The 2009 Nobel Prize in Chemistry: Thomas A. Steitz and the structure of the ribosome

Over the past 200 years, there have been countless groundbreaking discoveries in biology and medicine at Yale University. However, one particularly noteworthy discovery with profoundly important and broad consequences happened here in just the past two decades. In 2009, Thomas Steitz, the Sterling Professor of Molecular Biophysics & Biochemistry, was awarded the Nobel Prize in Chemistry for "studies of the structure and function of the ribosome," along with Venkatraman Ramakrishnan of the MRC Laboratory of Molecular Biology and Ada E. Yonath of the Weizmann Institute of Science. This article covers the historical context of Steitz's important discovery, the techniques his laboratory used to study the ribosome, and the impact that this research has had, and will have, on the future of biological and medical research.     

Reaction of incompatible isolate of Pyricularia grisea on rice leaves stripped of epicuticular wax reflects blast conduciveness of soils

Upland rice cultivars were evaluated in the greenhouse for susceptibility to the rice blast disease caused by Pyricularia grisea Sacc., on two upland soils from the Philippines previously considered to be “blast conducive” and “blast non-conducive”. Under monocyclic inoculation tests plants grown in conducive soil showed significantly greater lesion development than plants of the same cultivar grown in non-conducive soil: cultivars considered to be susceptible to the isolates used showed increased number of susceptible-type lesions; resistant cultivars showed increased number of hypersensitive resistant-type lesions. A similar effect was observed under polycyclic tests where several generations of the pathogen were allowed to develop on the test plants. Dilution of conducive soil with non-conducive soil resulted in a corresponding reduction of disease severity, although this was most pronounced on resistant cultivars. Removal of leaf epicuticular waxes (LEW) using organic solvents increased the number of resistant-type lesions on resistant cultivars grown in both soils following inoculation. Susceptible plants were not suitable for quantifying the relative blast conduciveness of a soil because of the extreme environmental sensitivity of the bioassay and the tendency of lesions to coalesce. Comparing numbers of resistant-type lesions on leaves of plants stripped of LEW and inoculated with an incompatible P. grisea isolate among plants grown in different soils proved to be a satisfactory means of distinguishing the relative blast conduciveness of soils under controlled conditions. This method was field tested in eastern India and results corroborated farmer assessment of which soils were blast conducive. Using incompatible isolate-cultivar combinations and LEW-free leaves is proposed as a simple bioassay for assessing blast conduciveness of soils and should prove useful in regional characterization of rice blast risk.     

籼稻品种浙辐802抗瘟性遗传研究

通过与８个日本鉴别品种杂交和利用日本代表菌系研５４－０４及我国南方稻区菌系９５-ｈ接种鉴定,研究了籼稻品种浙辐８０２的抗性遗传。研究结果表明,该品种对菌系研５４-０４的抗性由 ２对显性基因控制,对菌系 ９５-ｔ２的抗性由 １对显性基因控制。基因等位性分析确认,浙辐８０２中抵抗９５－ｔ２的抗病基因与Ｐｉ－ｉ基因等位,与Ｐｉ－ａ、Ｐｉ－ｓｈ、Ｐｉ－ｋ、Ｐｉ－ｚ、Ｐｉ－ｂ、Ｐｉ－ｔ、Ｐｉ－ｔａ等已知基因位点为非等位关系;抵抗菌系研５４－０４,感染菌系９５-ｔ２的基因与Ｐｉ－ｉ、Ｐｉ－ｋ、Ｐｉ－ｂ、 Ｐｉ－ｔ ４个已知基因位点为非等位关系。对这个基因与其他已知基因的等位性进行了分析,认为它可能是１个未被命名的新基因。     

Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder-order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.     

药物设计与同步辐射

当代药物设计是通过阐明药物与靶标相互作用的机理,对药物先导化合物进行改造和优化。利用晶体X射线衍射的方法获得药物与靶标复合物的结构,为药物设计提供最直接有力的依据。同步辐射凭借其高强度、低发散性、波长可调谐性等得天独厚的优势,实现了对药物与靶标复合物结构的高通量测定,大大提高了基于结构的药物设计效率。     

籼稻品种地谷抗稻瘟病基因的遗传

籼稻地谷是我国杂效裟育种上重要的稻瘟病抗源之一。利用我国稻区的稻瘟病菌系ＺＢ１３和ＺＢ１５对地谷与感病品种江南香糯的杂效Ｆ１、Ｆ２和Ｂ１Ｆ１群体,以及地谷与感病品种丽江新团黑谷、冈４６Ｂ和８９８７的Ｆ２群体进行接种鉴定,根据抗病性的分离确认地谷对ＺＢ１３和ＺＢ１５的抗性受显性基因控制。利用菌系ＺＢ１３接种地谷和１０个具有已知抗病基因的鉴别品种及其杂交Ｆ１和Ｆ２群体,进一步证明了地谷的抗温性受１对显     

靶向共价键药物的研究进展

药物与靶标的结合是启动药理作用的本源,共价键药物是以共享电子的方式来实现与靶标的结合,其中大多为抗感染、抗肿瘤以及心脑血管、神经系统和代谢类药物。简介共价键药物与非共价键药物的区别以及既往的重磅级共价键药物与靶标的结合特点,分类综述靶向共价键药物的理性设计及与靶标的结合反应。     

Structure-based efforts to optimize a non-β-lactam inhibitor of AmpC β-lactamase

β-Lactams are the most widely prescribed class of antibiotics, yet their efficacy is threatened by expression of β-lactamase enzymes, which hydrolyze the defining lactam ring of these antibiotics. To overcome resistance due to β-lactamases, inhibitors that do not resemble β-lactams are needed. A novel, non-β-lactam inhibitor for the class C β-lactamase AmpC (3-[(4-chloroanilino)sulfonyl]thiophene-2-carboxylic acid; K_i 26 μM) was previously identified. Based on this lead, a series of compounds with the potential to interact with residues at the edge of the active site were synthesized and tested for inhibition of AmpC. The length of the carbon chain spacer was extended by 1, 2, 3, and 4 carbons between the integral thiophene ring and the benzene ring (compounds 4, 5, 6, and 7). Compounds 4 and 6 showed minimal improvement over the lead compound (K_i 18 and 19 μM, respectively), and compound 5 inhibited to the same extent as the lead. The X-ray crystal structures of AmpC in complexes with compounds 4, 5, and 6 were determined. The complexes provide insight into the structural reasons for the observed inhibition, and inform future optimization efforts in this series.     

Apo and inhibitor complex structures of BACE (beta-secretase)

Human BACE, also known as beta-secretase, shows promise as a potential therapeutic target for Alzheimer's disease. We determined the apo structure of BACE to 1.75 A, and a structure of a hydroxyethylamine inhibitor complex derived by soaking. These show significant active-site movements compared to previously described BACE structures. Additionally, the structures reveal two pockets that could be targeted by structure-based drug design.     

A New Process for the Production of Coenzyme A

A new process has been described for the preparation of coenzyme A of high purity from the cultured broth of Brevibacterium ammoniagenes IFO 12071. The product was obtained in a high yield by the use of Duolite S–30, charcoal, and Dowex 1×2, and identified chemically and enzymatically. This method is simple, rapid, and compact, requires no special equipment, and has been shown to be adaptable for preparing large amounts of highly pure coenzyme A.     

Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC₅₀ values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.