Diversification of Ergot Alkaloids in Natural and Modified Fungi

Several fungi in two different families––the Clavicipitaceae and the Trichocomaceae––produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.     

Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site

We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC₅₀ versus TbFPPS ∼20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC₅₀ values in the range of ∼1–3 µM (as compared with ∼0.5 µM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg²⁺ to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (T_m), quite different to the large increases in ΔT_m seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads.Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of isopentenyl diphosphate (IPP; compound 1 in Fig. 1) with dimethylallyl diphosphate (DMAPP; compound 2 in Fig. 1) to form the C₁₀ isoprenoid geranyl diphosphate (GPP; compound 3 in Fig. 1), which then condenses with a second IPP to form the C₁₅ isoprenoid, farnesyl diphosphate (FPP; compound 4 in Fig. 1). FPP then is used in a wide range of reactions including the formation of geranylgeranyl diphosphate (GGPP) (1), squalene (involved in cholesterol and ergosterol biosynthesis), dehydrosqualene (used in formation of the Staphylococcus aureus virulence factor staphyloxanthin) (2), undecaprenyl diphosphate (used in bacterial cell wall biosynthesis), and quinone and in heme a/o biosynthesis. FPP and GGPP also are used in protein (e.g., Ras, Rho, Rac) prenylation, and FPPS is an important target for the bisphosphonate class of drugs (used to treat bone resorption diseases) such as zoledronate (compound 5 in Fig. 1) (3). Bisphosphonates targeting FPPS have activity as antiparasitics (4), act as immunomodulators (activating γδ T cells containing the Vγ2Vδ2 T-cell receptor) (5), and switch macrophages from an M2 (tumor-promoting) to an M1 (tumor-killing) phenotype (6). They also kill tumor cells (7) and inhibit angiogenesis (8). However, the bisphosphonates in clinical use (zoledronate, alendronate, risedronate, ibandronate, etidronate, and clodronate) are very hydrophilic and bind avidly to bone mineral (9). Therefore, there is interest in developing less hydrophilic species (10) that might have better activity against tumors in soft tissues and better antibacterial (11) and antiparasitic activity.Open in a separate windowFig. 1.Chemical structures of FPPS substrates, products, and inhibitors.The structure of FPPS (from chickens) was first reported by Tarshis et al. (12) and revealed a highly α-helical fold. The structures of bacterial and Homo sapiens FPPS (HsFPPS) are very similar; HsFPPS structure (13, 14) is shown in Fig. 2A. There are two substrate-binding sites, called here “S1” and “S2.” S1 is the allylic (DMAPP, GPP) binding site to which bisphosphonates such as zoledronate bind via a [Mg²⁺]₃ cluster (15) (Fig. 2B). S2 is the homoallylic site to which IPP binds, Fig. 2B. Recently, Jahnke et al. (10) and Salcius et al. (16) discovered a third ligand-binding site called the “allosteric site” (hereafter the “A site”). A representative zoledronate+A-site inhibitor structure [Protein Data Bank (PDB) ID code 3N46] (Nov_980; compound 6 in Fig. 1) showing zoledronate in S1 and Nov_980 (compound 6) in the A site is shown in a stereo close-up view in Fig. 2B, superimposed on a zoledronate+IPP structure (PDB ID code 2F8Z) in S2. Whether the allosteric site serves a biological function (e.g., in feedback regulation) has not been reported. Nevertheless, highly potent inhibitors (IC₅₀ ∼80 nM) have been developed (10), and the best of these newly developed inhibitors are far more hydrophobic than are typical bisphosphonates (∼2.4–3.3 for cLogP vs. ∼−3.3 for zoledronate) and are expected to have better direct antitumor effects in soft tissues (10).Open in a separate windowFig. 2.Structures of human FPPS. (A) Structure of HsFPPS showing zoledronate (compound 5) and IPP (compound 1) bound to the S1 (allylic) and S2 (homoallylic) ligand-binding sites (PDB ID code 2F8Z). (B) Superposition of the IPP-zoledronate structure (PDB ID code 2F8Z) on the zoledronate-Nov_980 A-site inhibitor structure (PDB ID code 3N46). Zoledronate binds to the allylic site S1, IPP binds to the homoallylic site S2, and the allosteric site inhibitor binds to the A site. Active-site “DDXXD” residues are indicated, as are Mg²⁺ molecules (green and yellow spheres, respectively). The views are in stereo.In our group we also have developed more lipophilic compounds (e.g., compound 7 in Fig. 1) (17, 18) as antiparasitic (19) and anticancer drug leads (18) and, using computational methods, have discovered other novel nonbisphosphonate FPPS inhibitors (e.g., compound 8 in Fig. 1) that have micromolar activity against FPPS (20). In this study, we extended our computational work and tried to discover other FPPS inhibitors that target the A site. Such compounds would be of interest because they might potentiate the effects of zoledronate and other bisphosphonates, as reported for other FPPS inhibitors (21), and have better tissue distribution properties in general.     

Preferential recognition of a microbial metabolite by human Vgamma2Vdelta2 T cells

Human Vgamma2Vdelta2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vgamma2Vdelta2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vgamma2Vdelta2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vgamma2Vdelta2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vgamma2Vdelta2 T cells in vivo upon engraftment into severe combined immunodeficiency-beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vgamma2Vdelta2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vgamma2Vdelta2 T cells to respond to a broad array of pathogens using this pathway.     

Licoflavone C attenuates the genotoxicity of cancer drugs in human peripheral lymphocytes

Flavonoids exhibit a wide spectrum of biological activities that can lead to beneficial effects for human health. The search for cytotoxic, genotoxic and/or antimutagenic natural compounds is therefore of great relevance, especially in cancer chemotherapy. In view of this, we screened the potential genotoxicity/antigenotoxicty of licoflavone C (LFLC) - a naturally occurring prenyl-flavone extracted from Genista ephedroides - using the micronucleus (MN) assay on stimulated and cytochalasin B-blocked human lymphocytes. LFLC did not increase the spontaneous MN level up to 600 microM final concentration where a strong toxicity was seen to occur. We therefore performed an antigenotoxicity assay against the two mutagenic anticancer drugs, mitomycin C (MMC) and daunorubicin (DAU), using two non-toxic LFLC concentrations (0.1 microM and 1.0 microM). The MN frequencies induced by 0.025 microg/ml or 0.05 microg/ml DAU were significantly lowered by 45.4% or 46.6% and 41.8% or 44.8% at LFLC 0.1 and 1.0 microM, respectively. After treatment with 0.085 microg/ml or 0.17 microg/ml MMC, we detected a reduction in genotoxicity of 35.1% or 37.0% and of 38.0% or 35.8% at LFLC 0.1 and 1.0 microM, respectively. In conclusion, LFLC was proven to be protective toward the chromosome damage induced by DAU or MMC in cultured human peripheral lymphocytes.     

核磁共振波谱法快速鉴定异戊烯基异黄酮类化合物

目的通过比较异戊烯基异黄酮类化合物核磁共振谱的特征,归纳核磁共振波谱法快速鉴定该类化合物的规律。方法应用核磁共振波谱法。结果从中药刺桐(ErythrinavariegataL .var .orientalis(L .)Merr.)的茎皮中分离得到8个异戊烯基异黄酮类化合物,即scandenolone( 1)、alpinumisoflavone( 2 )、6 ,8 diprenylgenistein( 3)、osajin( 4 )、isoerysenegalenseinE( 5 )、wighteone( 6 )、labumetin( 7)、lupiwighteone( 8)。结论总结出核磁共振波谱法快速鉴定异戊烯基异黄酮类化合物的规律     

柔毛淫羊藿叶黄酮类成分研究

对柔毛淫羊藿Epimedium pubescens叶的化学成分进行研究.采用硅胶,Sephadex LH-20,MCI柱色谱及制备、半制备HPLC等方法进行分离和纯化,根据理化性质和波谱数据鉴定化合物结构.从柔毛淫羊藿叶的70%乙醇提取物中分离得到11个化合物,分别鉴定为脱水淫羊藿素(1),淫羊藿次苷Ⅱ(2),2'-O-鼠李糖基淫羊藿次苷Ⅱ(3),去甲基脱水淫羊藿素(4),宝藿苷Ⅱ(5),朝霍素B(6),粗毛淫羊藿苷(7),苜蓿素(8),山柰酚(9),大豆素(10),对羟基苯甲酸乙酯(11).其中化合物 11 为首次从淫羊藿属植物中分离得到,其余10个化合物为首次从该植物中分离得到.     

异戊烯基口占吨酮类天然产物的抗肿瘤活性研究进展

近年来有研究显示,含有异戊烯基结构的天然咕吨酮类化合物对多种肿瘤具有显著抑制活性,故而从此类化合物中寻找具有开发价值的抗肿瘤先导化合物,已成为一个研究热点。按来源分别介绍异戊烯基咕吨酮类天然化合物及其抗肿瘤活性研究新进展,并对其中部分化合物的作用机制及构效关系进行了初步探讨。     

A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer‐aided Drug Discovery

With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis‐farnesyl diphosphate synthase (cis‐FPPS) and cis‐decaprenyl diphosphate synthase (cis‐DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis‐FPPS, cis‐DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis‐DPPS, finding ~1 μm inhibitors, and used the receiver operating characteristic–area under the curve (ROC‐AUC) method to test the predictive power of X‐ray and MD‐derived cis‐DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC₅₀ ~0.5‐ to 20‐μm inhibitor (depending on substrate) of cis‐DPPS, a ~660‐nm inhibitor of Rv3378c as well as a 4.8‐μm inhibitor of cis‐FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation.     

异戊烯基黄酮的生物活性及构效关系

异戊烯基黄酮是黄酮类化合物中的一个分支,特点是在黄酮类化合物的骨架上存在异戊烯基侧链。根据异戊烯基结合位点、数量及结构类型,异戊烯基黄酮分为:3号位异戊烯基黄酮,5号位异戊烯基黄酮,6号位异戊烯基黄酮,8号位异戊烯基黄酮、多侧链异戊烯基黄酮、吡喃环异戊烯基黄酮和薰衣草基异戊烯基黄酮7种。异戊烯基黄酮存在广泛的生物活性,特别是在促进干细胞分化、抗炎和免疫调节、心血管保护、改善代谢性疾病、改善骨质疏松、神经保护、抗肿瘤、抗衰老及生殖作用等方面都有显著疗效。异戊烯基黄酮的化学结构与生物活性之间存在显著的关联性。因此,异戊烯基黄酮的构效关系显得尤为重要,为新化合物的合成及功能预测提供理论依据。     

Phase I pharmacokinetic and pharmacodynamic study of the prenyl transferase inhibitor AZD3409 in patients with advanced cancer

AZD3409 is an orally active double prodrug that was developed as a novel dual prenyltransferase inhibitor. The formation of the active metabolite AZD3409 acid is mediated by esterases in plasma and cells. The aim of this phase I study was to determine the maximum tolerated dose, toxicities, pharmacokinetics and pharmacodynamics of AZD3409. AZD3409 was administered orally to patients with advanced solid malignancies using an interpatient dose-escalation scheme starting at 500 mg AZD3409 once daily. Twenty-nine patients were treated at seven dose levels. The MTD of part A was defined as 750 mg b.i.d. in the fasted state. Adverse events were mainly gastrointestinal and the severity was on average mild to moderate and reversible. The dose-limiting toxicities were vomiting, diarrhoea and uncontrolled nausea. Pharmacokinetic studies of the prodrug and the active metabolite indicated dose proportionality. Pharmacodynamic studies showed that farnesyltransferase (FTase) was inhibited at all dose levels. In conclusion, chronic oral dosing with AZD3409 is feasible and results in significant inhibition of FTase activity. Pharmacodynamic studies revealed that the maximal FTase inhibition, estimated at 49+/-11%, appeared to be reached at AZD3409 acid plasma concentrations at which the occurrence of drug-related toxicity was low. This study supports the rationale to implement biological effect studies in clinical trials with biologically active anticancer drugs to define optimal dosing regimens.