Characterisation of cisplatin binding sites in human serum proteins using hyphenated multidimensional liquid chromatography and ESI tandem mass spectrometry

Cisplatin binding sites in human serum proteins have been characterised by using combined multidimensional liquid chromatography and ESI tandem mass spectrometry (MudPIT). Following incubation periods of 3 h for cisplatin-blood serum mixtures and subsequent trypsin digestion, MS-MS spectra were recorded for individual peptides that had been separated by SCX and RP liquid chromatography. Matching of the MS-MS spectra to theoretical sequences that were generated for human proteins in the SWISS-PROT database led to the identification of specific binding sites in human serum albumin (HSA), serotransferrin (Trfe) and other abundant serum proteins (A2mg, A1at, Apoa1, Apoa2). The cisplatin coordination sites in HSA and Trfe were confirmed by independent MudPIT studies on cisplatin reaction mixtures with the individual proteins. A total of five specific binding sites were identified for HSA, including the cysteine residue C34, two methionine sites (M329, M548) and the tyrosine and aspartate O-donor sites Y150 (or Y148) and D375 (or E376). Methionine-256 was established as a cisplatin coordination site for Trfe in addition to the O-donor sites E265, Y314, E385 and T457. Inspection of the protein structures indicates that the preferred residues belong either to peripheral alpha helices or to flexible loops within the protein-binding pockets. O-donor residues dominate as cisplatin binding sites for other abundant serum proteins.     

Rational Design of Resorcylic Acid Lactone Analogues as Covalent MNK1/2 Kinase Inhibitors by Tuning the Reactivity of an Enamide Michael Acceptor

Recent biological and computational advances in drug design have led to renewed interest in targeted covalent inhibition as an efficient and practical approach for the development of new drugs. As part of our continuing efforts in the exploration of the therapeutic potential of resorcylic acid lactones (RALs), we report herein the design, synthesis, and biological evaluation of conveniently accessible RAL enamide analogues as novel covalent inhibitors of MAP kinase interacting kinases (MNKs). In this study, we have successfully demonstrated that the covalent binding ability of RAL enamides can be tuned by attaching an electron‐withdrawing motif, such as an acyl group, to enhance its reactivity toward the cysteine residues at the MNK1/2 binding sites. We have also shown that ¹H NMR spectroscopy is a convenient and effective tool for screening the covalent binding activities of enamides using cysteamine as a mimic of the key cysteine residue in the enzyme, whereas mass spectrometric analysis confirms covalent modification of the kinases. Preliminary optimization of the initial hit led to the discovery of enamides with low micromolar activity in MNK assays. Cancer cell line assays have identified RAL enamides that inhibit the growth of cancer cells with similar potency to the natural product L‐783 ,277.     

分子模拟和光谱法研究华法林与人血清白蛋白的结合机制

华法林(WF)是临床常用口服抗凝药物,有关它与人血清白蛋白(HSA)结合位点处的热点残基、作用力类型以及对HSA构象影响报道较少.通过在模拟人体生理条件下,利用分子模拟、内源荧光光谱、三维荧光光谱和同步荧光光谱,研究了WF与HSA之间作用机理.WF能使HSA构象发生改变,导致Tyr残基微环境极性降低、Trp残基周围亲水...     

Triplex Crosslinking through Furan Oxidation Requires Perturbation of the Structured Triple‐Helix

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan‐based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan‐oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan‐containing probes for the selective detection of single‐stranded regions within nucleic acids containing a variety of structural motifs.     

Imprinted β‐cyclodextrin polymers using naringin as template

The objective of this study was to identify a kind of molecular imprinting polymer (MIP) which was suitable for recognizing naringin (NG) in aqueous medium. Based on two crosslinkers (hexamethylene diisocyanate and epichlorohydrin) and two polymerization methods (solution polymerization and emulsion polymerization), four non‐covalent naringin‐β‐cyclodextrine (NG‐β‐CD) imprinted polymers were prepared by using β‐CD as a functional monomer and NG as a template molecule. The binding property and selectivity were evaluated by equilibrium binding experiments. These demonstrated that all the sites in the MIPs show good selective binding ability for NG from naringin dihydrochalcone, a structurally similar molecule. Of the four MIPs, rod‐like 3# MIP which was prepared by emulsion polymerization using hexamethylene diisocyanate as crosslinker exhibited the highest selectivity, its imprinting factor α being 1.53. Scatchard analysis of 3# MIP suggests that there are two classes of binding sites during the MIP's recognition of NG. Additionally, the 3# MIP could be used at least five times without any loss in sorption capacity. Copyright © 2011 Society of Chemical Industry     

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence,FRET, Atomic Force Spectroscopy and Computational Modelling

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 10⁴ M⁻¹ has been assessed through a Stern–Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.     

Noncovalent Association Thermodynamics of Turn-On Fluorescent Probes with Human Serum Albumin: Dual-Concentration Ratio Method

Efficient quantification of the affinity of a drug and the targeted protein is critical for strategic drug design. Among the various molecules, turn-on fluorescent probes are the most promising signal transducers to reveal the binding strength and site-specificity of designed drugs. However, the conventional method of measuring the binding ability of turn-on fluorescent probes by using the fractional occupancy under the law of mass action is time-consuming and a massive sample is required. Here, we report a new method, called dual-concentration ratio method, for quantifying the binding affinity of fluorescent probes and human serum albumin (HSA). Temperature-dependent fluorescence intensity ratios of a one-to-one complex (L ⋅ HSA) for a turn-on fluorescent probe (L), e. g., ThT (thioflavin T) or DG (dansylglycine), with HSA at two different values of [L]₀/[HSA]₀ under the constraint [HSA]₀>[L]₀ were collected. The van't Hoff analysis on these association constants further resulted in the thermodynamic properties. Since only two samples at different [L]₀/[HSA]₀ are required without the need of [L]₀/[HSA]₀ at a wide range, the dual-concentration ratio method is an easy way to greatly reduce the amounts of fluorescent probes and proteins, as well as the acquisition time.     

Flexible adaptations in the structure of the tRNA-modifying enzyme tRNA-guanine transglycosylase and their implications for substrate selectivity, reaction mechanism and structure-based drug design

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyses a base-exchange reaction that leads to anticodon modifications of certain tRNAs. The TGT enzymes of the eubacteria Zymomonas mobilis (Z. mobilis TGT) and Escherichia coli (E. coli TGT) show a different behaviour in the presence of competitive inhibitors. The active sites of both enzymes are identical apart from a single conservative amino acid exchange, namely Tyr106 of Z. mobilis TGT is replaced by a Phe in E. coli TGT. Although Tyr106 is, in contrast to Phe106, hydrogen bonded in the ligand-free structure, we can show by a mutational study of TGT(Y106F) that this is not the reason for the different responses upon competition. The TGT enzymes of various species differ in their substrate selectivity. Depending on the applied pH conditions and/or induced by ligand binding, a peptide-bond flip modulates the recognition properties of the substrate binding site, which changes between donor and acceptor functionality. Furthermore interstitial water molecules play an important role in these adaptations of the pocket. The flip of the peptide bond is further stabilised by a glutamate residue that operates as general acid/base. An active-site aspartate residue, presumed to operate as a nucleophile through covalent bonding during the base-exchange reaction, shows different conformations depending on the nature of the bound ligand. The induced-fit adaptations observed in the various TGT complex structures by multiple crystal-structure analyses are in agreement with the functional properties of the enzyme. In consequence, full understanding of this plasticity can be exploited for drug design.     

重组人血白蛋白与小分子药物结合功能研究

目的研究重组人血白蛋白与小分子药物的结合功能。方法在366nm下检测地西泮与人血白蛋白结合前后吸收值的变化,计算二者的结合常数;华法令与人血白蛋白结合后,用SephadexG-25HPLC柱进行分离,对游离华法令和二者结合产物分别积分,得到华法令与人血白蛋白的结合率。结果重组人血白蛋白与地西泮的结合常数为14·68mol Diaze-pam/mol rHSA,天然人血白蛋白与地西泮结合常数为12·82mol Diazepam/mol HSA。重组人血白蛋白中华法令的结合率为8·69%,而天然人血白蛋白中华法令的结合率为6·73%。结论重组人血白蛋白与天然人血白蛋白小分子药物结合功能基本一致。     

CdTe/CdS量子点对木犀草素与人血清白蛋白结合力的影响

用还原型谷胱甘肽(GSH)作硫源和稳定剂在水相中合成了生物相容性好、量子产率高的核壳型CdTe/CdS半导体量子点;同时模拟人体内生理环境,研究了CdTe/CdS量子点(QDs)对木犀草素与人血清白蛋白(HSA)相互作用的影响。结果表明,CdTe/CdS QDs可有规律地猝灭HSA荧光;在CdTe/CdS QDs存在条件下,木犀草素可使HSA荧光最大发射波长从341 nm红移到347 nm,红移程度明显大于无QDs存在时,同时木犀草素与HSA的表观结合常数和结合位点数均有增加,即CdTe/CdS QDs使木犀草素与HSA的结合力增强。     

分子印迹聚合物结合点的选择性修饰

以槲皮素为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,制备了分子印迹聚合物(MIP),并以重氮甲烷为修饰剂,对MIP的结合点进行了选择性修饰,探讨了模板分子浓度、溶剂及重氮甲烷用量等对修饰效果的影响。结果表明,选择性修饰能明显提高MIP的识别能力与吸附速度,以柚皮素为竞争分子,分离因子可从修饰前的1.60提高到修饰后的3.06,吸附平衡时间从修饰前的100 min降低至修饰后的60 min;在一定范围内提高模板分子浓度,增大重氮甲烷用量,采用形成氢键能力较弱的溶剂,均能提高修饰效果。测定了修饰前后MIP中不同结合点吸附过程的焓变、熵变和自由能变化,从热力学角度对修饰效果和修饰机理作了进一步的分析。     

Molecular Interactions between Thiostrepton and the TipAS Protein from Streptomyces lividans

In Streptomyces lividans, the expression of several proteins is stimulated by the thiopeptide antibiotic thiostrepton. Two of these, TipAL and TipAS, autoregulate their expression after covalently binding to thiostrepton; this irreversibly sequesters the antibiotic and desensitizes the organism to its effects. In this work, additional molecular recognition interactions involved in this critical event were explored by utilizing various thiostrepton analogues and several site‐directed mutants of the TipAS antibiotic binding protein. Dissociation constants for several thiostrepton analogues ranged from 0.19 to 12.95 μM , depending on the analogue. The contributions of specific structural elements of the thiostrepton molecule to this interaction have been discerned, and an unusual covalent modification between the antibiotic and a new residue in a TipAS mutant has been detected.     

Composite Microfiltration Membranes Imprinted with cAMP

Composite microfiltration membranes covered with a thin layer of molecularly imprinted polymer (MIP) selective to adenosine 3′:5′‐cyclic monophosphate (cAMP) were obtained and their separation properties were studied. MIP layers were prepared using photoinitiated copolymerization of dimethylaminoethyl methacrylate (DMAEM) as a functional monomer and trimethylopropane trimethacrylate (TRIM) as a crosslinker in the presence of cAMP as template in ethanol/water mixture. Blank membranes were prepared under the same conditions, but without cAMP. It was found out that pH of aqueous solution of the template has an effect on the binding of cAMP with MIP membranes. It was concluded that the ability of MIP membranes to bind cAMP is a result of both ionic interactions between charged dimethylamino groups of polymer matrix and the phosphorous residue of cAMP molecule and the specific shape of recognizing sites. These sites are complementary to cAMP in terms of three dimensional shape as well as correct position of functional groups involved in the template binding. This paper shows that the binding capability of MIP membranes can be adjusted by varying the values of degree of modification (DM). Atomic Force Microscopy (AFM) and Scanning Force Microscopy (SEM) were used to visualise surfaces and cross sections of membranes to gain better understanding in the analysis of MIP layer deposited on membranes.     

Covalent Inhibition of HIV‐1 Integrase by N‐Succinimidyl Peptides

We present a new approach for the covalent inhibition of HIV‐1 integrase (IN) by an LEDGF/p75‐derived peptide modified with an N‐terminal succinimide group. The covalent inhibition is mediated by direct binding of the succinimide to the amine group of a lysine residue in IN. The peptide serves as a specific recognition sequence for the target protein, while the succinimide serves as the binding moiety. The combination of a readily synthesizable peptide precursor with easy and efficient binding to the target protein makes this approach a promising new strategy for designing lead compounds.     

Thrombin‐Inhibiting Anticoagulant Liposomes: Development and Characterization

Many peptides and peptidomimetic drugs suffer from rapid clearance in vivo; this can be reduced by increasing their size through oligomerization or covalent conjugation with polymers. As proof of principle, an alternative strategy for drug oligomerization is described, in which peptidomimetic thrombin inhibitors are incorporated into the liposome surface. For this purpose, the inhibitor moieties were covalently coupled to a palmitic acid residue through a short bifunctionalized ethylene glycol spacer. These molecules were directly added to the lipid mixture used for liposome preparation. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements and anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine‐derived inhibitors are located on the surface of the liposomes. This concept should be applicable to other drug molecules that suffer from rapid elimination and allow covalent modification with a suitable fatty acid residue.     

Interaction of human serum album and c(60) aggregates in solution

An important property of C(60) in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC(60). Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C(60) is substantially altered in this aggregate form. Herein, we investigated the interaction of nC(60) and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC(60) with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.     

Exploiting bioorthogonal chemistry to elucidate protein-lipid binding interactions and other biological roles of phospholipids

Lipids play critical roles in a litany of physiological and pathophysiological events, often through the regulation of protein function. These activities are generally difficult to characterize, however, because the membrane environment in which lipids operate is very complex. Moreover, lipids have a diverse range of biological functions, including the recruitment of proteins to membrane surfaces, actions as small-molecule ligands, and covalent protein modification through lipidation. Advancements in the development of bioorthogonal reactions have facilitated the study of lipid activities by providing the ability to selectively label probes bearing bioorthogonal tags within complex biological samples. In this Account, we discuss recent efforts to harness the beneficial properties of bioorthogonal labeling strategies in elucidating lipid function. Initially, we summarize strategies for the design and synthesis of lipid probes bearing bioorthogonal tags. This discussion includes issues to be considered when deciding where to incorporate the tag, particularly the presentation within a membrane environment. We then present examples of the application of these probes to the study of lipid activities, with a particular emphasis on the elucidation of protein-lipid binding interactions. One such application involves the development of lipid and membrane microarray analysis as a high-throughput platform for characterizing protein-binding interactions. Here we discuss separate strategies for binding analysis involving the immobilization of either whole liposomes or simplified isolated lipid structures. In addition, we present the different strategies that have been used to derivatize membrane surfaces via bioorthogonal reactions, either by using this chemistry to produce functionalized lipid scaffolds that can be incorporated into membranes or through direct modification of intact membrane surfaces. We then provide an overview of the development of lipid activity probes to label and identify proteins that bind to a particular lipid from complex biological samples. This process involves the strategy of activity-based proteomics, in which proteins are collectively labeled on the basis of function (in this case, ligand binding) rather than abundance. We summarize strategies for designing and applying lipid activity probes that allow for the selective labeling and characterization of protein targets. Additionally, we briefly comment on applications other than studying protein-lipid binding. These include the generation of new lipid structures with beneficial properties, labeling of tagged lipids in live cells for studies involving fluorescence imaging, elucidation of covalent protein lipidation, and identification of biosynthetic lipid intermediates. These applications illustrate the early phase of the promising field of applying bioorthogonal chemistry to the study of lipid function.     

Structural Analysis of Human Serum Albumin in Complex with the Fibrate Drug Gemfibrozil

Gemfibrozil (GEM) is an orally administered lipid-regulating fibrate derivative drug sold under the brand name Lopid^®, among others. Since its approval in the early 80s, GEM has been largely applied to treat hypertriglyceridemia and other disorders of lipid metabolism. Though generally well tolerated, GEM can alter the distribution and the free, active concentration of some co-administered drugs, leading to adverse effects. Most of them appear to be related to the ability of GEM to bind with high affinity human serum albumin (HSA), the major drug-carrier protein in blood plasma. Here, we report the crystal structure of HSA in complex with GEM. Two binding sites have been identified, namely Sudlow’s binding sites I (FA7) and II (FA3–FA4). A comparison of the crystal structure of HSA in complex with GEM with those of other previously described HSA–drug complexes enabled us to appreciate the analogies and differences in their respective binding modes. The elucidation of the molecular interaction between GEM and HSA might offer the basis for the development of novel GEM derivatives that can be safely and synergistically co-administered with other drugs, enabling augmented therapeutic efficacies.     

Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (K_a = approximately 10⁴ M⁻¹). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (K_EB = approximately 10⁴ M⁻¹). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.     

Discovery of a Cell-Active SuTEx Ligand of Prostaglandin Reductase 2

Sulfonyl-triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment-based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration-dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure-activity relationship studies identified the SuTEx ligand HHS-0701 as a cell-active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.