石蒜碱药理活性及构效关系研究进展

石蒜碱是药用石蒜科植物的有效成分之一,是重要的异喹啉类生物碱.石蒜碱拥有刚性的环系骨架、连续的手性中心、三级胺等独特的化学结构特征.同时其药理活性丰富多样,近年来,针对其抗癌、抗病毒、抗炎、抗寄生虫、抑制乙酰胆碱酯酶活性的研究越来越多,尤其在抗癌、抗病毒方面石蒜碱表现出较大潜力,特别是新型冠状病毒SARS-CoV-2研...     

Synthesis of 15-methylene-eburnamonine from (+)-vincamine,evaluation of anticancer activity,and investigation of mechanism of action by quantitative NMR

The biological role of installing a critical exocyclic enone into the structure of the alkaloid, (?)-eburnamonine, and characterization of the new chemical reactivity by quantitative NMR without using deuterated solvents are described. This selective modification to a natural product imparts potent anticancer activity as well as bestows chemical reactivity toward nucleophilic thiols, which was measured by quantitative NMR. The synthetic strategy provides an overall conversion of 40%. In the key synthetic step, a modified Peterson olefination was accomplished through the facile release of trifluoroacetate to create the requisite enone in the presence of substantial steric hindrance.     

2-Methoxyestradiol—Biology and mechanism of action

In the last decade the endogenous estradiol metabolite, 2-methoxyestradiol (2ME), has gained more and more interest due to its marked anticancerogenic properties and possible cardiovascular benefits, as shown in numerous animal and experimental investigations. Some promising results in terms of the usage of 2ME as a therapeutic agent were obtained by various clinical studies in patients with breast cancer and prostate cancer. However, one main problem appears to be the bioavailability of 2ME, therefore new formulations are now in the test phase. In this review, the most important aspects of the biology and molecular mechanisms of 2ME are summarized.     

C1,C2-ether derivatives of the Amaryllidaceae alkaloid lycorine: Retention of activity of highly lipophilic analogues against cancer cells

As a continuation of the studies aimed at the development of new anticancer agents derived from the Amaryllidaceae alkaloid lycorine, 35 C1,C2-ether analogues of this natural product were synthesized. The compounds were evaluated for antiproliferative activities in vitro in a panel of tumor cell lines with varied levels of apoptosis resistance. A strong correlation between the compound lipophilicity and anticancer activity was observed, indicating that cell permeability properties must be an important determinant in the design of lycorine-based anticancer agents. A theoretical docking model, consistent with the experimental observations, is presented.     

Activities of the peptidyl transferase center of ribosomes lacking protein L27

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.     

Metalloproteomics in conjunction with other omics for uncovering the mechanism of action of metallodrugs: Mechanism-driven new therapy development

Medicinal inorganic chemistry has been largely stimulated by the clinic success of platinum anticancer drugs. An array of metal-based drugs (e.g. platinum, gold, bismuth, and silver) are currently used clinically for the treatment of various diseases. Integrating multiomics approaches, particularly metalloproteomics, with other biochemical characterizations enables comprehensive understanding of cellular responses of metallodrugs, which in turn will guide the rational design of a new drug and modification of the presently used drugs. This review aims to summarize the recent progress in this area. We will describe the technology platforms and their applications for uncovering the mechanisms of action of metallodrugs, for which remarkable advances have been achieved recently. Moreover, we will also highlight the application of newly generated knowledge for the development of novel therapeutic strategies.     

Crosslinking of mRNA Analog,pUUAGUAUUUAUU Derivative with Photoactivatable Group at the Guanosine Residue,with Human 80S Ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg²⁺, or 4 mM Mg²⁺ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840–1849 of 18S rRNA, but in the complexes formed with participation of Phe-tRNA^Phe (where the G residue carrying the arylazido group occupied position –3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position –3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg²⁺ concentration and the presence of polyamines.     

益生菌抗真菌作用机制研究进展

真菌感染性疾病是普遍的全球性问题,随着感染人数的增多,已成为威胁人类健康的主要传染疾病之一。其对治疗药物出现的耐药性及药物本身的副作用,迫切需要开发新的安全、有效的替代品。益生菌作为对人体健康有益的微生物,已被实验证明对断发毛癣菌、须癣毛癣菌、酵母菌和霉菌等有明显的抑制作用。研究表明益生菌在繁殖过程中产生的有机酸以及产生的酸性物质使周围环境pH降低能有效地抑制真菌的生长。益生菌培养液的无细胞上清液及其自身具备的粘附性、疏水性等对浅部真菌和深部真菌都具有抑制作用。本研究对益生菌抗真菌作用的机制作一综述。     

Mutations of highly conserved bases in the peptidyltransferase center induce compensatory rearrangements in yeast ribosomes

Molecular dynamics simulation identified three highly conserved rRNA bases in the large subunit of the ribosome that form a three-dimensional (3D) "gate" that induces pausing of the aa-tRNA acceptor stem during accommodation into the A-site. A nearby fourth base contacting the "tryptophan finger" of yeast protein L3, which is involved in the coordinating elongation factor recruitment to the ribosome with peptidyltransfer, is also implicated in this process. To better understand the functional importance of these bases, single base substitutions as well as deletions at all four positions were constructed and expressed as the sole forms of ribosomes in yeast Saccharomyces cerevisiae. None of the mutants had strong effects on cell growth, translational fidelity, or on the interactions between ribosomes and tRNAs. However, the mutants did promote strong effects on cell growth in the presence of translational inhibitors, and differences in viability between yeast and Escherichia coli mutants at homologous positions suggest new targets for antibacterial therapeutics. Mutant ribosomes also promoted changes in 25S rRNA structure, all localized to the core of peptidyltransferase center (i.e., the proto-ribosome area). We suggest that a certain degree of structural plasticity is built into the ribosome, enabling it to ensure accurate translation of the genetic code while providing it with the flexibility to adapt and evolve.     

Comparison of fungal 80 S ribosomes by cryo-EM reveals diversity in structure and conformation of rRNA expansion segments

Compared to the prokaryotic 70 S ribosome, the eukaryotic 80 S ribosome contains additional ribosomal proteins and extra segments of rRNA, referred to as rRNA expansion segments (ES). These eukaryotic-specific rRNA ES are mainly on the periphery of the 80 S ribosome, as revealed by cryo-electron microscopy (cryo-EM) studies, but their precise function is not known. To address the question of whether the rRNA ES are structurally conserved among 80 S ribosomes of different fungi we performed cryo-electron microscopy on 80 S ribosomes from the thermophilic fungus Thermomyces lanuginosus and compared it to the Saccharomyces cerevisiae 80 S ribosome. Our analysis reveals general structural conservation of the rRNA expansion segments but also changes in ES27 and ES7/39, as well as the absence of a tertiary interaction between ES3 and ES6 in T. lanuginosus. The differences provide a hint on the role of rRNA ES in regulating translation. Furthermore, we show that the stalk region and interactions with elongation factor 2 (eEF2) are different in T. lanuginosus, exhibiting a more extensive contact with domain I of eEF2.     

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.     

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.     

Gold(III) compounds as anticancer agents: relevance of gold-protein interactions for their mechanism of action

Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. During the past decade a number of structurally diverse gold(III) complexes were reported to be acceptably stable under physiological-like conditions and to manifest very promising cytotoxic effects against selected human tumour cell lines, making them good candidates as anti-tumour drugs. Some representative examples will be described in detail. There is considerable interest in understanding the precise biochemical mechanisms of these novel cytotoxic agents. Based on experimental evidence collected so far we hypothesize that these metallodrugs, at variance with classical platinum(II) drugs, produce in most cases their growth inhibition effects through a variety of "DNA-independent" mechanisms. Notably, strong inhibition of the selenoenzyme thioredoxin reductase and associated disregulation of mitochondrial functions were clearly documented in some selected cases, thus providing a solid biochemical basis for the pronounced proapoptotic effects. These observations led us to investigate in detail the reactions of gold(III) compounds with a few model proteins in order to gain molecular-level information on the possible interaction modes with possible protein targets. Valuable insight on the formation and the nature of gold-protein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectrophotometric studies of appropriate model systems as it is exemplified here by the reactions of two representative gold(III) compounds with cytochrome c and ubiquitin. The mechanistic relevance of gold(III)-induced oxidative protein damage and of direct gold coordination to protein sidechains is specifically assessed. Perspectives for the future of this topics are briefly outlined.     

Correlation of the structure and conformational changes of selected fragments of plant small ribosomal RNA within the steps of polypeptide chain elongation

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for ribosome activity. We tested seven different deoxyoligonucleotides complementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-translocational states (with or without pre-hybridized oligonucleotides). We found a correlation between the level of oligomer hybridization and the inhibition of AA-tRNA binding. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis.     

Protein Environment of the Sense Codon of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking to Oligoribonucleotide Derivatives

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA^Phe, which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA^Phe and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.     

Arrangement of the Sense and Stop Codons of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking with Oligonucleotide Derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3-flanking sense or stop codon with a perfluoroarylazido group at G or U were used to study the positioning of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, tRNA^Phe cognate to UCC was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 and nucleotide A1825 in helix 44 close to the 3 end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem–loop fragment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in the ribosomal A site of various organisms.     

Modulating the activity of the peptidyl transferase center of the ribosome

The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.