HIV-1整合酶核心区可溶性表达及抑制剂筛选

为了实现HIV-1整合酶蛋白核心区 (central core domain of integrase, IN-CCD) 的可溶性表达,并建立以IN-CCD为靶点的抑制剂体外筛选方法,从包含F185K突变HIV-1 IN基因的质粒中经PCR扩增得到含有F185K突变的IN-CCD基因,克隆到pET28b载体上构建重组质粒pIN-CCD,转化pIN-CCD至E. coli BL21 (DE3)中经IPTG诱导、表达,Ni-亲和层析纯化,获得IN-CCD蛋白。修饰DNA底物,以链亲和素包被的磁珠为载体捕获DNA产物,结合酶联免疫吸附测定法(ELISA)检测IN-CCD的去整合活性,并筛选以IN-CCD为靶点的抑制剂。结果表明重组蛋白IN-CCD实现了高效可溶性表达,纯化后蛋白纯度达95%。建立的ELISA可以检测IN-CCD的去整合活性,且方法特异性和灵敏度好,可以实现高通量抑制剂筛选。从100个样品中筛选得到5个具有初步抑制IN-CCD去整合活性的样品。     

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn²⁺ binds tightly to the sulfur atoms of the cysteine residues, but Mg²⁺ does not. We found that Mn²⁺, but not Mg²⁺, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn²⁺ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.     

Minimal Core Domain of HIV-1 Integrase for Biological Activity

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.     

Monitoring of polymorphonuclear leukocyte functions in diabetes mellitus— a comparative study of conventional radiometric function tests and low-light imaging systems

In this study neutrophil (PMN) phagocytic capacity was investigated using a conventional radiometric ingestion assay (IN) in comparison with PMN respiratory burst activity assessed by luminol-enhanced chemiluminescence (LCL) in response to phorbolesters and LCL induction during phagocytosis of opsonized Staphylococous aureus (STLCL) in diabetes mellitus and healthy controls. PMN ingestion was measured with ³H-thymidine-labelled S. aureus in a kinetic radiometric assay. LCL and STLCL were assessed in a parallel detecting microtitre-plate luminometer (MTP-Reader). PMN of diabetic subjects showed a highly significant reduction of peak LCL in response to PMA as well as during phagocytosis of S. aureus (STLCL) compared to non-diabetic controls (p<0.001 respectively). PMN ingestion in diabetic patients (51.8±4.6%) was significantly reduced compared to controls (78.3±6.2%) (p<0.01). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations ? 13.8mmol/L, whereas PMN IN was significantly reduced at glucose levels ?27.75mmol/L. The control group showed a positive correlation of peak LCL response and IN (p<0.05) but not of STCL and IN; in diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN respiratory burst activity and markedly reduced phagocytic PMN functions in diabetic patients ex vivo and in vitro as measured by LCL and by ingestion of ³H-thymidine-labelled S. aureus suggesting inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning altered host defence.     

The form of the DNA substrate required for excisive recombination of bacteriophage lambda.

We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt²(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt²(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA.In vitro, starting with a hydrogen-bonded λatt² DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt² DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg²⁺ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.     

Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase

HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D₆₄, D₁₁₆ and E₁₅₂ of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V₁₅₁E₁₅₂S₁₅₃ region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3′-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V₁₅₁E₁₅₂S₁₅₃ region involvement in the integration reaction is more important than for the 3′-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.     

Effect of Docosahexaenoic Acid and Ascorbate on Peroxidation of Retinal Membranes of ODS Rats

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 ± 1.6 min compared to 49 ± 2.1 min).     

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg²⁺ cofactor. The DNA-binding was essentially non-cooperative with Mn²⁺ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg²⁺-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC₅₀ = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn²⁺-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg²⁺ cofactor and the zinc effector.Integration of HIV-1 DNA into the host genome ensures stable maintenance of the viral genome in the host organism and, therefore, is a key process in the virus life cycle. Integrase (IN) is responsible for two distinct, consecutive catalytic steps in the integration process (1). The first of these two reactions is 3′-processing, which corresponds to the specific cleavage of two nucleotides from the 3′-ends of the linear viral DNA. The hydroxyl groups of newly recessed 3′-ends are then used in the second reaction— strand transfer—for the covalent joining of viral and cellular (or target) DNAs, resulting in full-site integration. For both reactions, IN functions as a multimer, most likely a dimer for 3′-processing and a tetramer (dimer of a dimer) for concerted integration (2–7). Two other reactions occur in vitro, a disintegration reaction that represents, in first approximation, the reversal of the half-site integration process (8) and a specific internal cleavage occurring on a symmetrical DNA site (9). All reactions require a metallic cofactor, Mg²⁺ or Mn²⁺, and, except for disintegration (10,11), all reactions require the full-length IN. There are several experimental evidences to suggest that Mg²⁺ is more physiologically relevant as a cofactor, particularly because Mg²⁺-dependent catalysis exhibits weaker non-specific endonucleolytic cleavage and the tolerance of sequence variation at the ends of the viral DNA is much greater in the presence of Mn²⁺ than in the presence of Mg²⁺ (12–15).The emergence of viral strains resistant against available drugs and the dynamic nature of the HIV-1 genome support a continued effort towards the discovery and characterization of novel targets and anti-viral drugs. Due to its central role in the HIV-1 life cycle, IN represents a promising therapeutic target. In the past, in vitro IN assays were extensively used to find IN inhibitors (16). Current inhibitors can be separated into two main classes, depending on their mechanisms of action: (i) Compounds that competitively prevent the DNA binding of IN to the viral DNA. These compounds are mainly directed against the 3′-processing reaction as they bind to the donor site within the catalytic site—i.e. the ‘specific’ DNA-binding site for the viral DNA -. This group is referred to as ‘integrase DNA-binding inhibitors’ (INBI) and includes styrylquinoline compounds (17,18). (ii) The second class includes compounds that cannot bind to the DNA-free IN. They bind to the pre-formed IN–viral DNA complex. These compounds preferentially inhibit strand transfer over the 3′-processing reaction [this family of compounds is referred to as ‘integrase strand transfer inhibitors’ (INSTI)], probably by displacing the viral DNA end from the active site (7,19–21). It is not clear whether this mechanism alone accounts for the inhibitory properties of INSTIs or whether these compounds also prevent the binding of the target DNA to the acceptor site—i.e. the ‘non-specific’ DNA binding site. INSTI compounds have generally good ex vivo activity against HIV replication, probably due to their ability to inhibit pre-assembled viral DNA/IN complexes. Raltegravir which is currently used in clinical treatment of HIV-1 belongs to this class. For both anti-IN classes, resistance mutations were identified (17,20,22,23). Difficulties in deeply understanding their mechanisms of action are closely related to the absence of structural data that clearly delineate the donor and the acceptor DNA binding sites in the active site. Although structural information is now available regarding the IN–viral DNA interaction, based on the recent crystal structure of the full-length primate foamy virus (PFV-1) IN in complex with its cognate processed viral DNA, the target DNA binding mode and the precise location of the acceptor site remains open to debate (7). Moreover, it is a difficult task to experimentally discriminate between the two DNA binding sites and no significant or only modest difference can be evidenced in vitro (depending on the method used for monitoring IN–DNA interactions) between the HIV-1 IN binding to the cognate viral DNA sequence and a non-specific random sequence in terms of overall affinity, suggesting that the specific and the non-specific DNA-binding modes display similar binding free energies (5,24). The basis of DNA binding specificity remains essentially unknown.HIV-1 IN (288 amino acids) contains three functional domains. The central domain or catalytic core domain (IN^50–213 or CC) contains the catalytic triad (DDE) that coordinates one or two metallic cofactors [probably a pair coordinated by three carboxylate groups of the triad, based on the X-ray structure of the PFV-1 IN (7)] and is essential for enzymatic activity; this domain alone can perform the disintegration reaction (10,11). This domain is flanked by the N-terminal (IN^1-49) and the C-terminal (IN^214–288) domains. The C-terminal domain is involved in IN–DNA contacts, together with the CC domain (25,26). The N-terminal domain contains a conserved non-conventional HHCC motif that binds zinc to ensure proper domain folding and promotes IN multimerization (27–29). It is worth noting that the integrity of the HHCC motif is crucial in the stringent Mg²⁺-context but appears dispensable under the less stringent Mn²⁺ condition (30), suggesting, at least, an indirect role of the zinc-binding domain in the establishment of specific and physiologically relevant IN–DNA complexes. In the structure of the PFV-1 IN–viral DNA complex, the N-terminal domain is also involved in the interaction with DNA (7).In this article, we found that IN binds cooperatively to the cognate viral DNA sequence only in the presence of Mg²⁺. The presence of Mn²⁺ or, most importantly, the use of non-specific random sequences, regardless of the metallic cofactor, dramatically reduced the Hill coefficient. This finding suggests that the cooperative DNA-binding mode of IN is strongly related to the formation of specific IN–DNA contacts. To gain deeper insight into the role of the zinc-binding domain in the cooperative/multimerization process, in relationship with the establishment of specific protein–DNA contacts, we studied the effect of DIBA-1 (2,2′-dithiobisbenzamide-1) (Figure 1A) on IN activity. This compound is a zinc ejector affecting many proteins containing zinc fingers, including HIV-1 nucleocapsid or estrogen receptor (31–34). Here, we found that DIBA-1 induced zinc ejection from the IN N-terminal domain by covalently targeting the HHCC motif. In the presence of Mg²⁺, DIBA-1 did not affect significantly the overall affinity of IN for the DNA substrate but dramatically reduced the Hill coefficient. Concomitantly, DIBA-1 strongly inhibited the catalytic step, with IC₅₀ values against the 3′-processing reaction of 11–15 nM. Interestingly, we found a secondary DIBA-1 binding site in the catalytic core (involving residue Cys 56), suggesting a second mechanism of action of DIBA-1, independent of zinc ejection. The prevalence of the two distinct mechanisms was dependent on the cofactor context, with the second one accounting for the weaker DIBA-1 inhibitory effect under Mn²⁺ conditions (IC₅₀ = 115–126 nM). DIBA-1 behaves as a non-competitive/catalytic inhibitor that did not disturb the fractional saturation of DNA sites, regardless of the mechanism considered. Open in a separate windowFigure 1.DIBA-1 induces the ejection of zinc from IN. (A) Structure of 2,2′-dithiobisbenzamide-1 (DIBA-1) (MW, 614 Da). (B) Ejection of zinc was measured by optical absorbance at 495 nm using a sample containing full-length IN (1 µM) and PAR (10⁻⁴ M) in a Tris buffer (20 mM pH 7.0) containing 15% DMSO (v/v) in the absence of reducing agent (black squares) or in the presence of 4 mM β-mercaptoethanol (white circles). The concentration of DIBA-1 for complete zinc release—[DIBA-1]_eq—was estimated graphically. This value was determined as a function of the initial concentration of IN (C). The slope of the straight line indicates that the zinc ejection coincides with the reaction of two DIBA-1 molecules per IN protomer.Altogether, our results show that, although it is a difficult task to discriminate between the specific viral sequence and a non-specific random sequence in terms of overall affinity, these sequences lead to distinct DNA-binding properties in terms of cooperativity. Moreover, our results highlight that the Mg²⁺-dependent catalytic activity of IN is strongly sensitive to the loss of cooperative DNA binding. Such a cooperative DNA-binding mode accounts for specific activity and requires: (i) the cognate viral DNA sequence, (ii) Mg²⁺ as a catalytic cofactor and (iii) zinc which can be considered as a positive allosteric effector. Development of non-competitive compounds acting on the N-terminal domain may be of interest for anti-IN pharmacology.     

Clinical Use of Aromatase Inhibitors: Current Data and Future Perspectives

Abstract

A systematic procedure for the kinetic study of irreversible inhibition when the enzyme is consumed in the reaction which it catalyses, has been developed and analysed. Whereas in most reactions the enzymes are regenerated after each catalytic event and serve as reusable transacting effectors, in the consumed enzymes each catalytic center participates only once and there is no enzyme turnover. A systematic kinetic analysis of irreversible inhibition of these enzyme reactions is presented. Based on the algebraic criteria proposed in this work, it should be possible to evaluate either the mechanism of inhibition (complexing or non-complexing), or the type of inhibition (competitive, non-competitive, uncompetitive, mixed non-competitive). In addition, all kinetic constants involved in each case could be calculated. An experimental application of this analysis is also presented, concerning peptide bond formation in vitro. Using the puromycin reaction, which is a model reaction for the study of peptide bond formation in vitro and which follows the same kinetic law as the enzymes under study, we have found that: (i) the antibiotic spiramycin inhibits the puromycin reaction as a competitive irreversible inhibitor in a one step mechanism with an association rate constant equal to 1.3 × 10⁴M^-1s^-1 and, (ii) hydroxylamine inhibits the same reaction as an irreversible non-competitive inhibitor also in a one step mechanism with a rate constant equal to 1.6 × 10^-3 M^-1s^-1.     

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)⁺-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO⁻ aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd²⁺, Co²⁺, Hg²⁺, Mg²⁺, Pb²⁺ and Zn²⁺ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD⁺-GDH activity was negligible. Among the applied ions, only Ca²⁺ activated NADH-GDH.     

Comparative effects of different cellulosic-based directly compressed orodispersable tablets on oral bioavailability of famotidine

Famotidine is a potent H₂-receptor antagonist most commonly used by elderly patients. Orodispersible tablets (ODT) are gaining popularity over conventional tablets due to their convenience and suitability for patients having dysphagia. The purpose of this study is to prepare famotidine ODT using the economic direct-compression method.A 3² full factorial design was used to evaluate the influence of different excipients on the properties and in vitro dissolution of famotidine ODT. Two factors were studied for their qualitative effects, namely, disintegrants and diluents. Disintegrants were studied in three levels viz. Ac-Di-Sol, sodium starch glycolate (Primojel) and low-substituted hydroxypropyl cellulose (L-HPC). Fillers were studied in three levels viz. mannitol, spray dried lactose and Avicel PH 101. The ODTs were prepared by direct compression and were evaluated for hardness, drug content, uniformity of weight, in vitro disintegration time, oral disintegration time, wetting time and in vitro dissolution. Maximum dissolution and minimum oral disintegration time (11.4 s) were observed in F7 prepared using L-HPC and mannitol. Furthermore, in human volunteers it showed significant increase in bioavailability compared to Servipep^® with mean AUC_(0–∞) 117.1 ng/ml and 82.71 ng/ml, respectively, and its relative bioavailability was 141.57%. Hence, ODT (F7) could possibly be used to overcome the drawbacks of conventional famotidine tablets in elderly patients with significant increase in oral bioavailability.     

Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA,Inhibition of Telomerase Activity and Cellular Uptake

Two ruthenium(II) complexes, Λ-[Ru(phen)₂(p-HPIP)]²⁺ and Δ-[Ru(phen)₂(p-HPIP)]²⁺, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)₂(p-HPIP)]²⁺ against telomerase, suggesting that Λ-[Ru(phen)₂(p-HPIP)]²⁺ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)₂(p-HPIP)]²⁺ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.     

Involvement of calcium in the in vitro sensitivity change of the plasma membrane H+ ATPase to indole-3-acetic acid

The proton pumping activity of phase-partitioning purified plasma membrane fraction from spinach leaves was tested in vitro in the presence of exogenous indole-3-acetic acid. The sensitivity of the H+ pumping activity to the auxin was changed after flowering induction. We investigated the effect of whole spinach leaf treatments with substances affecting the phosphatidylinositol diphosphate transduction pathway on the in vitro sensitivity modification by photoperiodic induction. A role of calcium ions was supported by studies on leaves treated with a specific Ca²⁺ chelator (EGTA), a synthetic Ca²⁺ ionophore (A23187) or with calcium channel blokers (verapamil, lanthan chloride). An experiment using the transduction pathway inhibitor, lithium chloride, indicated that the intracellular concentration of Ca²⁺ was increased by inositol triphosphate.     

Raltegravir flexibility and its impact on recognition by the HIV‐1 IN targets

HIV‐1 IN is a pertinent target for the development of AIDS chemotherapy. The first IN‐specific inhibitor approved for the treatment of HIV/AIDS, RAL, was designed to block the ST reaction. We characterized the structural and conformational features of RAL and its recognition by putative HIV‐1 targets – the unbound IN, the vDNA, and the IN?vDNA complex – mimicking the IN states over the integration process. RAL binding to the targets was studied by performing an extensive sampling of the inhibitor conformational landscape and by using four different docking algorithms: Glide, Autodock, VINA, and SurFlex. The obtained data evidenced that: (i) a large binding pocket delineated by the active site and an extended loop in the unbound IN accommodates RAL in distinct conformational states all lacking specific interactions with the target; (ii) a well‐defined cavity formed by the active site, the vDNA, and the shortened loop in the IN?vDNA complex provide a more optimized inhibitor binding site in which RAL chelates Mg²⁺ cations; (iii) a specific recognition between RAL and the unpaired cytosine of the processed DNA is governed by a pair of strong H‐bonds similar to those observed in DNA base pair G‐C. The identified RAL pose at the cleaved vDNA shed light on a putative step of RAL inhibition mechanism. This modeling study indicates that the inhibition process may include as a first step RAL recognition by the processed vDNA bound to a transient intermediate IN state, and thus provides a potentially promising route to the design of IN inhibitors with improved affinity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Fluorimetric Analysis of Phospholipase Activity in Tetrahymena pyriformis GL.

The unicellular Tetrahymena enzymatically split the synthetic phosphodiester, 4-methylum-belliferyl phosphocoline substrate. The enzyme activity was completely blocked in vitro and drastically inhibited in vivo by G-protein activating fluorides (NaF; AlF₄ ^– and BeF₃ ^–). The phospholipase A₂ inhibitor, quinacrine, and the protein phosphatase inhibitor, neomycin, inhibited the enzyme activity in vitro and activated it in vivo. Another phospholipase A₂ inhibitor 4-bromo phenacyl bromide was ineffective in vivo and in vitro alike, as well as the cyclooxygenase inhibitor indomethacin. Results of these experiments indicate that some treatments could be specific for a well defined activity (e.g., phospholipase A₂, G-protein) but subject to influence by other enzymes (e.g., phospholipase C, sphingomyelinase). The experiments call attention to the differences in the results of the in vivo and in vitro studies.     

Antioxidant Properties of 2-O-β-D-Glucopyranosyl-L-ascorbic Acid

The antioxidant activity of a provitamin C agent, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was compared to that of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^?+)-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2βG slowly and continuously scavenged DPPH radicals and ABTS^?+ in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2βG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2βG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2βG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2βG appeared to be comparable not only to that of AA-2G but also to that of AA.     

Reversal of antizyme-induced inhibition of ornithine decarboxylase by cations in barley seedlings

Ornithine decarboxylase (ODC) forms a stable complex with its antizyme (Az), a non-competitive protein inhibitor of ODC. The complex formation of ODC with Az occurs very rapidly and is dissociated by high salt concentrations e.g., 10% ammonium sulfate. When ODC and Az were mixed in the presence of increasing concentrations of Mg²⁺, a relief of ODC inhibition by Az was obtained. Complete relief of inhibition occurred at 2.0 mM of MgCl₂. Other bivalent cations Ca²⁺, Ba²⁺, Co²⁺, Mn²⁺, Zn²⁺ as well as the monocations Na⁺ and K⁺ caused similar effect. The polyamines putrescine, spermidine and spermine also caused relief of the in vitro inhibition of ODC by Az. Therefore, the in vivo inactivation of ODC by forming the ODC-Az complex is dependent on the intracellular amounts of salt and polyamines.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.