NMR Determination of the Absolute Configuration of a Macrophomate Synthase Inhibitor by Using an Axial Chiral Reagent

Macrophomate synthase catalyzes an extraordinary four-step transformation from oxalacetate and 2-pyrone to macrophomic acid by an intermolecular DielsAlder reaction. The absolute configuration of the most potent macrophomate synthase inhibitor; (?)-2-carboxylmethyl-1-methoxybicyclo[2.2.2]oct-5-ene-2-carboxylic acid, was determined to be (1S,2R,4R) by using an axial chiral reagent.     

Inhibition of Thioredoxin Reductase by Curcumin Analogs

Curcumin analogs were first investigated for their inhibitory effects on thioredoxin reductase (TrxR). Most of them were more potent TrxR inhibitors than natural curcumin. The structure-activity relationship was summarized, and the curcumin analog was found to inhibit TrxR irreversibly in a time-dependent manner. The action was caused by covalent modification of the redox-active residues Cys⁴⁹⁷ and Sec⁴⁹⁸ in TrxR.     

Characterization of l-Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[¹⁴C]arginine and the NOS inhibitor [¹⁴C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[¹⁴C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N ^G-methyl- l -arginine, N ^G-amino- l -arginine, and N ⁵-imidoethyl- l -ornithine. l -[¹⁴C]Arginine uptake was not inhibited by aminoguanidine or N ^G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [¹⁴C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[¹⁴C]arginine.     

Intracellular Thymidylate Synthase Inhibition by Trifluorothymidine in FM3A Cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5‐Fluorouracil (5FU). TS activity reduced to 9% (0.1 µM TFT) and 58% (1 µM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK^?) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Improved Cassava Starch by Antisense Inhibition of Granule-bound Starch Synthase I

Cassava is a poor man's crop which is mainly grown as a subsistence crop in many developing countries. Its commercial use was first as animal feed (also known as tapioca), but has shifted since the late sixties to a source of native starch. The availability of native starches, which on the one hand do not require substantial chemical derivatisation and on the other hand have improved properties, would make cassava also for small farmers a potentially attractive cash crop. Since breeding is difficult in this polyploid, vegetatively propagated, crop a transgenic approach would be ideal to improve certain characteristics. We have created a cassava genotype producing amylose-free starch by genetic modification. The absence of amylose increased the clarity and stability of gels made with the transgenic starch, without requiring treatment with environment-unfriendly chemicals such as epoxides (propylene oxide, ethylene oxide) and acetic anhydride, which are normally used to improve stability. The amylose-free starch showed no changes in particle size distribution, chain length distribution or phosphorous content when compared to amylose-containing starch, but the granule melting temperature was increased by almost 2°C. Furthermore, the amylose-free cassava starch shows enhanced clarity and stability properties. These improved functionalities are desired in technical applications in paper and textile manufacturing, but also in the food industry for the production of sauces, dairy products and noodles.     

Inhibition of photosystem II electron transport by acyl derivatives of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid)

In the course of the synthesis of γ-pyrones, well-known inhibitors of photosystem II electron transport, it turned out that the starting material, acyl derivatives of 2,2-dimethyl-1,3-dioxane-5,6-dione (Meldrum's acid) are potent inhibitors of photosystem II electron transport. Thus, in a simple one-step synthesis from commercial available substances, highly potent photosystem II inhibitors are generated. The biological activity of the acyl derivatives is in a parabolic fashion dependent from the length of the alkyl side chain.     

Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.     

脂肪酸合成酶在肿瘤中表达的调节机制

脂肪酸合成酶（fatty acid synthase,FASN）是肿瘤脂质生成的一种关键酶,在催化脂肪酸合成的最后一步中发挥重要作用。FASN在许多肿瘤细胞中过表达而在相应的正常细胞中却不表达。有证据表明FASN是一个代谢性癌基因,在癌细胞中高表达,在肿瘤生长和存活中有重要的作用。FASN在肿瘤中的表达调节是一个很复杂的过程,包括转录水平、翻译后控制和微环境状态的影响。正确认识FASN在肿瘤中的表达调节机制和研究新的FASN抑制剂,为成功治疗肿瘤提供一种新的思路。     

Functional Inactivity and Mutations of p53 Differentially Affect Sensitivity to 5-Fluorouracil and Antifolate Inhibitors of Thymidylate Synthase (TS) by Altering TS Levels in Colorectal Cancer Cells

The role of p53 in altering TS expression and chemosensitivity was studied in colorectal cancer cells with wildtype, mutated, or functionally inactive p53. Cytotoxicity of TS inhibitors was studied by MTT, while PCR, Western blot, and activity assays assessed whether p53 status influenced TS expression. Lovo-175X2 cells showed increased resistance to TS inhibitors and significantly greater than wildtype expression and activity of TS. In contrast, Lovo-273X17 and Lovo-li were more sensitive to TS inhibitors and had reduced TS expression, due either to reduced TS mRNA or altered regulation of TS activity. Thus, functional inactivity and mutations of p53 differentially affect TS, potentially influencing response to TS inhibitor-based treatment.     

多巴胺类似物抑制二氢蝶啶还原酶的研究

多巴胺类似物对二氢蝶啶还原酶有明显的非竞争性抑制作用(Ki或I_(50)值为10~(-5)—10~(-6)mol/L)。其中阿朴吗啡是最强的抑制剂之一(Ki或I~(50)=1-2×10~(-6)mol/L)。由于酪氨酸羟化酶和二氢蝶啶还原酶包含于同一酶促反应过程中,且限制了多巴胺合成的决定速度的那一步。这些结果可能提示出被多巴胺抑制的酪氨酸羟化作用包含着对这二种酶的抑制作用。     

何首乌提取物对脂肪酸合酶的抑制作用

最新报道脂肪酸合酶 (FAS)是治疗肥胖症的潜在靶部位 ,但目前已知的FAS抑制剂还很少 .测定表明 ,中药何首乌提取物对FAS同时具有很强的快结合可逆抑制和慢结合不可逆抑制作用 .萃取的最佳溶剂为 4 0 %乙醇水溶液 .该提取物对FAS全反应的半抑制浓度为 0 .0 0 5mg ml(以萃取时中药重量计 ) ;不可逆抑制过程为两相 ,在 0 4 6mg ml浓度下在 0 5min内快相失活超过 5 0 % ,慢相在 32min时失活达 90 % .该提取物对FAS中的酮酰还原反应有强抑制 ,半抑制浓度为 0 0 18mg ml,对烯酰还原反应有弱抑制作用 .抑制动力学分析表明 ,何首乌提取物对FAS的抑制和底物NADPH之间呈非竞争性关系 ,和丙二酰辅酶A接近竞争性关系 ,而与乙酰辅酶A为反竞争性关系 .推测何首乌还含有作用于FAS中的丙二酰转酰酶的抑制剂 .用何首乌提取物口服饲喂大鼠 ,可明显减低大鼠摄食量和降低大鼠体重 ;实验结束时实验组大鼠肝脏FAS活性低于对照组 .以上结果表明 ,中药何首乌提取物对FAS有很强的抑制作用 ,其抑制能力明显强于已知抑制剂 ,其动力学表现也和已知抑制剂完全不同 ,预计为新的抑制剂 ,对研究FAS的作用机理及在防治肥胖症的应用上可能具有重要的价值     

Inhibition of Neuronal Nitric Oxide Synthase by 7-Nitroindazole Protects Against MPTP-Induced Neurotoxicity in Mice

Abstract: Several studies suggest that nitric oxide (NO^•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7-nitroindazole (7-NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7-NI dose-dependently protected against MPTP-induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovanillic acid and 3,4-dihydroxyphenylacetic acid. 7-NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO^• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO⁻), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. Our results suggest that NO^• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.     

Inhibition of Depolarization-Induced Nitric Oxide Synthase Activation by NS-7, a Phenylpyrimidine Derivative, in Primary Neuronal Culture

Abstract: Neuronal nitric oxide synthase (NOS) is considered to be involved in the pathogenesis of ischemic brain damage. In the present study, the effect of a novel neuroprotective phenylpyrimidine derivative, 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), on depolarization-stimulated NOS activity was examined in cultured neurons of mouse cerebral cortex. Various depolarizing stimuli such as veratridine, KCl, and N -methyl- d -aspartate increased the NOS activity determined by cyclic GMP formation. NS-7 concentration-dependently inhibited both the veratridine- and KCl-induced NOS activation with IC₅₀ values of 9.3 and 9.6 µ M , respectively. The reversal of KCl-evoked NOS activity by NS-7 was also observed under blockade of both ionotropic glutamate receptors and the Na⁺ channel with MK-801, 6-cyano-7-nitroquinoxaline-2,3-dione, and tetrodotoxin. In contrast, NS-7, even at 100 µ M , did not affect N -methyl- d -aspartate-stimulated NOS activity, nor did it have any influence on NOS activity determined in the soluble fraction of rat hippocampus. Because NS-7 has already been shown to block both Na⁺ and Ca²⁺ channels, the present findings suggest that this compound inhibits depolarization-induced NOS activation by reducing Ca²⁺ influx through blockade of Na⁺ and Ca²⁺ channels in primary neuronal culture.     

6-Cyclopropylpurines as Novel Potent Analogs of Cytokinins

6-Alkynyl-, trans-6-alkenyl-, trans-6-cyclopropyl-and 6-alkylpurines structurally related to the cytokinin 6-benzylaminopurine (BAP) have been synthesized and examined with a radish cotyledon assay as plant growth stimulators. The growth stimulation obtained with the 6-alkylpurines trans-cyclopropylpurines was very close to that obtained with BAP, and the trans-styrylpurines were somewhat less effective. The fact that the conformationally locked cyclopropanes exhibit growth-stimulating effects comparable to the flexible 6-alkylpurines and to BAP, supports the hypothesis that the orientation of the NH-CH₂ bond in “the active conformation” of BAP is close to anti, which means that the torsion angle C(6)-N(6)-CH₂-C is approximately 180 degrees.     

引物悬挂延伸PCR扩增白藜芦醇合酶cDNA

为了得到葡萄白藜芦醇合酶(RS)基因的cDNA,实验以葡萄叶片为材料,利用引物悬挂延伸PCR法,以葡萄总DNA为模板,先分别扩增得到编码白藜芦醇合酶的第一外显子和第二外显子的序列,再以这两个序列为模板,进行第二轮的PCR,得到大小与RS基因大小相同的片段,经PCR产物测序证明,两个外显子已经准确无误地拼接,此片段就是RS基因。     

Hormonal Control of Sucrose Phosphate Synthase and Sucrose Synthase in Sugar Beet

We studied the effects of synthetic analogs of phytohormones (benzyladenine, IAA, and GA) on the activities of the enzymes catalyzing sucrose synthesis and metabolism, sucrose phosphate synthase (SPS, EC 2.4.1.14) and sucrose synthase (SS, EC 2.4.1.13), and on the content of chlorophyll and protein during the sugar-beet (Beta vulgaris L.) ontogeny. Plant spraying with phytohormonal preparations activated SPS in leaves; direct interaction between phytohormones and the enzyme also increased its activity. The degree of this activation differed during the ontogeny and in dependence on the compound used for treatment. Analogs of phytohormones maintained high protein level in leaves, retarded chlorophyll breakdown, and, thus, prolonged leaf functional activity during development. Phytohormonal preparations practically did not affect the SS activity both after plant treatment and at their direct interaction with the enzyme. It is supposed that the SS activity in sugar-beet roots is controlled by sucrose synthesized in leaves rather than by phytohormones. The effects of hormones on leaf metabolism were mainly manifested in growth activation.     

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.     

Synthesis and Biological Activity of Some Benzochromenoquinolinones: Tacrine Analogs as Potent Anti‐Alzheimer's Agents

Alzheimer's disease (AD) is a well‐known neurodegenerative disorder affecting millions of old people worldwide and the corresponding epidemiological data emphasize the importance of the disease. As AD is a multifactorial illness, various single target directed drugs that have reached clinical trials have failed. Therefore, various factors associated with outset of AD have been considered in targeted drug discovery. In this work, various benzochromenoquinolinones were synthesized and evaluated for their cholinesterase and BACE1 inhibitory activities as well as neuroprotective and metal‐chelating properties. Among the synthesized compounds, 14‐amino‐13‐(3‐nitrophenyl)‐2,3,4,13‐tetrahydro‐1H‐benzo[6,7]chromeno[2,3‐b]quinoline‐7,12‐dione ( 6m ) depicted the best inhibitory activity toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC₅₀s of 0.86 and 6.03 μm , respectively. Also, the compound could inhibit β‐secretase 1 (BACE1) with IC₅₀=19.60 μm and showed metal chelating ability toward Cu²⁺, Fe²⁺, and Zn²⁺. In addition, docking study demonstrated desirable interactions of compound 6m with amino acid residues characterizing AChE, BChE, and BACE1.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.