环氧酮肽蛋白酶体抑制剂的构效关系及基于分子相似性的分子设计

针对56个环氧酮肽衍生物,分别采用比较分子场分析(comparative molecular field analysis,CoMFA)、比较分子相似性形状指数分析(comparative molecular similarity indices analysis,CoMSIA)、Topomer CoMFA、Holo-gram QSAR(HQSAR)以及基于一维和二维描述符的支持向量机(support vector machine,SVM)方法进行了细致的构效关系研究。研究显示:通过引入一维和二维描述符的SVM建模方法,避免了柔性分子在三维构效关系研究中的构象选择和叠合难题,亦可有效避免过拟合现象的发生。所建最优SVM模型的决定系数R2、均方根误差(RMS)、交互验证系数Q2和外部预测R2pred分别为0.681,0.436,0.572和0.641。分析结果显示:电性、拓扑特征、疏水性和分子体积是影响环氧酮肽蛋白酶体抑制活性的主要因素。在此基础上,以活性最高样本分子(CID:42638286的)为模板,基于相似性评价方法对其侧链进行设计,结合Lipinski"5规则"类药性筛选,共得到12个新颖目标分子,且预测活性均达到纳摩尔水平。     

Discovery of a potent and highly β1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are β5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent β1 selective compound active in living cells reported to date.     

Towards subunit-specific proteasome inhibitors: synthesis and evaluation of peptide alpha',beta'-epoxyketones

BACKGROUND: The proteasome is a large multicatalytic protease complex (700 kDa) involved in a number of highly regulated processes. It has three major catalytic activities: a chymotrypsin-like activity, a trypsin-like activity and a post-glutamyl peptide hydrolyzing (PGPH) activity. To be useful as molecular probes, which could help dissect the cellular functions of the proteasome, inhibitors should be specific for the proteasome, active in vivo and selectively block only one of the three catalytic activities. To date, few inhibitors fulfill these requirements so we set out to make novel proteasome inhibitors that incorporate these characteristics. RESULTS: A panel of amino-terminally acetylated peptide alpha',beta'-epoxyketones with leucine in P1 and various aliphatic or aromatic amino acids in P2-P4 were prepared and evaluated. Most compounds selectively inhibited the chymotrypsin-like activity, while only weakly inhibiting the trypsin-like and PGPH activities. After optimization, one inhibitor, Ac-hFLFL-epoxide, was found to be more potent and selective for the inhibition of the chymotrypsin-like activity than several previously described inhibitors. This inhibitor also exhibited strong in vivo anti-inflammatory activity. CONCLUSIONS: Optimization of amino-terminally acetylated peptide alpha',beta'-epoxyketones furnished a potent proteasome inhibitor, Ac-hFLFL-epoxide, that has an excellent selectivity for the chymotrypsin-like activity. The inhibitor also proved to be a potent antiproliferative and anti-inflammatory agent. The strong in vivo and in vitro activities suggest that this class of proteasome inhibitors could be both molecular probes and therapeutic agents.     

Synthesis and biological evaluation of substituted indazolyl amide derivatives as S-adenosyl-L-homocysteine hydrolase inhibitors

A series of novel amide derivatives bearing an indazole moiety were synthesized and evaluated for their in vitro S-adenosyl-L-homocysteine hydrolase (SAHase) inhibitory activity. Among these compounds, 8b, 8m, 8r and 8w showed better or similar inhibitory effects compared to the positive control aristeromycin. These results provide a novel lead for the discovery of more potent non-adenosine analogs as SAHase inhibitors.     

Systematic Comparison of Peptidic Proteasome Inhibitors Highlights the α‐Ketoamide Electrophile as an Auspicious Reversible Lead Motif

The ubiquitin–proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C‐terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α‐ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.     

A Minimal β‐Lactone Fragment for Selective β5c or β5i Proteasome Inhibitors

Broad‐spectrum proteasome inhibitors are applied as anticancer drugs, whereas selective blockage of the immunoproteasome represents a promising therapeutic rationale for autoimmune diseases. We here aimed at identifying minimal structural elements that confer β5c or β5i selectivity on proteasome inhibitors. Based on the natural product belactosin C, we synthesized two β‐lactones featuring a dimethoxybenzyl moiety and either a methylpropyl (pseudo‐isoleucin) or an isopropyl (pseudo‐valine) P1 side chain. Although the two compounds differ only by one methyl group, the isoleucine analogue is six times more potent for β5i (IC₅₀=14 nM ) than the valine counterpart. Cell culture experiments demonstrate the cell‐permeability of the compounds and X‐ray crystallography data highlight them as minimal fragments that occupy primed and non‐primed pockets of the active sites of the proteasome. Together, these results qualify β‐lactones as a promising lead‐structure motif for potent nonpeptidic proteasome inhibitors with diverse pharmaceutical applications.     

Nonpeptidic Selective Inhibitors of the Chymotrypsin‐Like (β5 i) Subunit of the Immunoproteasome

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide‐based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin‐like (β5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the β5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell‐based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as β5i‐selective probes for possible application in noncancer diseases related to the immunoproteasome.     

SPR imaging biosensor for the 20S proteasome: sensor development and application to measurement of proteasomes in human blood plasma

The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome’s catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng·mL-1 on average for control, and 42300 ng·mL-1 on average for leukemia patients).

Boron‐Based Drug Design

The use of the element boron, which is not generally observed in a living body, possesses a high potential for the discovery of new biological activity in pharmaceutical drug design. In this account, we describe our recent developments in boron‐based drug design, including boronic acid containing protein tyrosine kinase inhibitors, proteasome inhibitors, and tubulin polymerization inhibitors, and ortho‐carborane‐containing proteasome activators, hypoxia‐inducible factor 1 inhibitors, and topoisomerase inhibitors. Furthermore, we applied a closo‐dodecaborate as a water‐soluble moiety as well as a boron‐10 source for the design of boron carriers in boron neutron capture therapy, such as boronated porphyrins and boron lipids for a liposomal boron delivery system.     

TMC-95-based inhibitor design provides evidence for the catalytic versatility of the proteasome

TMC-95's natural cyclic tripeptide metabolites represent potent competitive proteasome inhibitors. The constrained conformation of TMC-95 proteasomal inhibitors provides the driving force for entropically high-affinity binding. Based on the crystal structure of the proteasome:TMC-95A complex, the synthetically challenging TMC-95 core structure was used for the design and synthesis of less demanding biphenyl-ether macrocycles, in which the biphenyl-ether moiety functions as an endocyclic clamp restricting its tripeptide backbone. These simplified analogs allowed us to identify high plasticity of the proteasomal tryptic-like specificity pocket. Biphenyl-ether compounds extended with an amide group were hydrolyzed by the proteasome, although the crystal structure of such proteasome:biphenyl-ether complexes revealed quenching of proteolysis at the acyl-enzyme intermediate. Our data reveal that biphenyl-ether derivatives bind noncovalently to the proteasomal tryptic-like active site in a reversible substrate-like manner without allosteric changes of active site residues.     

A brief and stereoselective synthesis of limonoid models, with antifeedant activity against Locusts migratoria

A short stereoselective preparation of havanensin-type limonoid models is reported. The synthesis is based on a radical domino reaction of an epoxyketone to a bicyclic hydroxyketone, and is achieved in six and nine steps from simple cyclohexenones. The epoxyhavanensin derivatives show significant antifeedant activity against Spodoptera littoralis and Spodoptera frugiperda, and the epoxyketone 21 shows potent antifeedant activity against Locusts migratoria.     

Mixing of peptides and electrophilic traps gives rise to potent, broad-spectrum proteasome inhibitors

The synthesis and evaluation of hybrid proteasome inhibitors that contain structural elements of the known inhibitors bortezomib, epoxomicin and peptide vinyl sulfones is described. From the panel of 15 inhibitors some structure activity relationships can be deduced with regard to inhibitory activity in relation to peptide recognition element, inhibitor size and nature of the electrophilic trap. Further, the panel contains one of the most potent peptide-based pan-proteasome inhibitors reported to date.     

Synthesis,Biological Evaluation,and In Silico Studies of New Acetylcholinesterase Inhibitors Based on Quinoxaline Scaffold

A quinoxaline scaffold exhibits various bioactivities in pharmacotherapeutic interests. In this research, twelve quinoxaline derivatives were synthesized and evaluated as new acetylcholinesterase inhibitors. We found all compounds showed potent inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 0.077 to 50.080 µM, along with promising predicted drug-likeness and blood–brain barrier (BBB) permeation. In addition, potent butyrylcholinesterase (BChE) inhibitory activity with IC₅₀ values of 14.91 to 60.95 µM was observed in some compounds. Enzyme kinetic study revealed the most potent compound (6c) as a mixed-type AChE inhibitor. No cytotoxicity from the quinoxaline derivatives was noticed in the human neuroblastoma cell line (SHSY5Y). In silico study suggested the compounds preferred the peripheral anionic site (PAS) to the catalytic anionic site (CAS), which was different from AChE inhibitors (tacrine and galanthamine). We had proposed the molecular design guided for quinoxaline derivatives targeting the PAS site. Therefore, the quinoxaline derivatives could offer the lead for the newly developed candidate as potential acetylcholinesterase inhibitors.     

Identification of Limonol Derivatives as Heat Shock Protein 90 (Hsp90) Inhibitors through a Multidisciplinary Approach

The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high‐affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α‐limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c‐Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol‐derivatives as promising scaffolds for the design of novel Hsp90α inhibitors.     

Assessing the Role of a Malonamide Linker in the Design of Potent Dual Inhibitors of Factor Xa and Cholinesterases

The rational discovery of new peptidomimetic inhibitors of the coagulation factor Xa (fXa) could help set more effective therapeutic options (to prevent atrial fibrillation). In this respect, we explored the conformational impact on the enzyme inhibition potency of the malonamide bridge, compared to the glycinamide one, as a linker connecting the P1 benzamidine anchoring moiety to the P4 aryl group of novel selective fXa inhibitors. We carried out structure–activity relationship (SAR) studies aimed at investigating para- or meta-benzamidine as the P1 basic group as well as diversely decorated aryl moieties as P4 fragments. To this end, twenty-three malonamide derivatives were synthesized and tested as inhibitors of fXa and thrombin (thr); the molecular determinants behind potency and selectivity were also studied by employing molecular docking. The malonamide linker, compared to the glycinamide one, does significantly increase anti-fXa potency and selectivity. The meta-benzamidine (P1) derivatives bearing 2′,4′-difluoro-biphenyl as the P4 moiety proved to be highly potent reversible fXa-selective inhibitors, achieving inhibition constants (K_i) in the low nanomolar range. The most active compounds were also tested against cholinesterase (ChE) isoforms (acetyl- or butyrylcholinesterase, AChE, and BChE), and some of them returned single-digit micromolar inhibition potency against AChE and/or BChE, both being drug targets for symptomatic treatment of mild-to-moderate Alzheimer’s disease. Compounds 19h and 22b were selected as selective fXa inhibitors with potential as multimodal neuroprotective agents.     

A Set of Activity‐Based Probes to Visualize Human (Immuno)proteasome Activities

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, β1c, β2c, and β5c. Lymphoid tissues also express the immunoproteasome subunits β1i, β2i, and β5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity‐based probes that enables simultaneous gel‐based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for β1c, β2c, β5c, and β2i, to compare the active‐site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of β5i and β1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.     

Stereoselective functionalization of pyrrolidinone moiety towards the synthesis of salinosporamide A

An important feature of the synthesis of salinosporamide A, a potent proteasome inhibitor, is the establishment of the quaternary stereocenter at C3. A new route has been developed based on the methylation of a functionalized pyrrolidinone. Direct methylation reaction led to the unwanted diastereomer; however, by means of a Corey–Chaykovsky reaction followed by LiAlH₄ epoxide opening, the desired alcohol was obtained. The pyrrolidinone was elaborated through a key allylation reaction between a tertiary allyltitanium reagent and an aldehyde bearing a spiroketal moiety in α-position.     

1,2,3-Triazolyl-tetrahydropyrimidine Conjugates as Potential Sterol Carrier Protein-2 Inhibitors: Larvicidal Activity against the Malaria Vector Anopheles arabiensis and In Silico Molecular Docking Study

Alteration of insect growth regulators by the action of inhibitors is becoming an attractive strategy to combat disease-transmitting insects. In the present study, we investigated the larvicidal effect of 1,2,3-triazolyl-pyrimidinone derivatives against the larvae of the mosquito Anopheles arabiensis, a vector of malaria. All compounds demonstrated insecticidal activity against mosquito larvae in a dose-dependent fashion. A preliminary study of the structure–activity relationship indicated that the electron-withdrawing substituent in the para position of the 4-phenyl-pyrimidinone moiety enhanced the molecules’ potency. A docking study of these derivatives revealed favorable binding affinity for the sterol carrier protein-2 receptor, a protein present in the intestine of the mosquito larvae. Being effective insecticides against the malaria-transmitting Anopheles arabiensis, 1,2,3-triazole-based pyrimidinones represent a starting point to develop novel inhibitors of insect growth regulators.     

Synthesis of chiral iodo-N,O-acetonide aminal scaffolds via an efficient cascade reaction of amino acid-derived epoxides

Novel amino acid-derived iodo-N,O-acetonide aminals were developed as chiral, non-epimerizable scaffolds to facilitate complex molecule synthesis. These scaffolds are readily prepared from commercially available amino acid derivatives in ?6 steps, contain an orthogonally-protected β-hydroxy amine moiety, and feature a directly reactive alkyl-iodide group for facile substitution chemistry. Further, a novel ring opening/cyclization cascade reaction was developed to prepare these compounds efficiently (59-72%) from readily available epoxide derivatives.