TMC-86A, B and TMC-96, new proteasome inhibitors from Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094. I. Taxonomy, fermentation, isolation, and biological activities

TMC-86A, B and TMC-96, new 20S proteasome inhibitors with an epoxy-beta-aminoketone moiety, were isolated from the fermentation broth of Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094, respectively. TMC-86A, B and TMC-96 inhibited the chymotrypsin-like and peptidylglutamyl-peptide hydrolyzing activities of 20S proteasome with the following IC50 values: TMC-86A, 5.1 microM and 3.7microM; TMC-86B, 1.1 microM and 31 microM; TMC-96, 2.9 microM and 3.5 microM, respectively. TMC-86A, B and TMC-96 exhibited the weak inhibitory activity against the trypsin-like activity of 20S proteasome with IC50 values of 51 microM, 250 microM, and 36 microM, respectively. They did not inhibit m-calpain, cathepsin L, and trypsin at 100 microM, suggesting their high specificity for proteasome. Taxonomy of the producing strains is also described.     

TMC-256A1 and C1, new inhibitors of IL-4 signal transduction produced by Aspergillus niger var niger TC 1629

New inhibitors of IL-4 signal transduction, designated as TMC-256A1 and C1, were discovered together with TMC-256B1, a previously known dihydronaphthopyrone, from the fermentation broth of Aspergillus niger var niger TC 1629 by using an IL-4 driven reporter gene assay. Based on spectroscopic analyses, TMC-256A1 and C1 were found to be new members of the naphthopyrone antibiotics. TMC-256A1, B1 and C1 inhibited the IL-4 driven luciferase activity with IC50 values of 25 microM, 30 microM and 1.7 microM, respectively in this assay system. Furthermore, these compounds inhibited the expression of germline C epsilon mRNA with IC50 values of 6.6 microM , 34 microM and 0.31 microM, respectively.     

TMC-264, a novel inhibitor of STAT6 activation produced by Phoma sp. TC 1674

A novel inhibitor of STAT6 activation, named as TMC-264 (1), was discovered from the fermentation broth of Phoma sp. TC 1674. Based on spectroscopic analyses, TMC-264 was found to be a novel tricyclic polyketide with chloro-1H-dibenzo[b,d]pyran-4,6-dione. TMC-264 suppressed expression of IL-4 driven luciferase and germline Cepsilon mRNA with IC50 values of 0.3 microM and 0.4 microM, respectively. TMC-264 exhibited a potent inhibitory activity against tyrosine phosphorylation of STAT6 with an IC50 value of 1.6 microM, whereas TMC-264 weakly inhibited tyrosine phosphorylation of STAT5 with an IC50 value of 16 microM, but did not inhibit the phosphorylation of STAT1 up to 40 microM. TMC-264 blocked formation of the complexes between phosphorylated STAT6 and STAT6 oligonucleotides in a dose dependent manner, while TMC-264 did not affect the formation of phosphorylated STAT1/STAT1 oligonucleotides complexes. These results suggested that TMC-264 selectively inhibited IL-4 signaling by interfering both of phosphorylation of STAT6 and binding of the phosphorylated STAT6 to the recognition sequence.     

TMC-69, a new antitumor antibiotic with Cdc25A inhibitory activity, produced by Chrysosporium sp. TC1068. Taxonomy, fermentation and biological activities

A new antibiotic designated TMC-69 has been isolated from the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69 exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69 prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25A and B phosphatases.     

TMC-95A, B, C, and D, novel proteasome inhibitors produced by Apiospora montagnei Sacc. TC 1093. Taxonomy, production, isolation, and biological activities

In our course of screening for novel proteasome inhibitors, TMC-95A and its diastereomers, TMC-95B to D, were isolated from the fermentation broth of Apiospora montagnei Sacc. TC 1093. TMC-95A inhibited the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of 20S proteasome with IC50 values of 5.4nM, 200nM, and 60nM, respectively. TMC-95B inhibited these activities to the same extent as TMC-95A, while the inhibitory activities of TMC-95C and D were 20 to 150 times weaker than that of TMC-95A and B. TMC-95A did not inhibit m-calpain, cathepsin L, and trypsin at 30 microM, suggesting its high selectivity for proteasome. Taxonomy of the producing strain is also described.     

TMC-66, a new endothelin converting enzyme inhibitor produced by Streptomyces sp. A5008.

A new endothelin converting enzyme (ECE) inhibitor, TMC-66 was isolated from the fermentation broth of Streptomyces sp. A5008. The structure of TMC-66 was elucidated by spectroscopic analyses to be a new member of benzo[a]naphthacenequinone class of antibiotics. TMC-66 had a highly selective inhibitory activity for ECE with an IC50 value of 2.9 microM. Taxonomy of the producing strain is also described.     

TMC-114 (Tibotec)

Tibotec (formerly Tibotec-Virco) is developing TMC-114 as a potential treatment for HIV-1 infection. In February 2001, TMC-126 was revealed as the series prototype, from which TMC-114 was developed. Because of its improved antiviral and superior pharmacokinetic properties, TMC-114 was selected for clinical development. Phase II trials of TMC-114 are underway.     

Linear TMC-95-based proteasome inhibitors

We have designed and evaluated 45 linear analogues of the natural constrained cyclopeptide TMC-95A. These synthetically less demanding molecules are based on the tripeptide sequence Y-N-W of TMC-95A. Structural variations in the amino acid side chains and termini greatly influenced both the efficiency and selectivity of action on a given type of active site. Inhibition constants were submicromolar (Ki approximately 300 nM) despite the absence of the entropically favorable constrained conformation that is characteristic of TMC-95A and its cyclic analogues. These linear compounds were readily prepared and reasonably stable in culture medium and could be optimized to inhibit one, two, or all three proteasome catalytic sites. Cytotoxicity assays performed on a series of human tumor cell lines identified the most potent inhibitors in cells.     

Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (K(i)) of TMC-114 for mutants PR(D30N), PR(I50V), and PR(L90M) were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22-1.45 A) crystal structures of PR mutant complexes with TMC-114. In PR(D30N), the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PR(I50V) the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PR(L90M) structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.     

Lepidepyrone, a new gamma-pyrone derivative, from Neolentinus lepideus, inhibits hyaluronidase

In the course of screening for hyaluronidase (HAase) inhibitory agents, a new gamma-pyrone derivative, lepidepyrone, C(8)H(10)O(5), was isolated from the cultured mycelium of the mushroom Neolentinus lepideus TMC-1102 as a major HAase inhibitory compound (IC(50) 3.3 mM). The structure of lepidepyrone was established on the basis of spectroscopic investigation.     

NDP kinase reactivity towards 3TC nucleotides

Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.     

171例高血压患者血脂,脂蛋白及载脂蛋白水平的分析

本文就171例HBP患者血脂、脂蛋白及载脂蛋白水平与155例正常人进行比较分析发现:HBP患者WI、TG及apoB较对照组显著升高,TC无明显差别,HDL-C、HDL_2-C及apoAI显著降低、正常男性TC、TG和apoB随年龄增长呈升高趋势,女性TC、TG和LDL-C则在50岁以后开始升高。而HBP患者除男性apoB随年龄增加而升高外,其余各项,不分男女,均无随年龄增减而变化的趋势。     

胆固醇/高密度脂蛋白-胆固醇比值在脑梗死中的价值研究

目的:探讨胆固醇/高密度脂蛋白-胆固醇比值(TC/HDL-C比值)与脑梗死发病危险性的关系以及这一比值在脑梗死患者合并高血压或糖尿病时的变化。方法:选择197例脑梗死患者分为三组:单纯脑梗死组、合并高血压组、合并糖尿病组。58例健康者作为正常对照组,探讨这一比值的变化。结果:胆固醇(TC)水平及TC/HDL-C比值在脑梗死患者与正常对照组间有显著性差异(P<0.05);脑梗死患者的HDL-C水平较正常对照组降低,但两者差异无显著性(P>0.05);不同病例组间TC、HDL-C水平及TC/HDL-C比值无显著性差异(P>0.05)。结论:TC/HDL-C比值是脑梗死的危险因素,与高血压、糖尿病无关。     

TC/GLU和LDL-C/HDL-C比值与结直肠息肉的相关性研究

目的:研究总胆固醇与空腹血糖比值(TC/GLU)和低密度脂蛋白胆固醇与高密度脂蛋白胆固醇比值(LDL-C/HDL-C)对结直肠息肉的预测价值.方法:选取2017年2月至2018年12月在江苏省人民医院经肠镜检查的消化内科患者共2150例,收集患者肠镜前空腹静脉血中的TC、HDL-C、LDL-C、甘油三酯(TG)、GLU...     

Binding of the highly toxic tetracycline derivative, anhydrotetracycline, to bovine serum albumin

Tetracycline (TC) derivatives are extensively used as antibiotics in human and animal medicine and, very recently, they have been screened as anti-amyloidogenic drugs. Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug. We evaluated the interaction of AHTC with bovine serum albumin (BSA), one of the main carriers of amphiphilic molecules in blood, using three complementary analytical methods: fluorescence spectroscopy, isothermal titration calorimetry and differential scanning calorimetry. AHTC bound to BSA with an association constant in the order of 10(5) M(-1). Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.     

我院抗菌药使用情况回顾性分析

随机抽取我院 ９７年１～６月份出院病历 １５８份，其中 １２２份（７７．２％）使用了抗菌药，本文仅对此１２２份使用抗菌药情况进行综合分析。我们发现：有６８例病人（５５．７％）抗菌药使用不合理；有８１例为预防性使用抗菌药，治疗用药仅３５例，防治兼顾有６例；有７例发生医院感染；住院期间只使用过１种抗菌药的有４２例，用过２种有３２例，２种以上有４８例，最多为７种；共用抗菌药４５种，１９６疗程，其中１联１１１疗程，２联５９疗程，３联２０疗程，３联以上６疗程，最多１例为６联，共计用药２００７ｄ，平均每疗程１０．２ｄ，每例平均用药１６．５ｄ。本文还讨论了抗菌药滥用与院内感染的关系，以及目前预防用药存在的问题，并对如何合理使用抗菌药提出了几点建议。