PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.

The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched 'on' or 'off' in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.

Human Group IIA Phospholipase A2-Three Decades on from Its Discovery.

Phospholipase A2 (PLA2) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA2) enzymes were the first of the five major classes of human PLA2s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA2, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.

Structural and Functional Aspects of Targeting the Secreted Human Group IIA Phospholipase A2.

Human group IIA secretory phospholipase A2 (hGIIA) promotes the proliferation of cancer cells, making it a compelling therapeutic target, but it is also significant in other inflammatory conditions. Consequently, suitable inhibitors of hGIIA have always been sought. The activation of phospholipases A2 and the catalysis of glycerophospholipid substrates generally leads to the release of fatty acids such as arachidonic acid (AA) and lysophospholipid, which are then converted to mediator compounds, including prostaglandins, leukotrienes, and the platelet-activating factor. However, this ability of hGIIA to provide AA is not a complete explanation of its biological role in inflammation, as it has now been shown that it also exerts proinflammatory effects by a catalysis-independent mechanism. This mechanism is likely to be highly dependent on key specific molecular interactions, and the full mechanistic descriptions of this remain elusive. The current candidates for the protein partners that may mediate this catalysis-independent mechanism are also introduced in this review. A key discovery has been that selective inhibition of the catalysis-independent activity of hGIIA is achieved with cyclised derivatives of a pentapeptide, FLSYK, derived from the primary sequence of hGIIA. The effects of hGIIA on cell function appear to vary depending on the pathology studied, and so its mechanism of action is complex and context-dependent. This review is comprehensive and covers the most recent developments in the understanding of the many facets of hGIIA function and inhibition and the insight they provide into their clinical application for disease treatment. A cyclic analogue of FLSYK, c2, the most potent analogue known, has now been taken into clinical trials targeting advanced prostate cancer.

Demonstration of In Vitro Host-Guest Complex Formation and Safety of para-Sulfonatocalix[8]arene as a Delivery Vehicle for Two Antibiotic Drugs.

The macrocycle para-sulfonatocalix[8]arene, sCX[8], was examined with 2 antibiotic drugs, ciprofloxacin (CIP) and isoniazid. The drugs were shown to form complexes with sCX[8] using proton nuclear magnetic resonance, thermogravimetric analysis, fluorescence spectroscopy, and molecular modeling. Both drugs form 1:1 hydrated (H2O: 13%-14% w/w) host-guest complexes, with sCX[8] binding around the pyridine ring of isoniazid, and around the piperazine and cyclopropane rings of CIP. From proton nuclear magnetic resonance, the binding constant of isoniazid to sCX[8] was 6.8 (±0.3) × 103 M-1. Addition of 2 equivalents of sCX[8] to CIP resulted in a 58% decrease in fluorescence, and time-resolved fluorescence anisotropy of CIP doubles with sCX[8]. Each drug binds into the cavity of the macrocycle, with binding stabilized via combinations of hydrogen bonding, electrostatic interactions, π-π stacking, and hydrophobic effects. The safety of sCX[8] was examined in vitro with human embryonic kidney 293 cells. The IC50 of sCX[8] was 559 µM, which is a minimum of 5-fold higher than the concentration that would be used in the clinic. The in vitro effect of sCX[8] on the action of CIP was examined on a panel of bacterial lines. The results showed that sCX[8] has no inherent antibiotic activity and had no negative effect on the action of CIP.

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2.

Human Group IIA phospholipase A2 (hGIIA) promotes inflammation in immune-mediated pathologies by regulating the arachidonic acid pathway through both catalysis-dependent and -independent mechanisms. The hGIIA crystal structure, both alone and inhibitor-bound, together with structures of closely related snake-venom-derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis-independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A2 of Daboia russelli pulchella (svPLA2 ). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827-842. © 2016 Wiley Periodicals, Inc.

Host-Guest Complexes of Carboxylated Pillar[n]arenes With Drugs.

Pillar[n]arenes are a new family of nanocapsules that have shown application in a number of areas, but because of their poor water solubility their biomedical applications are limited. Recently, a method of synthesizing water-soluble pillar[n]arenes was developed. In this study, carboxylated pillar[n]arenes (WP[n], n = 6 or 7) have been examined for their ability to form host-guest complexes with compounds relevant to drug delivery and biodiagnostic applications. Both pillar[n]arenes form host-guest complexes with memantine, chlorhexidine hydrochloride, and proflavine by 1H nuclear magnetic resonance and modeling. Binding is stabilized by hydrophobic effects within the cavities, and hydrogen bonding and electrostatic interactions at the portals. Encapsulation within WP[6] results in the complete and efficient quenching of proflavine fluorescence, giving rise to "on" and "off" states that have potential in biodiagnostics. The toxicity of the pillar[n]arenes was examined using in vitro growth assays with the OVCAR-3 and HEK293 cell lines. The pillar[n]arenes are relatively nontoxic to cells except at high doses and after prolonged continuous exposure. Overall, the results show that there could be a potentially large range of medical applications for carboxylated pillar[n]arene nanocapsules.