Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca²⁺ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.     

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide

The $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rabbit sarcoplasmic reticulum, when labelled at two Ca²⁺-protected sites with $\text{N-}\text{cyclohexyl}\text{-N′-(4-}\text{dimethylamino}\text{-α-}\text{naphthyl}\text{)}\text{carbodiimide}$ (NCD-4) retains Ca²⁺ binding capacity at the sites with $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca²⁺ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca²⁺. It retains the ability to form phosphoenzyme, and can most likely translocate Ca²⁺.     

Fast-HMQC using Ernst angle pulses: An efficient tool for screening of ligand binding to target proteins

The application of Ernst angle pulses in multidimensional NMR spectroscopy is theoretically and experimentally investigated. Theory shows that only for a few pulse sequences employed at high repetition rate, a remarkable gain in sensitivity is possible using Ernst angle pulses. As an example, a new variant of the heteronuclear multiple quantum coherence (HMQC) experiment, the fast-(¹H,¹⁵N)-HMQC, is described. This sequence allows, with a 1 mM protein sample in H₂O, the acquisition of a highly resolved two-dimensional (¹H,¹⁵N) correlated spectrum within 37 s. The high efficiency of the fast-HMQC to detect ligand binding to a target protein is demonstrated.     

Large-ligand adsorption to membranes: II. Disk-like ligands and shape-dependence at low saturation

Large-ligand adsorption to membranes or cells is considered in the absence of cooperative interactions. For the low-saturation regime, a general and exact treatment is given by means of the concept of excluded areas. With the help of this formalism, shape dependence of the adsorption behavior can be discussed quantitatively. In addition, a formalism is presented which allows to calculate binding curves at arbitrary saturation for ligands having a symmetric shape (disks, regular polygons). The underlying model is a modified version of the hard core fluid theory of Andrews (Andrews, F.C. (1976) J. Chem. Phys. 64, 1941–1947). Apart from applications to symmetric ligands, the results can be used to derive limiting conditions for ligands of any shape.     

On an erroneous view of fluxional behaviour of [Fe3(CO)12]

The idea that the mechanism of the very low energy fluxional behaviour of [Fe₃(CO)₁₂] may be based on the movement of the ligand icosahedron about the central Fe₃ triangle which corresponds to a rotational symmetry operation of the CO ligand icosahedron is discussed and rejected.     

An increase in side-group hydrophobicity largely improves the potency of ritonavir-like inhibitors of CYP3A4

Identification of structural determinants required for potent inhibition of drug-metabolizing cytochrome P450 3A4 (CYP3A4) could help develop safer drugs and more effective pharmacoenhancers. We utilize a rational inhibitor design to decipher structure-activity relationships in analogues of ritonavir, a highly potent CYP3A4 inhibitor marketed as pharmacoenhancer. Analysis of compounds with the R₁ side-group as phenyl or naphthalene and R₂ as indole or naphthalene in different stereo configuration showed that (i) analogues with the R₂-naphthalene tend to bind tighter and inhibit CYP3A4 more potently than the R₂-phenyl/indole containing counterparts; (ii) stereochemistry becomes a more important contributing factor, as the bulky side-groups limit the ability to optimize protein-ligand interactions; (iii) the relationship between the R₁/R₂ configuration and preferential binding to CYP3A4 is complex and depends on the side-group functionality/interplay and backbone spacing; and (iv) three inhibitors, 5a-b and 7d, were superior to ritonavir (IC₅₀ of 0.055–0.085 μM vs. 0.130 μM, respectively).     

Ligand based design and synthesis of pyrazole based derivatives as selective COX-2 inhibitors

The design and synthesis of novel pyrazole based derivatives has been carried out using the ligand based approach like pharmacophore and QSAR modelling of reported pyrazoles from the available literature to investigate the chemical features that are essential for the design of selective and potent COX-2 inhibitors. Both pharmacophore and QSAR models with good statistical parameters were selected for the design of the lead molecule. Also by exploiting the chemical structures of selective and marketed COX-2 inhibitors, celecoxib and SC-558 were used in designing the molecules which are used in the treatment of inflammation and related disorders. The therapeutic action of the Non-Steroidal Anti-inflammatory Agents (NSAIDs) is based primarily on the COX-2 inhibition. With this background we have synthesized some azomethine derivatives of 3-methyl-1-substituted-4-phenyl-6-[{(1E)-phenylmethylene}amino]-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile 6(a-o) and were characterized by ¹HNMR, ¹³CNMR and Mass spectral techniques. All the synthesized pyrazole derivatives were tested for in vitro membrane stability property in both COX-1 & COX-2 inhibition studies and in vivo anti-inflammatory activity by carrageenan induced rat paw edema model. Among them, compound 6k showed very good activity by in vivo anti-inflammatory activity with 0.8575 mmol/kg as ED₅₀. Similarly compounds 6m, 6o, 6i and 6h exhibited comparable anti-inflammatory activity to standard drugs. Also the active compounds were further screened for ulcerogenic activity and were found be safer with less ulcer index compared to the marketed drugs like aspirin, ibuprofen and celecoxib.     

Evaluation of the therapeutic potential effect of Fas receptor gene knockdown in experimental model of non-alcoholic steatohepatitis

Aim: Stimulation of Fas death receptor is introduced as a major cause of non-alcoholic steatohepatitis (NASH) progression through suppression of cell viability. Therefore, the blocking of death pathways is hypothesised to be express new approaches to NASH therapy. For this purpose, current experiment applied synthetic small interference RNA (SiRNA) to trigger Fas death receptor and to show its potential therapeutic role in designed NASH model.

Methods: Male mice were placed on a western diet (WD) for 8 weeks and exposed to cigarette smoke during the last 4 weeks of feeding to induce NASH model. In the next step, Fas SiRNA was injected to mice aiming to examine specific Fas gene silencing, after 8 weeks. As a control, mice received scrambled SiRNA. Reversible possibility of disease was examined by 3 weeks of recovery.

Results: Analysis of data is accompanied with the significant histopathological changes (steatosis, ballooning and inflammation), increased lipid profile and hepatic enzyme activities (AST, ALT, ALP) plus TBARS as well as decreased antioxidants levels in NASH model. Upon Fas-SiRNA injection, almost all measured parameters of NASH such as overexpression of Fas receptor, caspase3, NF-kB genes and marked increase of hepatic TNF-α were significantly restored and were remained nearly unchanged following recovery liking as scrambled groups.

Conclusions: The suppression of Fas receptor signalling subsequent RNAi therapy may represent an applicable strategy to decline hepatocyte damages and so NASH progression in mice.     

NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance

Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity.     

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.