W, Estrada A, Genze F, Syrovets T, Paetz C, Schneider B, Simmet T. Characterization of 3a-acetyl-11-keto-a-boswellic acid, a pentacyclic triterpenoid inducing apoptosis in vitro and in vivo. Planta Med. 2006; 72:1285-9

Syrovets T, Gschwend JE, Büchele B, Laumonnier Y, Zugmaier W, Genze F, Simmet Th. Inhibition of IkB kinase activity by acetyl-boswellic acids promotes apoptosis in androgen-independent PC-3 prostate cancer cells in vitro and in vivo. J Biol Chem. 2005; 280:6170-80

Syrovets T, Büchele B, Krauß C, Laumonnier Y, Simmet Th. Acetyl-boswellic acids inhibit lipopolysaccharide-mediated TNF-a induction in monocytes by direct interaction with IkB kinases. J Immunol. 2005; 174:498-506

Büchele B, Zugmaier W, Genze F, Simmet T. High-performance liquid chromatographic determination of acetyl-11-keto-α-boswellic acid, a novel pentacyclic triterpenoid, in plasma using a fluorinated stationary phase and photodiode array detection: application in pharmacokinetic studies. J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 829:144-8

Sterk V, Büchele B, Simmet T. Effect of food intake on the bioavailability of boswellic acids from a herbal preparation in healthy volunteers. Planta Med. 2004; 70:1155-60

Belsner K, Büchele B, Werz U, Syrovets T, Simmet Th. Structural analysis of pentacyclic triterpenes from the gum resin of Boswellia serrata by NMR spectroscopy. Magn Reson Chem. 2003; 41:115-22

Syrovets T, Büchele B, Gedig E, Slupsky JR, Simmet Th. Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIa. Mol Pharmacol. 2000; 58:71-81

Doares SH, Syrovets T, Weiler EW, Ryan CA. Oligogalacturonides and chitosan activate plant defensive genes through the octadenoid pathway. Proc Natl Acad Sci USA. 1995; 92:4095-8