Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer.

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

Molecular epidemiology of isolates with multiple mcr plasmids from a pig farm in Great Britain: the effects of colistin withdrawal in the short and long term.

Background: The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans. Objectives: To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5 month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ∼20 months. Methods: mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined. Results: E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5 months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ∼20 months after colistin cessation. Conclusions: We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.

Epidemiological evidence that garden birds are a source of human salmonellosis in England and Wales.

The importance of wild bird populations as a reservoir of zoonotic pathogens is well established. Salmonellosis is a frequently diagnosed infectious cause of mortality of garden birds in England and Wales, predominantly caused by Salmonella enterica subspecies enterica serovar Typhimurium definitive phage types 40, 56(v) and 160. In Britain, these phage types are considered highly host-adapted with a high degree of genetic similarity amongst isolates, and in some instances are clonal. Pulsed field gel electrophoresis, however, demonstrated minimal variation amongst matched DT40 and DT56(v) isolates derived from passerine and human incidents of salmonellosis across England in 2000-2007. Also, during the period 1993-2012, similar temporal and spatial trends of infection with these S. Typhimurium phage types occurred in both the British garden bird and human populations; 1.6% of all S. Typhimurium (0.2% of all Salmonella) isolates from humans in England and Wales over the period 2000-2010. These findings support the hypothesis that garden birds act as the primary reservoir of infection for these zoonotic bacteria. Most passerine salmonellosis outbreaks identified occurred at and around feeding stations, which are likely sites of public exposure to sick or dead garden birds and their faeces. We, therefore, advise the public to practise routine personal hygiene measures when feeding wild birds and especially when handling sick wild birds.

Multiplexing terbium- and europium-based TR-FRET readouts to increase kinase assay capacity.

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKbeta, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.

TR-FRET biochemical assays for detecting posttranslational modifications of p53.

The p53 tumor suppressor protein plays a pivotal role in suppressing oncogenesis by regulating a range of cellular functions, including DNA repair, cell growth, cell cycle progression, and cellular death. A network of different pathways converge upon p53, ultimately regulating the response of the tumor suppressor protein by posttranslational modifications. The authors have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput compatible assay to analyze the critical posttranslational modifications of p53, including phosphorylation, acetylation, and ubiquitination. By using full-length p53 protein fused with GFP (GFP-p53) as the substrate, they were able to measure all 3 different posttranslational modifications with a single substrate. In addition, with a few additional steps, the GFP-p53 substrate can also be used to assay deacetylation to aid in the discovery of inhibitors for sirtuins or other deacetylase enzymes. The flexibility of the assay to measure a diverse range of posttranslational modifications allows one to further dissect the complex regulating mechanisms of p53 and enable the discovery of specific inhibitors for these processes.

A toolbox approach to high-throughput TR-FRET-based SUMOylation and DeSUMOylation assays.

The posttranslational modification of target substrates by the ubiquitin-like proteins, specifically the small ubiquitin-like modifier (SUMO), has emerged as an essential mechanism to regulate protein function and control intracellular trafficking. Traditional methods for monitoring either the attachment or removal of SUMO, such as gel electrophoresis or western blot, are effective but typically suffer from a lack of throughput. Here, we report the development and application of time-resolved Förster resonance energy transfer (TR-FRET)-based assays capable of detecting SUMOylation or deSUMOylation in a high-throughput screening (HTS) format. Using Ran GTPase-activating protein (RanGAP1) as a model target substrate, we have demonstrated that the SUMOylation of this protein can be detected using LanthaScreen (Invitrogen, Carlsbad, CA) TR-FRET technology. Additionally, we have generated reagents useful for assessing the deSUMOylation activity of a sentrin-specific protease. All assays are performed in 384-well format and display excellent statistical data (Z' > 0.7) with high signal-to-background levels. Together, this collection of tools can be utilized in a modular approach to develop HTS assays for inhibitors of SUMOylation or deSUMOylation.

High-throughput cellular assays for regulated posttranslational modifications.

We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific posttranslational modifications in a mammalian cell-based format. We chose the NFkappaB signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of posttranslational modifications in response to pathway stimulation. In this pathway, TNFalpha induces the phosphorylation, ubiquitination, and proteasomal degradation of IkappaBalpha, which leads to the release and translocation of the NFkappaB transcriptional complex into the nucleus. To measure these cellular processes, we describe the use of a stable cell line expressing a fusion of green fluorescent protein (GFP) with IkappaBalpha that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a time-resolved Förster resonance energy transfer (TR-FRET)-based readout. Concurrently, we have engineered a beta-lactamase-IkappaBalpha reporter cell line that can be used to quantify proteasomal degradation of IkappaBalpha in living cells. Both TR-FRET and beta-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFkappaB signaling in a physiologically relevant cellular context without the need to overexpress any enzyme involved in this pathway. Cellular HTS assays that interrogate such processes will provide a unique integrated approach to dissecting intermediate steps in NFkappaB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery.

A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.

Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.

Optically trapping confocal Raman microscopy of individual lipid vesicles: kinetics of phospholipase A(2)-catalyzed hydrolysis of phospholipids in the membrane bilayer.

Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.

Detection of drug-membrane interactions in individual phospholipid vesicles by confocal Raman microscopy.

Optical-trapping confocal Raman microscopy is developed as a method to study the interactions of drugs or other compounds with the membranes of individual phospholipid vesicles. This technique allows membrane disorder, permeability, and drug localization to be assessed without the need for labeling of the membrane or the compounds of interest. We have applied this technique to study the interactions of two nonsteroidal antiinflammatory drugs, salicylate and ibuprofen, with vesicles prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The results show that both salicylate and ibuprofen increase membrane disorder, as determined from increases in the Raman scattering from gauche conformers in the phospholipid acyl chains. By monitoring the Raman scattering of the drug molecules in optically trapped DMPC vesicles, the membrane permeability and partitioning of the drugs could be determined; the spatial distributions of the drugs were also measured by scanning the laser focus through surface-adhered 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles, producing a profile of the vesicle and its contents. Though the membrane is permeable to both drugs, ibuprofen preferentially accumulates in the membrane, whereas salicylate does not. The measured ibuprofen accumulation agrees quantitatively with the water/octanol partition coefficient of the drug and the estimated volume of the lipid membrane. The results suggest that ibuprofen localizes in the hydrophobic acyl chain region of the membrane, whereas salicylate weakly associates with the phospholipid headgroups.

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the gamma-glutamyl cycle.

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid gamma-glutamyl acceptor substrates for gamma-glutamyl transpeptidase (gamma-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the gamma-glutamyl cycle through interaction with gamma-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a gamma-glutamyl acceptor for both murine and bovine renal gamma-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a gamma-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the gamma-glutamyl cycle.

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity in CD-1 mice: I. Enhancement of acetaminophen nephrotoxicity by acetaminophen-cysteine.

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Recent studies suggest a contributory role for glutathione (GSH)-derived conjugates of APAP in the development of nephrotoxicity. Inhibitors of either gamma-glutamyl transpeptidase (gamma-GT) or the probenecid-sensitive organic anion transporter ameliorate APAP-induced nephrotoxicity but not hepatotoxicity in mice and inhibition of gamma-GT similarly protected rats from APAP nephrotoxicity. Protection against APAP nephrotoxicity by disruption of these GSH conjugate transport and metabolism pathways suggests that GSH conjugates are involved. APAP-induced renal injury may involve the acetaminophen-glutathione (APAP-GSH) conjugate or a metabolite derived from APAP-GSH. Acetaminophen-cysteine (APAP-CYS) is a likely candidate for involvement in APAP nephrotoxicity because it is both a product of the gamma-GT pathway and a probable substrate for the organic anion transporter. The present experiments demonstrated that APAP-CYS treatment alone depleted renal but not hepatic glutathione (GSH) in a dose-responsive manner. This depletion of renal GSH may predispose the kidney to APAP nephrotoxicity by diminishing GSH-mediated detoxification mechanisms. Indeed, pretreatment of male CD-1 mice with APAP-CYS before challenge with a threshold toxic dose of APAP resulted in significant enhancement of APAP-induced nephrotoxicity. This was evidenced by histopathology and plasma blood urea nitrogen (BUN) levels at 24 h after APAP challenge. APAP alone was minimally nephrotoxic and APAP-CYS alone produced no detectable injury. By contrast, APAP-CYS pretreatment did not alter the liver injury induced by APAP challenge. These data are consistent with there being a selective, contributory role for APAP-GSH-derived metabolites in APAP-induced renal injury that may involve renal-selective GSH depletion.

Synthesis and characterization of a cobalamin-colchicine conjugate as a novel tumor-targeted cytotoxin.

Colchicine was derivatized at C7 with p-alkoxyacetophenone and conjugated to cobalamin (vitamin B(12)) through an acid-labile hydrazone linker. The cobalamin moiety leads to preferential uptake of the cobalamin-colchicine prodrug by cancer cells, whereupon the hydrazone linker undergoes hydrolysis in the lysosome to unmask colchicine, which acts as a potent cytotoxin by stabilizing microtubules and causing cell death. The bioconjugate is stable in cell culture media and at neutral pH but undergoes hydrolysis with a half-life of 138 min at pH 4.5. The colchicine-cobalamin bioconjugate exhibits nanomolar LC(50) values against breast, brain, and melanoma cancer cell lines in culture. Attachment of colchicine to cobalamin is expected to increase the therapeutic index of the drug by limiting the side effects caused by the current nonselective administration of tubulin-targeted chemotherapeutic drugs.

Structural determination of 5'-OH alpha-ribofuranoside modified cobalamins via 13C and DEPT NMR.

A protocol for the rapid NMR characterization of cobalamin (vitamin B(12)) analogues with 5'-hydroxy-alpha-ribofuranoside modification is reported. The structure of cyanocobalamin in DMSO-d(6) has been assigned using COSY, NOESY, HSQC, and HMBC NMR methods. The robust precision of (13)C NMR assignments in DMSO-d(6) allows for the rapid structural determination of 5'-hydroxy-alpha-ribofuranosyl cyanocobalamin derivatives with solely 1-D (13)C and DEPT NMR spectra and only 10 mg of derivatized cobalamin. Using this method, the (13)C NMR resonances of four cobalamin analogues were determined with the most significant variance of (13)C chemical shifts occurring in the alpha-ribofuranoside ring. In DMSO-d(6), cobalamin concentrations greater than 30 mM can be achieved for an improved signal-to-noise ratio.