TAN-1057A: A Translational Inhibitor with a Specific Inhibitory Effect on 50S Ribosomal Subunit Formation

The inhibitory activities of a novel antibiotic compound have been investigated. A synthetic version of the natural product TAN-1057A was examined for its effects on translation and ribosomal subunit formation. The antibiotic at 6 μg/ml reduced the growth rate of wild-type Staphylococcus aureus cells by 50%. The IC₅₀ for inhibition of protein synthesis in these cells was 4.5 μg/ml. Pulse and chase labeling kinetics showed a strong inhibitory effect on 50S ribosomal subunit formation as well. The IC₅₀ for this process was 9 μg/ml, indicating an equivalent inhibitory effect of the antibiotic on translation and 50S synthesis. The post-antibiotic effect of the drug was investigated. Protein synthesis resumed rapidly after removal of the drug from cells, but full recovery of the normal 50S subunit complement in treated cells required 1.5 h. The dual inhibitory effects of this compound are compared with other antimicrobial agents having similar effects on cell growth. Received: 27 December 2000 / Accepted: 22 March 2001     

A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.     

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

Background

The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.

Methodology/Principal Findings

In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC₅₀) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC₅₀ of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.

Conclusions

Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.     

Specific binding to adrenal particulate fraction of cyclo(histidyl-proline), a TRH metabolite

Histidyl-proline diketopiperazine (cyclo(His-Pro), a metabolite of the neuropeptide thyrotropin releasing hormone, has been shown to possess intrinsic biological activities. The binding of this peptide to various tissue particulate preparations was investigated. While the peptide showed no apparent binding to particulate fractions derived from brain, pituitary, and some other tissues, binding to adrenal and liver was demonstrated. The binding of cyclo(His-Pro) to bovine adrenal cortical particles was further characterized. Binding at equilibrium was greater at 4 degrees C than at 37 degrees C. The binding was dependent on tissue concentration, showed a pH optimum between 7 and 8, and was inactivated by treatment of the particulate fraction with trypsin or by boiling. The interaction of cyclo(His-Pro) with the tissue was not associated with any metabolism of the peptide. Kinetic studies of association of cyclo(His-Pro) with adrenal cortical particles indicated a single class of binding sites with a KD of approximately 900 nM and a maximum number of sites of 92 pmoles/mg protein. The binding was stereospecific and the histidine moiety of the peptide was the major determinant of the binding. A variety of catechols, serotonin and histamine competed with cyclo(His-Pro) for binding with IC50's ranging from 17-450 muM. Cyclo(His-Pro) did not affect monoamine oxidase or adenylate cyclase activity in adrenal cortical particulate preparations.     

Intracellular Metabolism of β-L-ddAMP-bis(tbutylSATE), a Potent Inhibitor of Hepatitis B Virus Replication

Abstract

β-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC₅₀ = 0.1 μM. Following a 0-to72-hrs exposure of human hepatocytes to a 10 μM [2′,3′^?3H] β-L-ddAMP-bis(tbutylSATE), the pharmacologically active β-L-ddATP was the predominant metabolite attaining a concentration of 268.53 ± 107.97 pmoles/10⁶ cells at 2 hrs. In Hep-G2 cell, β-L-ddATP accounted for 146.8 ± 29.8 pmoles/10⁶ cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of β-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.     

Thermolysin as a catalyst in enzymatic synthesis of asparagine-containing peptides

The following di- and tripeptides were synthesized to study the potential utility of thermolysin as a catalyst in reactions of incorporation of N_α-acyl-l-asparagine into esters of amino acids and peptides: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu-OBzl, Z-Asn-Leu-OBzl, Moz-Asn-Leu-OBzl, Boc-Asn-Phe-OBzl, Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Asn-Val-OBzl, Moz-Asn-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl, and Moz-Asn-Ile-Phe-OBzl. All of these peptides were obtained in pure form in good yield and characterized by thin-layer chromatography, melting point, elemental analysis, amino acid analysis, and proton magnetic resonance. The use of benzyloxycarbonyl (Z) and p-methoxybenzyloxycarbonyl (Moz) as protecting groups for asparagine gave excellent yields of the dipeptides. Relative to the dipeptides, the synthesis of the tripeptides was found to require lower enzyme concentrations and reaction times. Since the yields of the tripeptides failed to exhibit significant differences, it was not possible to establish the existence of a secondary specificity of thermolysin for the residue P′₂. A methodological study was also performed to determine the optimum conditions for synthesis of Boc-Asn-Ile-OBzl. This study consisted of an analysis of the influence of pH, enzyme concentration, volume and concentration of the solution of sodium acetate, relative proportions of carboxyl and amine components, temperature, and addition of organic solvent to the reaction medium.     

广谱趋化因子受体结合物-vMIP-II的体内抗SIV功能研究

病毒巨噬细胞炎症蛋白II(vMIP-II)是一种广谱趋化因子受体拮抗剂,它所拮抗的趋化因子受体被认为是不同的人免疫缺陷病毒株(Human immunodeficiency virus,HIV)进入靶细胞的辅受体。虽然理论上vMIP-II是一个广谱的HIV抑制剂,但vMIP-II的抗HIV感染作用却少有报道,特别是体内研究。本研究利用一个有效的SIV-mac251感染食蟹猴模型来评价vMIP-II的体内抗HIV感染作用,结果显示vMIP-II能够有效地并呈剂量依赖性地降低食蟹猴血浆病毒载量,同时对宿主免疫功能具有保护作用。这些结果表明vMIP-II是一种有效的抗HIV物质,可以作为一类新型的抗HIV先导药物,也为研发靶向病毒进入的新药提供了进一步的理论支持。     

Thermolysin as a catalyst in enzymatic synthesis of asparagine-containing peptides II

Thermolysin was used as a catalyst to obtain the following protected di- and tripeptide esters: Z-Asn-Leu-OEt, Z-Asn-Phe-OEt, Moz-Asn-Leu-Gly-OEt, Boc-Asn-Leu-Gly-OEt, Z-Asn-Leu-Gly-OEt, Moz-Asn-Leu-Gly-OBzl, Moz-Asn-Leu-Gly-OtBu, Moz-Gln-Leu-Gly-OEt, Moz-Asn-Ile-Gly-OEt, and Moz-Asn-Leu-Ala-OEt. These compounds were obtained in pure form and the yields exceeded 50%, except for Moz-Asn-Leu-Gly-OtBu and Boc-Asn-Leu-Gly-OEt. H-Cys(Bzl)-OtBu and H-Cys(Bzl)-Pro-Leu-Gly-NH2 were both inadequate as amino components for obtaining Moz-Asn-Cys(Bzl)-OtBu, Z-Asn-Cys(Bzl)-OtBu and Moz-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 in the thermolysin-catalyzed reactions. In the attempted synthesis of the protected pentapeptide amide, this protease cleaved the Pro-Leu bond of the amino component H-Cys(Bzl)-Pro-Leu-Gly-NH2 and catalyzed the coupling between the resulting dipeptide amide and Moz-Asn-OH, thus yielding Moz-Asn-Leu-Gly-NH2 as the main product.     

2-Nitro-5-Thiosulfobenzoic Acid as a Novel Inhibitor Specific for Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase I) contains four cysteine residues forming two disulfide bonds. Though there are no free sulfhydryl groups, incubation of bpDNase I with 2-nitro-5-thiosulfobenzoic acid (NTSB) in the presence of Ca(2+) or Mg(2+) at pH 7.5 results in inactivation of the enzyme. Amino acid analysis shows that NTSB-treated bpDNase I still contains all 4 half-cystine residues. The only amino acid residues having reduced values are threonine and serine, indicating that these may be the reaction sites for NTSB. Plasmid scission assay and circular dichroism analysis reveal the structural integrity of the inactivated enzyme. Treatment of bpDNase I with NTSB does not result in fragmentation, as demonstrated by SDS-PAGE analysis. NTSB binds bpDNase I through covalent modification, since dialysis and gel filtration can not reverse the inactivation reaction. However, after dilution into an acid buffer of pH 4.7, the inactivated enzyme regains about 40% of its initial activity, suggesting a reversible inactivation by acid treatment. NTSB does not inactivate DNase II, ribonuclease, chymotrypsin and lysozyme, while it effectively inactivates rat parotid DNase I. These results strongly suggest that NTSB can be considered as a novel inhibitor specific for DNase I.     

FASCAPLYSIN as a Specific Inhibitor for CDK4: Insights from Molecular Modelling

Cyclin-dependent kinases (CDKs) play a key role in the cell cycle and are important anti-cancer drug targets. The natural product fascaplysin inhibits CDK4 with surprising selectivity (IC₅₀ = 0.4 µM) compared to the close homolog CDK2 (IC₅₀ = 500 µM). Free energy calculations of the positively charged fascaplysin and an uncharged iso-electronic derivative in the CDK2 and CDK4 inhibitor complexes indicate that the positive charge of fascaplysin is crucial for selectivity. This finding will guide further improvements in the design of fascaplysin-based selective inhibitors for CDK4.     

Kinetics of binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluorescence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (kon)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (kon)app.     

赤小豆抑制剂对胰蛋白酶不可逆抑制作用的动力学探讨

本文从中药赤小豆(Phaseolus Calcaratus,Roxb)中分离出一种胰蛋白酶抑制剂。通过一系列酶促反应动力学的研究表明,赤小豆抑制剂对胰蛋白酶有较强的不可逆竞争性抑制作用。其Km和ki值分别为1.43×10~(-3)mmol/L和2.4×10~(-6)mmol/L。     

Experience-Dependent Rewiring of Specific Inhibitory Connections in Adult Neocortex

Although neocortical connectivity is remarkably stereotyped, the abundance of some wiring motifs varies greatly between cortical areas. To examine if regional wiring differences represent functional adaptations, we have used optogenetic raster stimulation to map the laminar distribution of GABAergic interneurons providing inhibition to pyramidal cells in layer 2/3 (L2/3) of adult mouse barrel cortex during sensory deprivation and recovery. Whisker trimming caused large, motif-specific changes in inhibitory synaptic connectivity: ascending inhibition from deep layers 4 and 5 was attenuated to 20%–45% of baseline, whereas inhibition from superficial layers remained stable (L2/3) or increased moderately (L1). The principal mechanism of deprivation-induced plasticity was motif-specific changes in inhibitory-to-excitatory connection probabilities; the strengths of extant connections were left unaltered. Whisker regrowth restored the original balance of inhibition from deep and superficial layers. Targeted, reversible modifications of specific inhibitory wiring motifs thus contribute to the adaptive remodeling of cortical circuits.     

新型血管内皮细胞抑制因子VEGI_(151)的基因克隆表达及其活性

血管内皮细胞生长抑制因子 (vascularendothelialcellgrowthinhibitor,VEGI)是近年发现的一类肿瘤坏死因子超家族成员 ,具有抑制内皮细胞增殖的作用。从人脐静脉内皮细胞株 (ECV30 4)克隆到其基因 ,构建N端缺失 2 3个氨基酸的表达载体 ,并通过原核表达系统进行表达 (命名为VEGI151) ,表达量为 2 5 .5 % ,纯化后纯度达92 .5 %。通过生物学效应检测 ,发现VEGI151可明显抑制无血清培养基中内皮细胞的增殖 ,2 4h时VEGI151对内皮细胞的IC50 为 10mg/L ,0 .6 13mg/L时使内皮细胞在 36h内完全凋亡。通过检测体外培养肿瘤细胞 (A5 49、HepG2、Hela等 )的存活率 ,未发现明显的增殖或抑制效应。提示VEGI是一种主要作用于血管内皮细胞 ,在新生血管性疾病及肿瘤的治疗中有潜在的应用前景。     

Specific binding of N-allylnormetazocine (SKF 10047), a ligand of sigma-opioid receptors, with liver membranes

A sigma-opioid receptor ligand, N-allylnormetazocine (SKF 10047), binds specifically and reversibly to rat liver membranes. The rat liver binding sites for SKF 10047 are similar to sigma-opioid CNS receptors. They fail to interact with classical opiates (morphine, naloxone) and opioid peptides but bind with high affinity benzomorphans (bremazocine, SKF 10047) and various psychotropic drugs (haloperidol, imipramine, phencyclidine etc).