Structural basis for the interaction between carbonic anhydrase and 1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamides

Isoquinolinesulfonamides inhibit human carbonic anhydrases (hCAs) and display selectivity toward therapeutically relevant isozymes. The crystal structure of hCA II in complex with 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide revealed unusual inhibitor binding. Structural analyses allowed for discerning the fine details of the inhibitor binding mode to the active site, thus providing clues for the future design of even more selective inhibitors for druggable isoforms such as the cancer associated hCA IX and neuronal hCA VII.     

Structural basis for activation of G-protein-coupled receptors

Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where disruption of intramolecular interactions between TM3 and TM6 leads to a major conformational change of TM6 relative to the rest of the receptor. This conclusion was based on experiments in which environmentally sensitive, sulfhydryl-reactive fluorophores were site-selectively incorporated into wild-type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co-expression with the cAMP sensitive Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.     

Structural basis of NR2B-selective antagonist recognition by N-methyl-D-aspartate receptors

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors endowed with unique pharmacological and functional properties. In particular, their high permeability to calcium ions confers on NMDARs a central role in triggering long term changes in synaptic strength. Under excitotoxic pathological conditions, such as those occurring during brain trauma, stroke, or Parkinson's or Huntington's diseases, calcium influx through NMDAR channels can also lead to neuronal injury. This argues for the use of NMDAR antagonists as potential therapeutic agents. To date, the most promising NMDAR antagonists are ifenprodil and derivatives, compounds that act as noncompetitive inhibitors selective for NMDARs containing the NR2B subunit. Recent studies have identified the large N-terminal domain (NTD) of NR2B as the region controlling ifenprodil sensitivity of NMDARs. We present here a detailed characterization of the ifenprodil binding site using both experimental and computational approaches. 3D homology modeling reveals that ifenprodil fits well in a closed cleft conformation of the NRB NTD; however, ifenprodil can adopt either of two possible binding orientations of opposite direction. By studying the effects of cleft mutations, we show that only the orientation in which the phenyl moiety points deep toward the NTD hinge is functionally relevant. Moreover, based on our model, we identify novel NTD NR2B residues that are crucial for conferring ifenprodil sensitivity and provide functional evidence that these residues directly interact with the ifenprodil molecule. This work provides a general insight into the origin of the subunit-selectivity of NMDAR noncompetitive antagonists and offer clues for the discovery of novel NR2B-selective antagonists.     

Structural studies of racecadotril and its process impurities by NMR and mass spectroscopy

Three unknown impurities in racecadotril bulk drug at levels below 0.5% were detected by simple reverse phase isocratic high performance liquid chromatography (HPLC). Structures for these impurities were proposed by molecular ion information and their fragmentation pattern obtained by LC-MS and these impurities were confirmed by NMR spectroscopy. The impurities I, II and III were characterized as benzyl 2-methyl carboximido acetate, benzyl 2-phenyl ethyl carboximido acetate, and benzyl 2-(1-benzyl vinyl carboximido) acetate. These structures were further confirmed by co-injecting of synthetic standards of impurities with racecadotril. The mechanism of the formation of these process related impurities is discussed.     

Structural basis for the interaction between casein kinase 1 delta and a potent and selective inhibitor

Casein kinase 1 delta (CK1δ) and its closest homologue CK1ε are key regulators of diverse cellular growth and survival processes such as Wnt signaling, DNA repair, and circadian rhythms. We report three crystal structures of the kinase domain of human CK1δ, one apo and two complexed with a potent and selective CK1δ/ε inhibitor PF670462 in two different crystal forms. These structures provide a molecular basis for the strong and specific inhibitor interactions and suggest clues for further development of CK1δ/ε inhibitors.     

Structural basis and therapeutic implication of the interaction of CCN proteins with glycoconjugates

The CCN family of growth factors is composed of six structurally related proteins including the cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), nephroblastoma overexpressed (NOV), Wnt-1 induced secreted protein-1 (WISP-1), WISP-2 and WISP-3. Each family member consists of four conserved cysteine rich modular domains with sequence similarity to the insulin like growth factor binding proteins, von Willebrand factor, thrombospondin repeat and the growth factors cysteine knot. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. Both cancer promoting and inhibiting roles were proposed for several CCN proteins suggesting that contextual factors could regulate their activities. Consistent with this hypothesis, structural and experimental evidence indicate that the function of these proteins is modulated by their interaction with sulfated glycosaminoglycans. Because the CCN proteins are implicated in the tumorigenic process, they are potential targets for the development of cancer therapeutics. Modulation of their glycosaminoglycan interaction by exogenous, highly sulfated polysaccharides, oligosaccharides or glycosaminoglycan mimetics could prevent their participation in cancer progression. Understanding the structural requirements for their polysaccharide interaction should provide important information to generate glycosaminoglycan-based cancer therapeutics targeting the CCN family of proteins.     

荧光光谱法研究芒柄花素与牛血清白蛋白的相互作用

在模拟动物体生理条件下,用荧光光谱法研究芒柄花素(hq-2)与牛血清白蛋白(BSA)的结合作用.结果 表明,芒柄花素对BSA荧光猝灭属于静态猝灭;并求得结合常数Ka为5.27×104L·mol-1,结合位点数为1.热力学数据表明该药物与牛血清白蛋白的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的主要作用力类型为...     

荧光光谱法研究忍冬苷与牛血清白蛋白的相互作用

本文首次采用荧光光谱法研究了忍冬瞢与牛血清白费白（BSA）在模拟人体生理条件下的相互作用。结果表明忍冬苷对BSA的荧光猝灭作用属于静态猝灭．且296K下结合常数Ka为3．82×10^-5L·mol^-1,结合位点数n为1．16;热力学分析表明二者主要靠氢键和范德华力结合;位点竞争实验则显示忍冬苷主要通过Site I与BSA相结合;最后还考察了几种常见的共存离子对相互作用的影响。     

荧光光谱法研究塞曲司特与牛血清白蛋白的相互作用

目的:研究不同pH条件下塞曲司特与牛血清白蛋白的相互作用机制.方法:利用荧光光谱法,并以Stern-Volmer方程确定药物与蛋白的作用类型.结果:根据Stern-Volmer方程求出了不同二者pH条件下塞曲司特与牛血清白蛋白之间的猝灭常数,并依据 Foster能量转移理论确定了生理条件下药物与蛋白的结合距离为 2.53 nm.结论:在pH 5.0和人体生理pH条件下塞曲司特对牛血清白蛋白具有荧光猝灭作用且为动态猝灭过程,在pH 8.4时塞曲司特与牛血清白蛋白之间的猝灭为静态猝灭;人体生理pH条件下塞曲司特与牛血清白蛋白之间相互作用力主要为范德华力.     

木犀草素与牛血清白蛋白相互作用的荧光光谱法研究

本文首次采用荧光光谱法研究传统中药白毛夏枯草中活性成分木犀草素与牛血清白蛋白（BSA）的猝灭机制。实验研究了木犀草素在291 k和310 k条件下与BSA的猝灭情况,通过计算求得不同温度下的热力学参数。结果表明木犀草素对牛血清白蛋白荧光的猝灭机制属于静态猝灭过程,二者之间的作用力主要为范德华力和氢键。此外,实验还考察了木犀草素对BSA构象的影响。     

Structural and molecular basis of carbohydrate-protein interaction systems as potential therapeutic targets

The sugar chains covalently modifying proteins and lipids are recognized by a variety of proteins, thereby mediating a broad range of physiological and pathological events on cell surfaces as well as in cells. Hence, these carbohydrate-protein interaction systems could be potential therapeutic targets for various diseases, including viral infections, autoimmune diseases and neurodegenerative disorders. Cumulative crystallographic data of lectins complexed with their cognate carbohydrate ligands have elucidated the sugar recognition modes of these proteins, offering a structural basis for the design of drugs targeting carbohydrate-lectin interaction systems. In particular, structural and functional studies of animal L-type lectins, which possess a carbohydrate recognition domain with a structural resemblance to that of leguminous lectins such as concanavalin A, have demonstrated the molecular mechanisms underlying their distinct roles in sorting and trafficking of glycoproteins in cells, exemplifying the structure-based engineering that manipulates the sugar-binding properties of lectins. Furthermore, structural basis has been provided for the functional interplay between the L-type lectin ERGIC-53 and the EF-hand Ca2?-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII. This article also deals with pathological carbohydrate-protein interactions involving ganglioside clusters on cell surfaces, particularly focusing on the interaction between amyloid β (Aβ) and GM1 ganglioside. This interaction triggers conformational transition and consequent aggregation of Aβ, and therefore, is considered to be a key step in Alzheimer's disease. The recently reported structural information of the Aβ-GM1 interaction is presented, underscoring the significance of assemblages of glycoconjugates as therapeutic targets.     

Structural basis of the antagonism between inorganic mercury and selenium in mammals

Mercuric chloride toxicity in mammals can be overcome by co-administration of sodium selenite. We report a study of the mutual detoxification product in rabbit plasma, and of a Hg-Se-S-containing species synthesized by addition of equimolar mercuric chloride and sodium selenite to aqueous, buffered glutathione. Chromatographic purification of this Hg-Se-S species and subsequent structural analysis by Se and Hg extended X-ray absorption fine structure (EXAFS) spectroscopy revealed the presence of four-coordinate Se and Hg entities separated by 2.61 A. Hg and Se near-edge X-ray absorption spectroscopy of erythrocytes, plasma, and bile of rabbits that had been injected with solutions of sodium selenite and mercuric chloride showed that Hg and Se in plasma samples exhibited X-ray absorption spectra that were essentially identical to those of the synthetic Hg-Se-S species. Thus, the molecular detoxification product of sodium selenite and mercuric chloride in rabbits exhibits similarities to the synthetic Hg-Se-S species. The underlying molecular mechanism for the formation of the Hg-Se-S species is discussed.     

The isolation of Nepsilon-formyl-L-lysine from the reaction between formaldehyde and L-lysine and its identification by OPLC and NMR spectroscopy

From the reaction between l-lysine and formaldehyde, Nepsilon-formyl-L-lysine was isolated by means of ion-exchange column chromatography. The identification of Nepsilon-formyl-L-lysine was carried out by ion-exchange overpressured-layer chromatography (OPLC) and 1H NMR and 13C NMR spectroscopies. The m.p. and mixed m.p. values, the retention characteristics and the chemical shifts of the isolated product were identical with those of an authentic sample of Nepsilon-formyl-L-lysine.     

NMR spectroscopy in pharmaceutical analysis. VII. Determination of the component composition of gentamicin by13C NMR spectroscopy

The preceding communication described the identification and quantitative analysis of the relative contents of the Z- and E-isomers of derivatives of 2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetic acid by¹H and¹³C NMR [A].     

金合欢素-7-O-葡萄糖苷与BSA相互作用的荧光光谱法研究

应用荧光光谱法研究了生理条件下金合欢素-7-O-葡萄糖苷与牛血清白蛋白（BSA）的相互作用。结果表明,其对BSA荧光的猝灭机制属于形成复合物的静态猝灭过程,并求得结合常数Ka为8.57×104L.mol-1,结合位点数n为1。根据热力学参数确定了其与BSA之间的主要作用力类型为静电作用力。采用同步荧光考察了药物对BSA构象的影响。此外,讨论了共存离子Cu2＋,Al3＋,Zn2＋,Mg2＋对药物与BSA结合作用的影响。     

香烟提取物对血栓调节蛋白与凝血酶结合力的影响

目的利用单分子力谱法检测香烟提取物(cigarette smoke extract,CSE)对血栓调节蛋白(thrombomodulin,TM)与凝血酶间单分子水平相互作用,探讨吸烟致血管内血栓形成机制。方法构建TM-GFP质粒并转染至COS-7细胞,应用原子力显微镜(atomic force microscope,AFM)分组测力:(1)空白质粒对照组(GFP-Thr);(2)TM-Thr对照组;(3)5%CSE孵育TM-Thr细胞组(CSE-TM-Thr);(4)5%CSE孵育空白质粒细胞组(CSE-GFP-Thr)。结果 (1)TM与凝血酶的相互作用力为(60.90±0.82)pN,成键几率为(22.58±3.95)%,抗体阻断后成键几率为(2.58±2.0)%。(2)与TM-Thr组比较,GFP-Thr组、CSE-TM-Thr组、CSE-GFP-Thr组,3组AFM成键几率明显减低,P<0.05;但3组间两两比较差异无统计学意义。在定量TM修饰的硅片表面检测证实CSE同样可减少TM与凝血酶的成键几率。结论吸烟可能通过减少TM与凝血酶结合几率,抑制TM的抗凝作用,从而导致血管内血栓形成。     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.