New cyclic peptide proteasome inhibitors

Here we report the study of a new series of vinyl ester cyclopeptide analogues synthesized on the basis of our previous development of a class of cyclopeptides derived from our linear prototype inhibitors. In these compounds, the exocyclic pharmacophoric unit Leu-VE was linked to the γ-carboxyl group of the glutamic acid residue at the C-terminal. The best analogues of the series have been shown to inhibit the caspase-like activity of the proteasome at nanomolar concentrations and have also demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.     

Trypanocidal activity of peptidyl vinyl ester derivatives selective for inhibition of mammalian proteasome trypsin-like activity

Nine vinyl ester tripeptides selective for inhibition of mammalian proteasome trypsin-like activity were tested for in vitro activity against Trypanosoma brucei. Interestingly, two compounds showed trypanocidal activity in the low micromolar range without displaying cytotoxicity against human cells. However, the compounds did not inhibit the trypsin-like activity of the trypanosome proteasome although their effect correlates with inactivation of the chymotrypsin-like activity. This finding shows that the inhibitor sensitivities between mammalian and trypanosome proteasome are distinct. This difference may be exploited for rational anti-trypanosomal drug development.     

Synthesis and biological properties of C-terminal vinyl ketone pseudotripeptides

The ubiquitin-proteasome pathway responsible for the turnover of many cellular proteins represents an attractive target in the development of new drug therapies: In particular, modulation of the proteasome activity by specific inhibitors may represent a useful tool for the treatment of tumours. Here, we report synthesis and activity of a new series of oligopseudopeptide analogues bearing a vinyl ketone pharmacophoric unit at the C-terminal position. Some derivatives showed inhibition in the µM range of the trypsin-like (T-L) active site of the proteasome.     

Synthesis and activity of isoxazoline vinyl ester pseudopeptides as proteasome inhibitors

The ubiquitin–proteasome pathway (UPP) influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses. The main proteolytic component of the UPP is the 26S proteasome, which is responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered. We report herein the synthesis, proteasome inhibition and modelling studies of novel C‐terminal isoxazoline vinyl ester pseudopeptides. Some new compounds that contain a C‐terminal extended conjugation inhibit β1 and especially β5 proteasomal catalytic subunits with IC₅₀ values ranging from 10 to 100 µm . These results will permit further optimization based on these structural moieties to develop more active and selective molecules. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Novel CADD-based peptidyl vinyl ester derivatives as potential proteasome inhibitors

A series of peptidyl vinyl ester derivatives bearing three different P1 substitutions as potential proteasome inhibitors were studied. The target molecules were designed based on CADD (computer aided drug design) protocol and synthesized. Their activities toward proteasome and four human cancer cell lines (including hepatoma cell line (Bel-7402), myeloid leukemic cell line (HL-60), gastric cancer cell line (BGC-823) and nasopharyngeal cancer cell line (KB)) were tested using fluorescence assay. Two compounds showed proteasome inhibitory activities, and four compounds showed weak antiproliferative activities toward HL-60 and BGC-823.     

P3 and P4 position analysis of vinyl ester pseudopeptide proteasome inhibitors

Two small libraries of tripeptidic-based vinyl ester derivative proteasome inhibitors were synthesized and tested, starting with the Hmb-Val-Gln-Leu-VE prototype. The P3 and P4 positions were investigated with a complete set of amino acid residues, some of which showed remarkable selective inhibition of the trypsin-like (beta2) subunit. In both positions, aromatic and hydrophobic residues were preferred.     

Capillary electrophoresis for screening of 20S proteasome inhibitors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional “helper” molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 μm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC₅₀ values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.     

Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach

The ubiquitin-proteasome pathway is particularly important for the regulated degradation of various proteins which control a vast array of biological processes. Therefore, proteasome inhibitors are promising candidates for anti-tumoral or anti-inflammatory drugs. N-Acetyl-Leu-Leu-Norleucinal (Ac-LLN-al, also termed calpain inhibitor I) was one of the first proteasome inhibitors discovered and has been widely used to study the 20S proteasome core particle (CP) function in vivo, despite its lack of specificity. Vinyl sulfones, like Ac-PRLN-vs, show covalent binding of the beta-carbon atom of the vinyl sulfone group to the Thr1Ogamma only of subunit beta2. However, vinyl sulfones have similar limitations as peptide aldehydes as they have been reported also to bind and block intracellular cysteine proteases. A more specific proteasome inhibitor is the natural product lactacystin, which can be isolated from Streptomyces. It was found that this compound forms an ester bond only to the Thr1Ogamma of the chymotrypsin-like active subunit beta5 due to specific P1 interactions. In contrast to most other proteasome inhibitors, the natural alpha',beta'-epoxyketone peptide epoxomicin binds specifically to the small class of N-terminal nucleophilic (Ntn) hydrolases (CPs belong to this protease family) with the formation of a morpholino adduct. All previously described proteasome inhibitors bind covalently to the proteolytic active sites. However, as the proteasome is involved in a variety of biological important functions, it is of particular interest to block the CP only for limited time in order to reduce cytotoxic effects. Recently, the binding mode of the natural specific proteasome inhibitor TMC-95 obtained from Apiospora montagnei was investigated. The crystal structure revealed that the TMC-95 blocks the active sites of the CP noncovalently in the low nanomolar range. This review summarizes the current structural knowledge of inhibitory compounds bound to the CP, showing the proteasome as a potential target for drug development in medical research.     

C-terminal trans,trans-muconic acid ethyl ester partial retro-inverso pseudopeptides as proteasome inhibitors

The development of specific inhibitors of the proteasome represents an important opportunity for new drug therapies. The central role of the multicatalytic complex in the intracellular proteolysis mediated by ubiquitin-proteasome pathway goes to discovery many molecules able to selectively inhibits enzymatic active subsites. Now, we report synthesis and activity of a new partial retro-inverso oligopseudopeptide derivatives bearing a trans,trans-muconic acid ethyl ester pharmacophoric unit at the C-terminal. Some analogues selectively inhibited in µM range the caspase-like (C-L) activity in the β1 subunit of the proteasome.     

Proteasome inhibition by new dual warhead containing peptido vinyl sulfonyl fluorides

The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long lasting ties. We have introduced peptido sulfonyl fluorides for this purpose. Tuning the reactivity of this electrophilic trap may be crucial for modulating the biological action. Here we describe incorporation of a vinyl moiety into a peptido sulfonyl fluoride backbone, which should lead to a combined attack of the proteasome active site threonine on the double bond and the sulfonyl fluoride. Although this led to strong proteasome inhibitors, in vitro studies did not unambiguously demonstrate the formation of the proposed seven-membered ring structure. Possibly, formation of a seven-membered covalent adduct with the proteosomal active site threonine can only be achieved within the context of the enzyme. Nevertheless, this dual warhead concept may provide exclusive possibilities for duration and selectivity of proteasome inhibition.     

松香树脂酸乙烯酯的研究Ⅰ：歧化松香乙烯酯组成与结构的研究

本文研究了国产歧化松香乙烯酯的组成与各组分的分子结构。结果表明:国产歧化松香乙烯酯的主要成分是二氢枞酸和脱氢枞酸的乙烯酯,二者重量比为3:4;其乙烯酯上乙烯基在核磁共振谱图中呈现为很好的ABX系统;因为二氢枞酸有三个异构体,所以仅用光谱方法无法确定二氢枞酸乙烯酯环内双键的位置。     

蛋白酶体结构和活性调节机制的研究进展

蛋白酶体负责细胞内绝大多数蛋白质的降解,几乎对生物体所有的生命活动都具有调控作用.蛋白酶体功能异常能够导致很多疾病.近期,研究者们在蛋白酶体的结构分析和活性调节机制等方面的研究都获得了重要的突破.本文综述了有关蛋白酶体结构和活性调控机制,包括转录调控、翻译后修饰、组装机制等的研究进展,这些对蛋白酶体新的认识将为蛋白酶体相关疾病的研究及相应药物的开发带来新的思路.对于目前蛋白酶体抑制剂的研发本文也做了简要的介绍.     

Dopamine induces proteasome inhibition in neural PC12 cell line

The autoxidation and enzymatic catabolism of dopamine results in the generation of reactive oxygen species (ROS), which may possibly contribute to oxidative stress in multiple neurodegenerative disorders. Recent studies indicate that proteasome inhibition occurs in numerous neurodegenerative conditions, possibly as the result of oxidative stress, although the effects of dopamine on proteasome activity have not been determined. In the present study we examined the effects of dopamine on proteasome activity in the neural PC12 cell line. Application of dopamine induced a dose- and time-dependent decrease in proteasome activity, which occurred prior to cell death. Application of an antioxidant (gluthathione monoethyl ester), monoamine oxidase inhibitors (deprenyl, clogyline, paragyline), or an inhibitor of dopamine uptake (nomifensine) attenuated dopamine toxicity and dopamine-induced proteasome impairment. Application of the proteasome inhibitor lactacystin increased the toxicity of dopamine and the levels of protein oxidation following administration of dopamine. Together, these data indicate that dopamine induces proteasome inhibition that is dependent, in part, on ROS and dopamine uptake, and suggest a possible role for proteasome inhibition in dopamine toxicity.     

Proteasome inhibitors: Dozens of molecules and still counting

The discovery of the proteasome in the late 80’s as the core protease of what will be then called the ubiquitin–proteasome system, rapidly followed by the development of specific inhibitors of this enzyme, opened up a new era in biology in the 90’s. Indeed, the first proteasome inhibitors were instrumental for understanding that the proteasome is a key actor in most, if not all, cellular processes. The recognition of the central role of this complex in intracellular proteolysis in turn fuelled an intense quest for novel compounds with both increased selectivity towards the proteasome and better bioavailability that could be used in fundamental research or in the clinic. To date, a plethora of molecules that target the proteasome have been identified or designed. The success of the proteasome inhibitor bortezomib (Velcade^®) as a new drug for the treatment of Multiple Myeloma, and the ongoing clinical trials to evaluate the effect of several other proteasome inhibitors in various human pathologies, illustrate the interest for human health of these compounds.     

Synthesis of fructose esters by Pseudomonas sp. lipase in anhydrous pyridine

Fructose esters were synthesized from fructose and vinyl esters by transesterification catalyzed by lipase AK in anhydrous pyridine. The efficacy of ester synthesis was enhanced by increasing the length of carbon chain in the vinyl ester. Fructose monoesters and diesters were synthesized and their relative production ratio depended on the chain length of vinyl esters. Vinyl esters with chain length longer than C10 produced only monoacyl fructose which has an acyl moiety attached to C1 carbon of fructose. The monoacyl fructose composed of fatty acid with C10 or longer chains had a strong emulsifying activity on various hydrocarbons and oils.     

Apoptosis meets proteasome, an invaluable therapeutic target of anticancer drugs

This report reviews the current status of extensive efforts directed towards the interpretation of crosstalk between apoptosis and proteasome to understanding the molecular mechanism of anticancer agents targeting proteasome, with particular focus on MG132 and PS-341. The discovery that all cancer cells have retained the apoptotic death program has offered to the researchers new biochemical targets to design anticancer drugs. Moreover, the demonstration that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents has proposed the proteasome as an attractive target for development of new anticancer drugs. Since then, a number of both naturally occurring and synthetic inhibitors of the proteasome have been identified. The best characterized and most widely used inhibitors of the proteasome are the peptide aldehydes; among these MG132, due to its broad spectrum of action, low cost and rapid reversibility of action, still remains the first choice to study proteasome function in cell and tissue cultures. Recently, a very potent new class of selective and reversible proteasome inhibitors which contains an inhibitory boronate group has been described. PS-341 represent the first of this promising class of agents that could have application in cancer therapy and it is the only that has progressed to clinical trials.     

The capture proteasome assay: A method to measure proteasome activity in vitro

Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i–β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome–Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO₄ in the proteasome–Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO₄ and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.     

Selective immunoproteasome inhibitors with non-peptide scaffolds identified from structure-based virtual screening

As a major component of the crucial nonlysosomal protein degradation pathway in the cells, the proteasome has been implicated in many diseases such as Alzheimer’s disease, Huntington’s disease, inflammatory bowel diseases, autoimmune diseases, multiple myeloma (MM) and other cancers. There are two main proteasome subtypes: the constitutive proteasome which is expressed in all eukaryotic cells and the immunoproteasome which is expressed in immune cells and can be induced in other cell types. Majority of currently available proteasome inhibitors are peptide backbone-based, having short half-lives in the body. It is highly desirable to identify novel, immunoproteasome-selective inhibitors with non-peptide scaffolds for development of novel therapeutics. Through combined virtual screening and experimental studies targeting the immunoproteasome, we have identified a set of novel immunoproteasome inhibitors with diverse non-peptide scaffolds. Some of the identified inhibitors have significant selectivity for the immunoproteasome over the constitutive proteasome. Unlike most of the currently available proteasome inhibitors, these new inhibitors lacking electrophilic pharmacophores are not expected to form a covalent bond with proteasome after the binding. These non-peptide scaffolds may provide a new platform for future rational drug design and discovery targeting the immunoproteasome.