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Three integrase strand transfer inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG), have been approved by the FDA. Resistance against these three INSTIs have been reported and cross-resistance among them has been documented. Due to extensive and dynamic genetic diversity in different HIV-1 variants, significant differences in susceptibility to the INSTIs have been observed among HIV subtypes. This review summarizes what is known about this topic and discusses possible clinical implications.

An MS guided fractionation of the alkaloidal constituents of Flueggea virosa has yielded nine new Securinega alkaloid oligomers, fluevirosinines B–J ( 1 – 9 ). The absolute structures of these oligomers have been characterized on the basis of spectroscopic data and biogenetic consideration. Fluevirosinines B–E ( 1 – 4 ) are four tetramers with new connecting sequences or new oligomerizing patterns, and fluevirosinines G–J ( 6 – 9 ) represent the first examples of pentamers of the Securinega alkaloid family. Among these alkaloids, fluevirosinine B ( 1 ) showed the best anti-HIV activity with an EC 50 value of 14.1±1.2 μM.

Integrase strand transfer inhibitors (INSTIs) are the latest class of potent anti-HIV drugs. Currently, three INSTIs have been approved by the US Food and Drug Administration: raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). Resistance mutations to RAL and EVG emerge rapidly, and significant cross-resistance between these compounds has been documented. In addition, limited cross-resistance has been observed among DTG, a newer INSTI, and RAL and EVG even though clinical resistance to DTG, or mutations associated with DTG resistance in treatment-naïve patients, has not yet been observed. This review summarises progress in studies on understanding resistance to DTG, mechanisms of possible resistance to DTG, and reasons for the absence of DTG-associated resistance mutations when the drug has been used in first-line therapy.

Three new Securinega alkaloids, flueggethers B and C (1 and 2) as dimers and flueggether D (3) as a trimer, were isolated from the twigs and leaves of a Chinese herb, Flueggea virosa. Their structures were characterized by spectroscopic means and NMR comparison with known analogues. Among all Securinega alkaloid oligomers reported to date, alkaloids 1 and 2 are rare examples linked via an ether bond, while 3 represents the first member with a mixed oligomerizing pattern incorporating both oxygen bridge and carbon–carbon linkage. An in vitro anti-HIV bioassay revealed moderate activity for 3.

Human immunodeficiency virus integrase (HIV-1 IN) inhibitors that are currently approved or are in advanced clinical trials specifically target the strand transfer step of integration. However, considerable cross-resistance exists among some members of this class of IN inhibitors. Intriguingly, though, HIV-1 IN possesses multiple sites, distinct from those involved in the strand transfer step, that could be targeted to develop new HIV-1 IN inhibitors. We have developed a fluorescent HIV-1 IN DNA binding assay that can identify small molecules termed IN binding inhibitors (INBIs) that inhibit IN binding to viral DNA. This assay has been optimized with respect to concentrations of each protein, long terminal repeat (LTR) DNA substrate, salt, and time, and has been used successfully to measure the HIV-1 IN DNA binding activity of a well-characterized INBI termed FZ41. In addition, we have used the assay to screen a small library of natural products, resulting in the identification of nigranoic acid as a new INBI. The proposed fluorescence assay is easy and inexpensive, and provides a high-throughput detection method for determination of HIV-1 IN DNA binding activity, monitoring of enzyme kinetics, and high-throughput screening for the identification of new INBIs. (C) 2013 Elsevier B.V. All rights reserved.

Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth, primarily via regulation of protein synthesis. In Saccharomyces cerevisiae, TOR can also suppress the transcription of stress response genes by a mechanism involving Tap42, a serine/threonine phosphatase subunit, and the transcription factor Msn2. A physical association between mammalian TOR (mTOR) and the transcription factor signal transducer and activator of transcription-1 (STAT1) was recently identified in human cells, suggesting a similar role for mTOR in the transcription of interferon-gamma-stimulated genes. In the current study, we identified a macromolecular protein complex composed of mTOR, STAT1, the Tap42 homologue alpha4, and the protein phosphatase 2A catalytic subunit (PP2Ac). Inactivation of mTOR enhanced its association with STAT1 and increased STAT1 nuclear content in PP2Ac-dependent fashion. Depletion of alpha4, PP2A, or mTOR enhanced the induction of early (i.e. IRF-1) and late (i.e. caspase-1, hiNOS, and Fas) STAT1-dependent genes. The regulation of IRF-1 or caspase-1 by mTOR was independent of other known mTOR effectors p70 S6 kinase and Akt. These results describe a new role for mTOR and alpha4/PP2A in the control of STAT1 nuclear content, and the expression of interferon-gamma-sensitive genes involved in immunity and apoptosis.

Suspension cultures of Sussurea medusa produce jaceosidin and hispidulin. In an attempt to increase productivity, silver nitrate and glutathione (GSH) elicitors were tested. Both dosage and addition time as well as time course studies were carried out on the effects of the elicitors on the formation of the metabolites. Silver nitrate and glutathione elicitors increased the production of jaceosidin and hispidulin (from ca. 32.01 to 51.25 mg l −1 and 3.11 to 5.13 mg l −1 ) at optimum dosage and addition time, respectively. Silver nitrate and glutathione in a combination were added at concentrations of 0.01 and 1 mmol l −1 on days 0 and 5 to cultures of S. medusa , jaceosidin and hispidulin production reached 84.3 and 7.9 mg l −1 , respectively, which were 2.6 and 2.5 times higher than that in controls and was higher than for each elicitor alone. It was also found that glutathione enhanced phenylalanine ammonialyase (PAL) activity while silver nitrate did not.

In higher plants, there are two pathways leading to the formation of isoprenoids: the mevalonate pathway and the recently discovered methyl- d -erythritol 4-phosphate (MEP) pathway. It was shown that the isoprenoid moiety, constituting ring C of anthraquinones (AQs) in the plant family Rubiaceae, is formed from the MEP pathway. 1-Deoxy- d -xylulose 5-phosphate synthase (DXS), catalyzing the first step of the MEP pathway, is suggested to be a regulatory enzyme. In order to evaluate the potential regulatory role of the DXS in AQ biosynthesis, we identified a full-length cDNA encoding DXS from Morinda citrifolia, a member of the Rubiaceae family, by a PCR strategy. The cDNA encodes a protein of 722 amino acids with a calculated molecular mass of 78 kDa. The deduced amino acid sequence shares high homology with amino acid sequences of DXSs from other higher plants. Southern blot indicated the existence of a small family of DXS genes in M. citrifolia. Northern blot analysis showed that the DXS transcript levels were correlated with AQ accumulation, suggesting that AQ biosynthesis is regulated at the transcriptional level of the DXS gene.

A high-performance liquid chromatography assay for activity of 1-deoxy- d -xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl- d -erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy- d -xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN–water–1-butylamine–tetrahydrofuran–H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy- d -xylulose. This assay was successfully applied to measure the 1-deoxy- d -xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present.