Synthesis and biological evaluation with plant cells of new fosmidomycin analogues containing a benzoxazolone or oxazolopyridinone ring

Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45-85% at 125 microM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.     

Effects of fosmidomycin on plant photosynthesis as measured by gas exchange and chlorophyll fluorescence

In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of individual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V _cmax and J _max. Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.     

Structure of the E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) from Thermus thermophilus

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe–4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe–4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)₈ barrel. The great [4Fe–4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.

Structured summary

gcpEbinds to gcpE by x-ray crystallography (View interaction)     

Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona`Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.     

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.     

Synthesis and antibacterial properties of new N4-acetylated hexahydro-2,7-dioxopyrido[2,3-f]quinoxaline-8-carboxylic acids

Direct interaction between 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid and primary α-amino acids (exemplified by glycine, alanine, and l-valine) in aqueous ethanolic NaHCO₃ at 70–80°C for 24–72?h produced the respective N-(4-oxoquinolin-7-yl)-α-amino acids (6a–c). The latter derivatives underwent reductive lactamization upon treatment with Na₂S₂O₄ in aqueous ethanol to afford moderate yields of the corresponding pyrido[2,3-f]quinoxaline-8-carboxylic acids (8a–c). Acetylation of 8a–c using acetyl chloride afforded N₄-acetylated hexahydro-2,7-dioxopyrido[2,3-f]quinoxaline-8-carboxylic acids (9a–c). The structures, assigned to these new heterocyclic products, are supported by analytical and spectral data. The synthesized compounds (6a–c/9a–c) showed appreciable antibacterial activity as compared with ciprofloxacin.     

The Pharmacological effects of novel 5-fluoro-N-(9,10-dihydro-9,10-dioxoanthracen-8-yl)-1H-indole-2-carboxamide derivatives on plasma lipid profile of Triton-WR-1339-induced Wistar rats

A novel series of 5-fluoro-N-(9,10-dihydro-9,10-dioxoanthracen-8-yl)-1H-indole-2-carboxamides (3c–3g) were synthesized. The present study was undertaken to investigate the possible antihyperlipidemic effect of these novel compounds on hyperlipidemic rats. Hyperlipidemia was induced by a single intraperitoneal injection of Triton WR-1339 (300 mg/kg). The tested animals were divided into normal control (NCG), hyperlipidemic control (HCG), compounds 3c-, 3d-, 3e-, 3f-, 3g- and bezafibrate (BF)-treated groups. At a dose of 15 mg/kg, compounds 3c–3g and BF (100 mg/kg) significantly (p < 0.0001) reduced elevated plasma triglycerides levels after 12 and 24 h compared to the hyperlipidemic control group. However, only compounds 3e and 3g obviously showed a significant (p < 0.0001) reduction in plasma total cholesterol levels after 12 and 24 h. Moreover, high-density lipoprotein cholesterol levels were significantly increased in all treated groups. The current study demonstrates that 5-fluoro-N-(9,10-dihydro-9,10-dioxoanthracen-8-yl)-1H-indole-2-carboxamides (3c–3g) have a definite antihyperlipidemic potential and these beneficial activities may contribute to their cardioprotective and antiatherosclerotic role.     

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.     

苏云金芽胞杆菌dxr1基因的转录受SigH控制

【目的】萜类化合物广泛分布在生物界,是重要的生命物质。目前发现有两条萜类化合物的生物合成途径,即甲羟戊酸(MVA)途径和2-甲基-D-赤藓糖醇-4-磷酸(MEP)途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶(DXR,EC1.1.1.267)催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶,而在苏云金芽胞杆菌(Bacillusthuringiensis,Bt)中有2个基因(dxr1和dxr2)编码DXR酶。通过分析BtHD73菌株的dxr1基因的转录活性和dxr1突变体表型,明确dxr1基因的转录调控机制和功能。【方法】通过5?RACE分析dxr1的转录起始位点;β-半乳糖苷酶活性测定分析dxr1基因启动子(Pdxr1)的转录活性;采用同源重组技术分别敲除BtHD73菌株的dxr1和dxr2基因;利用总蛋白定量确定Cry1Ac蛋白产量;利用DXR检测试剂盒检测Bt菌株的DXR活性。【结果】dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基;与出发菌株HD73相比,Pdxr1在sig H突变体中的转录活性明显降低;dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响,但使DXR活性下降。【结论】Bt中dxr1基因的转录受Sig H控制,dxr1基因的缺失影响DXR的活性。     

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K _m of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K _m of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g^?1 DW) than the EtOH control (0.183 mg g^?1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g^?1 DW) followed by in bark (0.161 mg g^?1 DW) and young leaf (0.130 mg g^?1 DW), and least in root (0.014 mg g^?1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.     

Towards new antimalarial drugs: synthesis of non-hydrolyzable phosphate mimics as feed for a predictive QSAR study on 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors

The conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP) is effectively blocked by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non-hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC(50)) in the range from 1 to >30 microM vs. E. coli Dxr and 0.4 to 20 microM against P. falciparum Dxr is reported.     

冬凌草1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆与表达分析

1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)是植物萜类代谢通路中2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径的第一个关键酶,在植物萜类物质的生物合成中发挥重要的作用.为了研究该基因在冬凌草二萜类成分合成中的作用,该研究在冬凌草转录组测序结果的基础上设计一对特异性引物,采用RT-PCR方法得到冬凌草IrDXS基因cDNA全长序列,并对其蛋白进行理化性质分析、信号肽预测、亚细胞定位预测、蛋白质二级结构、三级结构预测分析及跨膜域分析等生物信息学分析,同时利用实时荧光定量PCR的方法检测IrDXS基因在冬凌草不同部位中的表达情况.结果表明:从冬凌草叶片中分离得到了一条编码DXS的全长基因,通过生物信息学软件分析发现,该基因编码全长2169 bp,编码722个氨基酸,分子量为77.7 kD.多序列比对发现该基因编码的蛋白和其他植物中已知的DXS蛋白序列具有较高的同源性,N端均包含了一段质体转运肽序列,并均具有一个保守的焦磷酸硫胺素结构域和与吡啶结合相关的DRAG结构域.序列进化树分析显示,IrDXS基因属于植物DXS2家族.DXS基因在冬凌草根中表达量最高、愈伤组织中最低.该研究首次获得了IrDXS基因的全长cDNA序列,并揭示了其在不同组织中的表达差异,为后续的深入研究IrDXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础.     

Synthesis of novel selenium containing sulfa drugs and their antibacterial activities

Synthesis of 3-[4-(N-substituted sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyri-do[3′,2′:4,5]selenolo[3,2-d]pyrimidines,7-[4-(N-substituted sulfamoyl)phenyl]-7,8-dihydro-8-oxo-3,4-diphenylpyrimido[4′,5′:4,5]selenolo [2,3-c]pyridazines and 1-[4-(N-substituted sulfamoyl)phenyl]-1,11-dihydro 11-oxo-4-methylpyrimido[4′,5′:4,5]selenolo[2,3-b]quinolines is reported. 4-Amino-N-pyrimidine-2-ylbenzene sulfonamide (a), 4-amino-N-(2,6-dimethylpyrimidin-4-yl)benzene sulfonamide (b), N-[(4-aminophenyl)sulfonyl] acetamide (c) with N-ethoxymethyleneamino of selenolo pyridine, selenolo pyridazine and selenolo quinoline derivatives respectively were obtained starting from 1-amino-N ⁴-substituted sulfanilamides. Spectroscopic data (IR, ¹H NMR, ¹³C NMR and Mass spectral) confirmed the structure of the newly synthesized compounds. Substituted pyrimidines, pyridazines and quinolines were screened for antibacterial activity against gram-positive and gram-negative bacteria. Selenolo derivative of N-[(4-aminophenyl)sulfonyl] acetamide (substitutent of sulfacetamide c) showed strong bactericidal effect against all the tested organisms. Selenolo[3,2-d]pyrimidin (substitutent a) showed a good bactericidal effect against Serratia marcescens, Staphylococcus aureus and Escherichia coli. Compounds selenolo[2,3-c]pyridazine (substitutent b), selenolo[2,3-b]quinoline(substitutents c)) exhibited a moderate bactericidal effect against Serratia marcescens. None of the synthesized seleno pyridazines has a considerable antimicrobial activity against the tested organisms. The minimum inhibitory concentration (MIC) of the most active compound-3-[4-(N-acetyl sulfamoyl)phenyl]-3,4-dihydro-4-oxo-7,9-dimethylpyrido[3′,2′:4,5]selenolo [3,2-d]pyrimidine was 10 mg ml⁻¹.     

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-¹³C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.     

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) — an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) — most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82–92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes, well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.     

Synthesis of Novel 4′-Cyclopropyl-5′-norcarbocyclic Adenosine Phosphonic Acid Analogues

Novel 4′-cyclopropyl-5′-norcarbocyclic adenosine phosphonic acid analogues were designed and racemically synthesized from propionaldehyde 5 through a de novo acyclic stereoselective route using triple Grignard addition and ring-closing metathesis (RCM) as key reactions. To improve cellular permeability and enhance the anti-HIV activity of this phosphonic acid, SATE phosphonodiester nucleoside prodrug 23 was prepared. The synthesized adenosine phosphonic acids analogues 17, 18, 19, 21, and 23 were subjected to antiviral screening against HIV-1. Compound 23 exhibits enhanced anti-HIV activity than its parent nucleoside phosphonic acid 18.     

N-aryl 2-aryloxyacetamides as a new class of fatty acid amide hydrolase (FAAH) inhibitors

Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC₅₀ of 2.6?µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC₅₀ of 0.35?µM.