Epitope mapping of Der p 2, a clinically important dust-mite allergen is the first step in designing immunotherapy hypoallergen vaccine candidates. Twenty-one single alanine mutants of Der p 2 were generated and their secondary structure was analysed using circular dichroism spectra. Only one mutant, K96A resulted in a misfolded protein. All mutants were tested for serum IgE reactivity using serum from dust mite allergic individuals by immuno dot-blots. Mutations to five residues, N10, E25, K77, K96 and E102 consistently showed reduced IgE reactions compared to wild-type Der p 2, and therefore these residues constitute the major IgE epitopes of Der p 2. Two mutants with consistent low IgE binding, K96A and E102A, were subsequently evaluated as hypoallergen candidates. IgG antibodies raised in mice against both mutants could inhibit human IgE-binding to WT Der p 2. Both mutants had intact T-cell epitopes as they were able to stimulate peripheral blood mononuclear cell proliferation similar to WT Der p 2. However, a switch in Th1:Th2 cytokine profile was not observed. In summary, we have identified the major conformational epitopes of Der p 2, and evaluated two Der p 2 hypoallergen vaccine candidates for immunotherapy. House dust mites are the most important source of allergens in the indoor environment2, causing sensitizations in up to 90% allergic patients4. Currently 34 allergens originating from dust mites have been published . Of these, group 1 and 2 allergens, mainly from Dermatophagoides spp. are considered as major allergens, as they are recognized by more than 80% of the IgE from dust-mite sensitized individuals4.About 30% of the world population suffer from allergic-related diseases and its prevalence is increasing annually1. However, these measures are cumbersome and not long lasting.An allergic response occurs when allergens bind and crosslink IgE antibodies on the surfaces of mast cells and basophils via the FcεRI receptor, causing these cells to get activated and release inflammatory mediators such as histamine, leukotriene and cytokines. Exposure to dust mite allergens can be managed by reducing physical exposure using exclusion methods such as dust-mite impermeable bedding covers and particulate air filters, or by managing symptoms using topical steroids or anti-histamines5. Allergen SIT is based on repeated injections of increasing doses of allergen extracts and has been reported to have lasting effects, up to 3 years after discontinuation of treatment6. Conventionally, immunotherapy was performed using crude allergen extracts. This method risked allergic side effects, including anaphylaxis which could be fatal. To improve the safety of SIT, biologist are generating modified allergen proteins which have lesser IgE binding capacity, but retained T-cell proliferation7. Maintaining T-cell proliferation is important as studies on the mechanisms of immunotherapy have highlighted the role of allergen-specific CD4+ T-cells in tolerance mechanisms and control of allergic responses8.The only curative treatment for allergies currently known is by allergen specific immunotherapy (SIT)In this study, we identify the conformational IgE epitopes of Der p 2 using site-directed mutations, and test these mutants for their reduction in IgE-binding capacity using serum from dust-mite allergic individuals. We then test two site directed mutants of Der p 2 for their potential as prophylactic hypoallergen vaccine in a murine model.10. For the remaining surface residues, their side chain surface accessibility was calculated using the WHATIF algorithm11 based on the solved crystal structure of Der p 210, as these residues were more likely to participate in protein-protein interactions9. Based on these considerations, ten residues with the highest side chain solvent accessibility and with different chemical properties from hNPC2 were selected for epitope mapping of Der p 2. We also included mutants of 11 residues which were previously reported to be involved in the IgE epitope of Der p 215. Each residue was mutated to alanine and expressed as a recombinant protein , and found that hNPC2 did not show IgE binding Fig. . TherefoAll mutant recombinant proteins showed similar β-sheeted secondary structure as WT Der p 2, except for mutant K96A which resulted in a misfolded protein Fig. . InteresDermatophagoides pteronyssinus and Blomia tropicalis, the two predominant dust mite species in Singapore16. Among the dust mite allergic individuals, 78% had Der p 2-specific IgE  demonstrated IgE binding to crude dust mite extracts of IgE Fig. . We obseIgE Fig. . We nextSera from five allergic patients, from whom sera was abundant and fresh blood samples were available for cellular studies were then tested the IgE binding to WT Der p 2, WT Blo t 2, hNPC2 and 21 Der p 2 mutants. All individuals showed almost negligible levels of IgE reactivity to hNPC2 Fig. . Each in50 of mutant E102A was at least 4-fold higher, while for K96A it was 16-fold higher compared to WT Der p 2 in all allergic individuals tested  from dust-mite allergic individuals. Both E102A and K96A were able to stimulate PBMC proliferation comparable to WT Der p 2 in three allergic individuals tested, indicating the Der p 2 mutants retained functional T-cell epitopes Fig. . To dete17. Immunotherapy represents a form of curative treatment for allergies as it causes immune deviation or tolerance in a patient’s immune mechanism8. Although natural allergens were initially used for immunotherapy, they could potentially cause anaphylactic shock in some patients due to the presence of their IgE-binding epitopes. T-cells have been shown to play an important role in the tolerance mechanism for immunotherapy18. Therefore, to provide safer therapeutic reagents, immunotherapy molecules which lack the IgE epitopes  but retained T-cell proliferation are generated19.Der p 2 is regarded as an important allergen worldwide, and it is therefore of clinical importance to understand its epitopes, and to evaluate the potential hypoallergen vaccine candidates of this protein. Conventional therapy for allergies such as the use of anti-cytokines, anti-IgE or corticosteroids are only able to alleviate the symptoms experienced, but do not confer long term protection12. Our study showed that alanine mutation of K96 resulted in a drastic change in the secondary structure of the protein, from a β-sheeted structure to a misfolded structure, based on the circular dichroism (CD) spectra data, indicating that it is a crucial residue in the refolding of Der p 2. No significant changes in secondary structures were observed when residues adjacent to K96 were mutated . In a study involving a cherry allergen, Pru av 1, mutation of a single amino acid residue also resulted in a misfolded protein structure that showed nearly complete loss of IgE binding20. However, not all amino acid residues seem to be critical for protein refolding, as seen from our data on mutants such as N10A, E25A, K77A and E102A, which retained their secondary protein structure comparable to the wild type protein. In these cases, the reduction in IgE binding could be a result of the local changes in surface accessibility or charge.Single alanine mutants of surface exposed amino acids of Der p 2 were generated to evaluate their contribution to the IgE reactivity of Der p 2. The IgE epitopes of Der p 2 were mapped in 116 dust mite-sensitized individuals from Singapore. Five mutants, N10A, E25A, K77A, K96A and E102A showed a consistent reduction in IgE binding across all sensitization groups in our study, and therefore constitute major IgE epitopes of Der p 2 in our study population. Of these, two mutants (K96A and E102A) have been identified earlier as IgE epitopes of Der p 2 using a different approach50 ratio of mutant:native was higher when K96A was used as the inhibitor compared to E102A, suggesting that some IgE binding epitopes were still present on mutant E102A. In contrast, the majority of the IgE epitopes of mutant K96A may have been destroyed due to the protein misfolding.Two mutants, K96A and E102A to further characterize their hypoallergen properties. Both mutants showed reduced IgE binding in all patients when compared to WT Der p 2. Both mutants were also able to inhibit serum IgE of allergic individuals from binding to WT Der p 2 allergen in inhibition ELISA assays, albeit to different degrees. The IC22. There have been several proposed mechanisms by which allergen specific IgG play a role in immunotherapy. First, allergen specific IgG ‘blocks’ IgE from binding to the allergen by direct competition, resulting in the lack of IgE cross-linking, reducing the release of mediators from mast cells and basophils24. Alternatively, the IgG generated may interfere with the IgE-allergen complex which binds to the low affinity IgE receptor on antigen presenting cells, CD23, which facilitates the allergen presentation to T-cells26. Due to the non-reversible nature of immunoglobulin gene recombination, cells which already produce IgE antibodies will remain doing so, even after immunotherapy. Hence, no change in the amounts of IgE would be observed when pre- and post-treatment levels are compared27. The increase in IgG production causes an increase in the IgG:IgE ratio, which then results in the immunological changes post-immunotherapy. In pollen allergy, immunotherapy shows a seasonal rise in IgG, particularly IgG428 while gradually attenuating the seasonal rise in IgE21.The production of allergen-specific IgG antibodies has been reported to be among the first signs of successful immunotherapy30 and birch pollen allergen, Bet v 131.In this study, sera from mice immunized with E102A or K96A were able inhibit human IgE binding to WT Der p 2 at similar intensity, which was only slightly lower than homologous inhibition using WT Der p 2. Since the mutants lack the IgE binding epitopes, this inhibition could be due to steric hindrance, and not direct competition in IgE binding. The production of blocking IgG antibodies have also been observed for other hypoallergen vaccine candidates from house dust miteset al.32 reported that birch pollen allergoid used for immunotherapy failed to change cytokine expression for IL-4, IL-5, IL-10 or IFN-γ in cell cultures but found expected increases in cytokines in nasal secretions on the immunotherapy patients. In a study of grass pollen immunotherapy, seasonal increase in the ratio of IFN-γ to IL-5 was found in the nasal mucosa, but not in the allergen-stimulated T-cell cultures33. Nevertheless, other studies have shown enhanced IFN-γ and reduced IL-4 production after allergen stimulation35. It appears that the switch from Th2 to Th1 cytokines is more prominent in later stages of immunotherapy, and less pronounced in a T-cell culture, as the cells are in contact with the allergen for a shorter time period.Although much emphasis has been placed on the production or expression levels of cytokines, which are indicative of the T-helper subtype following allergen stimulation, results from different studies have been conflicting. In this study, we observed that although mutants E102A and K96A was able to stimulate PBMC proliferation, a clear distinction in T-helper subtype could not be made. Similarly, Klimek Of the two hypoallergen candidates evaluated, the misfolded K96A showed almost negligible serum IgE binding but was able to induce blocking IgG antibody production that inhibited the binding of allergic-patient serum IgE to WT Der p 2. In addition, the T-cell epitopes of K96A remained intact as it was capable of stimulating PBMC proliferation. These characteristics suggests that mutant K96A is promising hypoallergenic vaccine candidate and should be further tested in pre-clinical trials as specific immunotherapy against house dust mite allergy.Dermatophagoides pteronyssinus using primers containing BamH I and Eco R I restriction sites. The DNA insert was ligated into a modified pET-32a plasmid (Novagen) and transformed into DH5-α competent cells. Colonies were screened by PCR, and the insert sequence was verified by DNA sequencing . Mutant constructs were generated using the Quikchange® kit (Statagene) with primers containing mismatches coding for alanine. Correct substitutions were verified by DNA sequence analysis. Mutated DNA insert was sub-cloned in the same manner as WT Der p 2.DNA encoding for Der p 2 were amplified from cDNA of E. coli  for protein expression. Cultures were induced overnight with 1.0 mM IPTG at 20 °C. The protein was expressed as a His-tagged soluble protein and purified using a Ni-NTA resin (Novagen) under denaturing conditions. Recombinant proteins were refolded by rapid dilution into 50 mM sodium acetate, pH 4.6 at 4 °C and concentrated using Amicon® Stir Cell .Plasmid containing DNA insert of WT or mutant Der p 2 was transformed into CD spectra was acquired using a J-180 Spectropolarimeter (Jasco) using a 1 mm path length quartz cuvette. The spectra were recorded at the resolution of 0.1 nm and averaged for 10 scans (50 nm/min) from 190 to 260 nm.Consecutive serum samples from patients from Singapore with clinical symptoms of allergies were used. Written informed consent were obtained from all participants to obtain blood samples. The human and animal studies were reviewed and approved by the Institutional Review Board of the National University Hospital, the Animal Research Ethics Committee of the National University of Singapore and the Hospital Ethics Committee of the KK Women’s and Children’s Hospital and Singapore General Hospital. All experiments were performed in accordance with relevant guidelines and regulations of the institutions and committees indicated above.One microgram of crude allergen extract or recombinant proteins were dotted on nitrocellulose membranes , air dried and blocked with PBS-0.1% Tween-20. Membranes were incubated with serum from allergic individuals overnight at 4 °C, followed by alkaline phosphatase conjugated anti-human IgE  for 2 hrs. Membranes were developed with NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) (Promega). Spot intensities were measured using an image analysis software . Spot intensities  were normalized by subtracting the background. Intensities greater than 2 SDs above the mean negative sera responses were considered positive. For comparison, a standard curve using the human serum IgE standard (75/502), purchased from the National Institute for Biological Standards and Control (NIBSC), United Kingdom, was used.WT or mutant Der p 2 were coated overnight at 4 °C onto microtiter plates (Nunc) at 250ng of protein per well. After blocking, wells were incubated with patients’ sera diluted 1:10 or 1:5, and incubated at room temperature (RT) for 2.5 hrs. After washing, plates were incubated with biotin conjugated anti-human IgE monoclonal antibody , followed by avidin conjugated HRP  for 30 min. Plates were developed by adding TMB  substrate . The colour reaction was stopped after 30 mins using 1 M HCl. Absorbance was measured at 450 nm using a microplate reader.−4 to 1 × 10−11 μg/mL of recombinant allergen. The degree of cross-reactivity was calculated by the percentage of inhibition based on the following formula: [(Iu − Ii)/(Iu − B)] × 100%, where Iu represents the reaction in the absence of inhibitor protein, Ii the reaction at a particular inhibitor concentration, and B represents background intensity when the allergen was incubated with blocking solution instead of sera.Inhibition ELISAs was performed according to the ELISA protocol described, except that the sera used were pre-absorbed with 1 × 105 cells per well. WT or mutant Der p 2 were added to the cell suspension , and incubated for 6 days at 37 °C under 5% CO2. Cell proliferation was measured using the Bromodeoxyuridine colorimetric method using the manufacturer’s protocol (Roche Applied Sciences). Proliferation is reported as stimulation index, calculated by the formula S/C . To measure cytokines, supernatants of the cell culture on day 6 were harvested, and cytokine concentrations were determined using human Th1/Th2 Bioplex cytokine assay (Biorad) according to the manufacturer’s protocol.Fresh blood was collected from three allergic individuals and PBMCs were isolated using the Ficoll-Hypaque gradient centrifugation. PBMCs were cultured in RPMI with 10% FBS, 50 units/mL penicillin, 50 μg/mL streptomycin and 2mM L-glutamine in 96-wells plate at 10Six-week old female Balb/c mice (n = 4) were injected intra-peritoneally with 10 μg of purified WT or mutant Der p 2, or hNPC2 with 1.25 mg/mL aluminium hydroxide gel  every 14 days. PBS buffer containing Alum was similarly injected into another group of 2 mice as controls. Pre- and post-immune blood samples after the 6th injection was drawn by orbital bleeding and stored at −20 °C until use.WT Der p 2 was coated overnight at 4 °C onto Maxisorp ELISA plate (Nunc) at 250ng of protein per well. After a washing and blocking step, wells were incubated with 1:10 diluted mouse sera for 2 hrs. Next, wells were incubated with diluted patients’ sera for another 2 hrs. The remaining steps were similar to that described in the IgE binding ELISA. Percentage inhibition of human IgE binding to Der p 2 was calculated using the following formula: % inhibition of IgE binding = 100 − (A − B) × 100%. A and B represent the absorbance values after pre-incubation with serum from immunized mouse and control mouse respectively.t-test.Statistical analysis was carried out using GraphPad Prism. Differences in IgE binding between wild type Der p 2 and mutant allergens of individual patients was calculated using two-way ANOVA with Dunnette’s multiple comparison test at a significance of p < 0.0001. Comparisons between allergen levels in the dust samples were performed using paired Supplementary Information


The supply of freshwater to fjord systems in Greenland is increasing as a result of climate change-induced acceleration in ice sheet melt. However, insight into the marine implications of the melt water is impaired by lack of observations demonstrating the fate of freshwater along the Greenland coast and providing evaluation basis for ocean models. Here we present 13 years of summer measurements along a 120 km transect in Young Sound, Northeast Greenland and show that sub-surface coastal waters are decreasing in salinity with an average rate of 0.12 ± 0.05 per year. This is the first observational evidence of a significant freshening on decadal scale of the waters surrounding the ice sheet and comes from a region where ice sheet melt has been less significant. It implies that ice sheet dynamics in Northeast Greenland could be of key importance as freshwater is retained in southward flowing coastal currents thus reducing density of water masses influencing major deep water formation areas in the Subarctic Atlantic Ocean. Ultimately, the observed freshening could have implications for the Atlantic meridional overturning circulation. A simultaneous freshening is expected in Greenland coastal waters where current rates of ice loss from the Greenland Ice Sheet have more than doubled compared to rates from 1983–20033. The annual net loss of ice is estimated at 186 Gt (i.e. 186 km3) whereas the total ice loss during the seasonal melt in summer exceeds 1200 km3 annually4. Although much of the ice loss to date can be attributed to glaciers in West and Southeast Greenland it is now evident that there is also accelerating ice loss in Northeast Greenland5. In addition to contributing to global sea level rise6, increasing freshwater contribution from Greenland may impact local and global circulation patterns. Transport of freshwater in Greenlandic coastal currents and into the surface waters of the subarctic Atlantic could influence deep water convection and potentially the Atlantic meridional overturning circulation8. This represents an important potential climatic feedback mechanism not included in current IPCC models.The Arctic Ocean has increased its freshwater content significantly the past decadehttp://www.g-e-m.dk) since 2003. The aim of this study was to analyse the dataset for evidence in changes in the freshwater budget. The hydrographic transect is sampled each year in early August and spans from the inner fjord and approximately 30 km off the coast  and sub-zero temperatures. Below the Polar Surface Waters, warm and saline re-circulating Atlantic Water is found typically at the depth of 250 m. For fjords such as Young Sound with a shallow sill, the dense Atlantic Water is prevented from entering the fjord as bottom water  of the surface waters (0–50) in the fjord. We used the average salinity (2003 to 2015) at 50 m in Young Sound as the reference salinity. In the fjord, FWC for this layer increased from 0.9 ± 1.1 (2003) to 3.7 ± 1.1 m in 2015 . In August, the salinity at 65  m is approximately equal to early spring conditions7  which was attributed to the release of river water temporarily stored on the Siberian shelf11. In 2011 to 2013 an increasing contribution from Pacific water to the freshwater content in the Denmark Strait was observed12. Both these studies sampled on the East Greenland shelf whereas this study is close to the coast line in what has been termed the Riverine Coastal Domain in the Arctic Ocean13 characterized by coastal trapped runoff from land. The observed freshening along our transect, therefore, likely represents a regional signal of increased ice mass loss from the Greenland Ice sheet north of our study area which is accelerating due to increasing air temperature5. Clearly the meltwater signal from the ice sheet is modified by other fresh water sources. For example, the 2007 peak in FWC and low salinities coincides with an exceptional amount of melting sea ice in outer Young Sound (monitored by daily images in Young Sound) and also coincided with higher than average sea ice cover along the east Greenland coast5.There are two potential sources for the observed freshening in the fjord: from the local terrestrial catchment or the coastal water outside the sill. Data on total discharge from the main river in the catchment (Zackenberg River) reveals no significant increasing linear trend during the study period Table . Neitherns7 Fig. , and sin FWC Fig . This su14 and nutrients15, influencing light availability16, productivity17 and pelagic18 and benthic19 ecosystem structure. Impacts of changing freshwater budgets in coastal waters is an additional manifestation of a warming climate, and in concert with changes in sea ice cover, ocean warming and acidification, freshening is likely an important driver for ecosystem change that remains poorly quantified in the Arctic.Meltwater from the Greenland Ice Sheet impacts the local marine ecosystem in numerous ways, providing organic carbon4) suggest that meltwater is efficiently retained near the coast as has been shown in other Arctic regions13. Recent modelling studies have shown that meltwater from East Greenland may be efficiently transported to the Labrador Sea22. Combined with the decreasing salinity trend in our data it suggests that a signal of ocean freshening is being transported downstream by a nearshore component of the East Greenland current and will contribute to the freshwater content in the subarctic Atlantic.The salinity time series presented are unique for the region and the first to document a decreasing trend in salinity on decadal scale from the coastal ocean surrounding the ice sheet. The presence of a freshening signal in Northeast Greenland despite a modest increase in ice sheet melt . An additional measure of the annual change of freshwater content (FWC_S) was defined by applying the annual averaged value of Sref in Eq. ref = 32.91, 32.88, 33.12, 32.70, 32.20, 32.39, 32.38, 32.25, 31.87, 32.00, 31.73, 32.19, 32.05 in 2003–2015). Wind speed was collected by the ClimateBasis component of Greenland Ecosystem Monitoring program (GEM). Average wind speed  collected at a height of 7.5 m available at the GEM database were used. The dates for sea ice melt and sea ice formations was also extracted from the GEM database, where an automatic camera system provides information on daily local ice conditions in the outer part of Young Sound.Hydrography profiles were obtained using a Sea-Bird SBE19plus CTD. The instruments were calibrated by Sea-Bird every year before fieldwork and data was averaged for 1 m intervals. For this analysis a database of about 300 CTD profiles has been accumulated and analysed. Salinity profiles in this analysis covers measurements during early August. The profiles were divided into two sectors; inside the fjord and outside in the coastal water masses. Only profiles with water depth larger than 50 m were considered. Freshwater content (FWC) was calculated as:The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 μM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications.The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from  Brieflyn-BuOH fraction from APE were mixed with 180 μL prepared DPPH solution (0.32 mM). The mixture was incubated in the dark for 20 min, then filtered through a 0.45-μm filter for HPLC analysis. The n-BuOH (20 mg/mL in MeOH) was used as a control. The extent of peak decrease is expressed as a quantitative reduction.The offline DPPH HPLC assay was performed by modifying a previously-described protocol . Thirty DL-glyceraldehyde and AR inhibitor in a total volume of 600 μL. Concentrations ranged from 0 to 25 mM for DL-glyceraldehyde and from 0 to 20 μM for the active compound. RHAR activity was assayed by measuring the decrease in absorption of NADPH after substrate addition at 340 nm using a Bio Tek Power Wave XS spectrophotometer  [Reaction mixtures consisted of 0.1 M potassium phosphate, 1.6 mM NADPH, and 2 mM of RHAR with varied concentrations of substrate VT, USA) .2 and 95% air at 37 °C. Samples were dissolved in DMSO. The lenses were divided into three groups and cultured in medium containing 30 mM glucose and RLAR-active compounds. Each lens was placed in a well containing 2.0 mL of medium. Sorbitol was determined by HPLC using the methods mentioned in the section of 4.7 after its derivatization by reaction with benzoic acid to a fluorescent compound [Lenses isolated from 10-week-old Sprague–Dawley rats using the registration number mentioned in the section of 4.5 were cultured for 6 d in TC-199 medium that contained 15% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin, under sterile conditions and an atmosphere of 5% COcompound .50 value was defined as the concentration at which 50% inhibition occurred. Data are expressed as mean values ± standard deviation of triplicate experiments.Inhibition rates were calculated as percentages (%) with respect to the control value, and the ICn-BuOH fraction of the APE and ten flavonoids known as ingredients of AP were evaluated for RLAR inhibitory activity and DPPH radical scavenging activity. Additionally, antioxidant compounds in the n-BuOH fraction of APE were investigated with a DPPH offline HPLC assay. Of the compounds tested, AM, LGN, QU, LT, and AZ showed strong inhibitory activity against RLAR and sorbitol accumulation. Consequently, we conclude that APE and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AP may potentially be used as a herbal drug to treat diabetic complications.In summary, seven compounds isolated from the


Drosophila melanogaster embryos and observed Bicoid forming transient “hubs” of locally high density that facilitate binding as factor levels drop.Mir et al. used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early  Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient “hubs” of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early  Drosophila melanogaster was the first identified morphogen  and its role in the regulation of chromatin accessibility  and in mExtant models cannot, for example, address how BCD is sufficient for activating its targets such as Knirps  and HairIn this study, we address gaps in our understanding of morphogen activity by performing direct measurements of BCD–DNA interactions in vivo by single-molecule imaging. Single-molecule imaging in living cells has been increasingly used in recent years to measure the dynamics of TF–DNA interactions . HoweverLattice light-sheet microscopy (LLSM) was developed recently to overcome these technical barriers . The priD. melanogaster embryos in order to characterize for the first time the single-molecule DNA-binding kinetics of BCD along its concentration gradient. We found that BCD binds to chromatin in a highly transient manner, with specific binding events lasting on the order of a second, in all portions of the embryo. Examination of the spatial distribution of BCD-binding events reveals spatiotemporal hubs of high local BCD concentration that increase its local DNA binding on rate and facilitate specific binding even with such a high off rate. This effect is most dramatic in posterior nuclei, where, given that there is minimal BCD, we were surprised to observe a significant number of binding events. Through genome-wide analysis of BCD–DNA binding on dissected posterior segments of embryos, we show that the binding that we observed via single-molecule imaging in posterior nuclei occurs at specific regulatory regions, a result that cannot be explained by classical models of BCD activity.Here we applied LLSM to developing We further found that the regions that are enriched for BCD in the posterior segments are highly correlated with binding of the maternal factor ZLD, which has been shown previously to affect the regulation of BCD targets, especially at lower concentrations. Through single-molecule imaging of BCD in ZLD-null embryos, we show that ZLD is required for the formation of BCD hubs in the posterior embryo. Together, these data advocate for a model in which ZLD mediates the formation of hubs of high local BCD concentration that facilitate BCD binding and enable the activation of BCD-dependent targets at all positions along the A–P axis of the embryo.Supplemental Fig. S1; Drosophila embryos over a large field of view with high temporal resolution . We used a yw; his2av-mrfp1; bcdE1, egfp-bcd fly line in which only the labeled BCD is expressed to indicate proper functionality and expression levels  and discrete characteristics  of the observed binding events.We constructed a lattice light-sheet microscope  exposure times, effectively blurring out the fast-moving (unbound) population . Fits to the survival probability distributions of BCD-binding events  in the anterior, middle, and posterior thirds of the embryo identified short-lived populations with average RTs (after photobleaching correction) on the order of hundreds of milliseconds and longer-lived populations with average RTs on the order of 1 sec, with no significant dependence on position along the A–P axis for either population . The validity of our RT estimation of specific binding on the order of 1 sec is supported by additional measurements at 500-msec exposure times . We note that the values reported here are the genome-wide averages for both the long-lived and short-lived binding populations. It is also likely that there are even shorter-lived BCD–DNA interactions that we cannot access due to the practical trade-off between temporal resolution and signal to noise ratio in single-molecule imaging.Previous single-molecule studies of TFs have consistently found two populations in the survival probability distributions: a short-lived population with RTs on the order of hundreds of milliseconds and a longer-lived population with RTs on the order of tens of seconds to minutes . These tSupplemental Fig. S5). The rapid dynamics of BCD indicated by the FRAP data are consistent with previous measurements by others using fluorescence correlation spectroscopy (FCS)  . Althougpy (FCS)  were prepy (FCS) , which tpy (FCS) . These rpy (FCS) C highlig studies . The obsSupplemental Movie 3). However, despite this reduced contrast, we were able to perform single-particle tracking and, through analyses of displacement distributions , estimated the fraction of BCD that is bound along the A–P axis  concentrations , motivatA–P axis A. SurpriSupplemental Movie S4) of binding events that became more pronounced toward posterior positions , with a greater fraction of binding events occurring within clusters . Remarkably, although the number of binding events per nucleus followed the trend dictated by the global concentration gradient across the embryo , a ubiquitous activator often described as a pioneer factor active during early embryonic development . The difference in the binding profiles of the posterior third segments compared with whole embryos emphasizes the need to perform genomic analysis in a spatially resolved manner across the A–P axis. While we provide a proof of principle on how to perform these measurements through manual dissection, improved methods are required in order to improve the throughput of these experiments.Remarkably, enrichment of ZLD is more predictive of BCD binding in the posterior than previously determined positions of enhancer activity for the loci shown in zelda-null embryos with BCD-eGFP and measured BCD binding at 100-msec exposure times. We found an abolishment of BCD hubs in the posterior embryo and a small decrease in RTs . Due to this loss of clustering, the same analysis that was performed in the wild-type case . Both the magnitude and correlation length indicate a diminishment of clustering in the posterior nuclei of the ZLD mutants. We also note that due to the lower labeling density of BCD in the ZLD-null embryos, the presumably ZLD-independent clustering in the anterior embryo now becomes more apparent . The loss of clustering in the ZLD mutants also confirms that the clustering that we originally observed is not due to aggregation of eGFP but simply nonhomogenous distribution of BCD within nuclei or other artifactual reasons. To provide further validation that the observed clustering is not occurring by chance, we calculated the pair correlation function for randomly distributed points in a disc approximately the diameter of the nuclei for comparison .The posterior third genomic data and the published evidence for ZLD's role in the regulation of chromatin accessibility  and its ype case C could nype case . This anAlthough the exact mechanism by which ZLD mediates BCD hub formation and binding remains unclear, we speculate that a combination of protein–protein interactions facilitated by intrinsically disordered low-complexity domains  of ZLD aOur initial observation of the low-affinity nature of BCD binding to chromatin is partially congruent with the classical view of BCD as a concentration-dependent morphogen. High BCD concentrations in the anterior embryo lead to high on rates along with high chromatin occupancy, with the high off rate enabling frequent sampling of both specific and nonspecific sites . As the Our demonstration that BCD overcomes the combination of low concentrations and high off rate by forming transient hubs highlights the power of dynamic, single-molecule studies on living embryos. Although most vivid in posterior nuclei, this phenomenon is likely to play a significant role in regulating the large number of important known BCD targets in the middle of the embryo. Unlike BCD targets in the most anterior region, a large number of BCD targets in the middle of the embryo are dependent on ZLD, and our in vivo imaging data provide an elegant model for how this is accomplished, in which ZLD binds to BCD targets, triggers the clustering of BCD (likely via low-complexity region-mediated interactions among ZLD molecules and between ZLD and BCD), thereby increasing the effective concentration of BCD at its targets. We speculate that this protein–protein interaction-mediated clustering acts in tandem with ZLD's known role in promoting chromatin accessibility to facilitate binding to low-affinity enhancers.The formation of such clusters or hubs has been reported for other TFs  and for All fly cages were prepared by combining males and females of the desired strains in a plastic cage left for at least 3 d at room temperature in light prior to imaging. The lids on the cages were filled with agar dissolved in apple juice . A paste of dry yeast was smeared on the lids to induce egg laying. Lids were exchanged once each day.yw; his2av-mrfp1; BcdE1, egfp-bcd. This fly line results in embryos in which only the labeled BCD is expressed, indicating proper functionality and expression levels .The fly strain used for all wild-type BCD imaging experiments was n levels . For theFor embryo collection, lids on fly cages were exchanged 1 h prior to imaging. After 1 h, embryos were collected from the lids using an inoculation loop. A 5-mm-diameter glass coverslip  was prepared by immersion in a small amount of glue (prepared by dissolving adhesive from about one-fifth of a roll of double-sided Scotch tape overnight in heptane) and left to dry for 5–10 min while collecting embryos. Collection was performed on a dissection scope with transillumination. Embryos were bathed in Halocarbon oil 27 (Sigma) for staging and then selected between developmental stages 1 and 4. Selected embryos were placed on a small square of paper towel and then dechorionated in 100% bleach for 1 min. Bleach was wicked off with a Kimwipe after 1 min, and then the square was washed with a small amount of distilled water. Excess water was wicked off the square, and the square was dipped in a small water bath. Unpunctured embryos that floated to the top of the bath were selected for imaging and placed on a small paper towel square to slightly dry. To prevent excess desiccation, embryos were immediately placed on the glass coverslip in rows and then immersed in a drop of phosphate-buffered saline (PBS).Supplemental Fig. S1) following the design described by Supplemental Fig. S1A). A pair of cylindrical lenses was then used to elongate and collimate the output Gaussian beam to illuminate a stripe on a spatial light modulator (SLM). The SLM was used to generate a coherent pattern of an array of 30 Bessel beams spaced such that they coherently interfered to create a two-dimensional (2D) optical lattice pattern with a maximum numerical aperture (NA) of 0.6 and minimum NA of 0.505. A 500-mm lens was used to project the Fourier transform of the SLM plane onto an annular mask conjugate to the back pupil plane (BPP) of the excitation objective to spatially filter the pattern . The BPP was then projected first onto a galvo scanning mirror for z-scanning and then onto a second galvo scanning mirror for x-dithering. The x-galvo scanning plane was projected onto the BPP of the excitation objective . The excitation objective focused the lattice pattern onto the sample, exciting any fluorophores within the axial range (∼400 nm) of the sheet. The emitted fluorescence was collected by the detection objective, which was oriented orthogonally to the excitation objective and projected onto an intermediate image plane by a 500-mm tube lens . An 80- and 200-mm lens pair was then used to demagnify the image further to provide a 100-nm sampling per pixel on each of the EMCCD sensors. A dichroic mirror  was placed between the last lens pair to allow for dual-color imaging in red and green with maximal spectral separation. An emission filter was placed in the path of each camera to both reject the excitation wavelengths and select the wavelength range of interest  . During each camera exposure, the x-galvo mirror was dithered twice over a 5.1-µm range in 100-nm steps to provide uniform illumination.Imaging was performed using a home-built lattice light-sheet microscope , and the positions of the anterior and posterior extremes of the embryo were then marked. For each data set acquired, the stage position was recorded to determine the position as a fraction of the embryonic length (EL) as the distance of the position from the anterior pole divided by the total length of the embryo.zelda embryos, lethality was confirmed after imaging.For the RT measurements on BCD-eGFP, a 488-nm excitation laser was used with a power of 2.9 mW measured at the back pupil plane of the excitation objective. Images were acquired with 100-msec exposure times and an EM gain setting of 300. At each location, at least 1000 frames were acquired, resulting in a total time of 105 sec with a frame rate of 105 msec. Prior to and after acquiring the BCD-eGFP data, an image was taken in His2-AVmRFP using a 561-nm excitation laser at an excitation power of ∼0.17 mW at the back pupil plane to determine the nuclear cycle phase; data not acquired during interphase were discarded upon examination of these images. For RT measurements at 500-msec exposure times, the 488-nm excitation laser power at the back pupil plane was reduced to 0.5 mW; all other settings were the same as above. For the displacement distribution measurements, the exposure time was set to 10 msec, resulting in a frame rate of 15 msec, and the excitation power was increased to 8.28 mW for the 488-nm laser line. All other settings were the same as described above. Viability of the embryos was determined by allowing them to develop until gastrulation after imaging. For the For all data sets, the following procedure was followed: First, for each movie, the corresponding before and after histone images were checked for any evidence of chromatin condensation to ensure that analysis was performed only in interphase nuclei. Data from mid to late nuclear cycle 14, where the nuclei exhibit an elongated shape, were also excluded. A metadata file was then created for each movie file containing the position as a fraction of the EL (0 for anterior and 1 for posterior), the nuclear cycle (determined by counting the number of mitoses before the 14th cycle). Visual examination of the data set was used to determine whether there was any motion of the nuclei during the acquisition period. Movies that contained any detectable motion were discarded or cropped to include only the time interval in which there was no motion. A rectangular region of interest was then drawn around each nucleus that was then used to crop areas around individual nuclei. The boundary of each nucleus was then marked using a hand-drawn polygon. A masked movie was then created for each nucleus in which regions outside the nucleus were set to 0 grayscale values so that all of the analyses described below were performed only on molecules within nuclear regions.−6. For tracking, the maximum expected diffusion coefficient was set to 5 µm2/sec, the maximum number of competitors was set to 1, and the maximum off/blinking frames was set to 1.Localization and tracking of single molecules were conducted using a Matlab implementation of the MTT algorithm . In brieThe RTs were estimated from the 100-msec data using the results from the single-particle tracking using the MTT algorithm. The data were pooled into bins corresponding to the position along the A–P axis of the embryo in one-third fractions of the EL (0–1 anterior to posterior) with the following number of nuclei and single-molecule trajectories per position bin: anterior: 34 nuclei, 17,735 trajectories; middle: 70 nuclei, 40,092 trajectories; and posterior: 83 nuclei, 20,823 trajectories.In the ZLD embryos, we measured the following number of nuclei and single-molecule trajectories per position bin: anterior: 23 nuclei, 11,415 trajectories; middle: 31 nuclei, 7572 trajectories; and posterior: 31 nuclei, 3606 trajectories.Supplemental Figs. S3, S4). The model used to fit the data and calculate the time constants and fraction of the population wasks and kns are the low (specific) and high (nonspecific) off rates, respectively. The total pooled data sets of 78,650 trajectories from the MTT results from 187 nuclei were also fit in the same manner.The survival probability distribution was then calculated as 1 − the cumulative distribution function of the trajectory lengths and was fit to both single- and double-exponential models . The douHis2Av-eGFP to estimate the bleaching constant (0.00426 sec−1) and correct the off rate as ks,corrected = ks−kbleach.. We note that the bleaching correction had a minimal effect on our estimated off rates. The fact that we were not limited by bleaching due to the transient nature of BCD binding was further validated through even longer, 500-msec exposure time measurements on 17 nuclei, which provided an estimate for the specific and nonspecific off rates that did not differ significantly from those measured at 100 msec . The results of the fits to all data are shown in Supplemental Table S1.To correct for photobleaching, we used Experiments were performed on a Zeiss LSM 800 laser scanning confocal system (coupled to a Zeiss Axio Observer Z1) using a plan-achromat 63×/1.4 NA oil immersion objective, a GaAsP-PMT detector, and a 488-nm laser. A circular bleach region with a diameter of 1.5 µm was used, the bleach location was selected manually in each nucleus at approximately the center, and a total bleach time of 78.1 msec was used. Data were acquired for at least 1 sec prior to bleaching and at least 6 sec after bleaching, with a time interval of 0.430 msec. Experiments were performed using live embryos from the same fly line as for lattice light-sheet imaging and were collected and prepared in the same manner as described above with the exception of the mounting procedure. For FRAP, the embryos were mounted between a semipermeable membrane  and a coverslip and then embedded in Halocarbon 27 oil (Sigma). As in the case of the single-molecule measurements, the embryos were staged using the HIS2-AV-MRFP channel, and all data were acquired on embryos in nuclear cycle 13. The data were acquired on 21 nuclei, all within the first 25% of the embryonic length from the anterior pole. FRAP experiments could not be performed at more anterior locations due to the low BCD concentrations and thus signal to noise ratio.For analysis, the spatial and temporal locations of the bleach region were retrieved from the metadata recorded by the microscope control software and manually verified by inspecting the data. Due to the short duration of the movies and rapid recovery, drift correction was not necessary. Each nucleus was manually segmented from the rest of the image by defining a polygon region of interest. A region of interest the same size as the bleach region was also marked in an area of each image outside of the nuclear region to be used to measure the background or dark intensity. The FRAP curve for each nucleus was then calculated as follows:Inuc(t), bleach region, Ibleach(t), and background region, Idark(t), was calculated at each frame.First, the mean intensity in the nuclear region, Iprebleach). The final FRAP curve for each nucleus was thus calculated as FRAP(t) = {[Ibleach(t) − Idark(t)]/[Inuc(t) − Idark(t)]}Iprebleach.The background intensity was then subtracted from both the mean nuclear and bleach region intensities. To correct for photobleaching from imaging, the bleach region intensity was then divided by the mean nuclear intensity at each time point. The resulting photobleaching-corrected intensity was then normalized to the mean prebleach intensity, calculated as the average intensity in all frames prior to bleaching (ka × t)] and double-exponential [1 − A × exp(−ka × t) − B × exp(−ka × t)] model  to measure the recovery time constants. There was no significant difference in the quality of fit between the two models. For comparison, the exact same experimental and analysis procedure was followed for His2AV-MRFP1  in the same embryos with the exception of using a 561-nm bleach laser; three nuclei were measured with these settings. For the histone measurements, the two-exponent fit was significantly better than the one-exponent, as expected.The calculated FRAP curves for each nucleus were then aligned to the bleach frame and averaged to generate the average FRAP curve shown in Single-molecule trajectories were analyzed as described above. A total of 158 nuclei from four embryos was analyzed. The data from nuclei were binned according to their position along the A–P axis in one-third fractions of the EL  with the following number of nuclei and single-molecule displacements per bin: In the anterior-most (0–0.2) positions, tracking could not be performed reliably due to the high concentrations of BCD at those locations at these high frame rates and presumably a large mobile population: anterior: 30 nuclei, 12,923 trajectories; middle: 67 nuclei, 23,640 trajectories; and posterior: 66 nuclei; 8600 trajectories.Supplemental Fig. S6); displacements corresponding to distances <225 nm were scored as part of the bound population. In the second approach, the probability density functions of the displacements were fit to a two-population model :Fbound is the fraction of the population that is considered bound, and r is the displacement distance. The two-population model fits the displacement data with R2 values of 0.97, 0.98, and 0.96 for the anterior, middle, and posterior distributions, respectively, and provides an estimate of the fraction present in the mobile and bound populations. The trend of an increase in the population-bound estimate in the middle and posterior positions relative to the anterior is similar from both approaches. Although the bound population includes nonspecific binding events as well, we were interested in the relative change across the A–P axis.The fraction of the population that is bound versus mobile was estimated using two approaches. First, a cumulative distribution function of the displacements was calculated for each EL bin  of the widely used DBSCAN was used with a minimum points setting of 10 points and an ε (maximum radius of neighborhood) setting of 0.8. These settings were empirically determined by iteratively changing parameters and examining the results. A balance had to be struck in the ability to accurately identify clusters in the high-density situations in anterior nuclei and also low-density situations in the posterior. Only data sets with time spans of at least 105 sec were included. Totals of 12, 49, and 48 nuclei and 436, 1168, and 367 clusters were analyzed in this manner for the anterior, middle, and posterior positions, respectively. Examples of clusters identified in nuclei at various positions are shown in Supplemental Figure S7. For comparing distributions, outliers were removed (<5th or >95th percentile) to disregard clusters that were significantly overpartitioned or underpartitioned. In the case of the ZLD− embryos, due to the loss of apparent clustering, DBSCAN was not able to detect clusters, so instead the spatial distribution of points was compared using pair correlation analysis as described below to provide insight into the change beyond visual examination.The clustering of BCD-binding events in the 100-msec wild-type embryo data set is readily apparent in nuclei across the A–P axis in the projection of all localizations from the MTT algorithm B. In ordzld embryos. A Matlab implementation was used to calculate the correlation function for the spatial distribution in each nucleus. The results were then averaged for each position  embryo for comparison . To generate simulations of randomly distributed points, spatial coordinates of detections were generated randomly (using a Matlab script and random draws from a uniform distribution) to lie within a 5-μm disc, and simulations were performed with 5000, 3000, and 1000 points to correspond to detections at anterior, middle, and posterior positions.The pair correlation function for the spatial distribution of particles computes the probability of finding a particle at the range of distances from other particles. In the case of complete spatial randomness, which can be represented by a Poisson process, the pair correlation function is equal to 1. The analysis is conducted on point lists generated from the MTT algorithm using the first spatial coordinate of each trajectory. Totals of 22, 48, and 42 nuclei were analyzed for the anterior, middle, and posterior positions, respectively, for the wild-type embryos and totals of 23, 31, and 31 nuclei were analyzed for the anterior, middle, and posterior positions, respectively, for the Drosophila pseudoobscura embryos at stage 5 (to serve as carrier) and homogenized in homogenization buffer containing 15 mM NaCl, 15 mM Tris-HCl (pH 7.5), 60 mM KCl, 1 mM EDTA, and 0.1% Triton X-100, with 1 mM DTT, 0.1 mM PMSF, and protease inhibit cocktail (Roche) added before use. After homogenization, 0.5% NP40 was added, and, following a 5-min incubation, samples were spun down at a low speed. Nuclei in the pellet were then washed once with the homogenization buffer containing 0.2 M NaCl. The low-speed centrifugation was repeated, and the recovered nucleus pellet was then resuspended in nuclear lysis buffer , 1% SDS, and 1.5% sarkosyl. The chromatin was recovered by spinning the sample at full speed in a microcentrifuge for 1 h at 4°C and was resuspended in a small volume of nuclear lysis buffer. Chromatin was sheared to an average size of 300 base pairs (bp) using a Covaris sonicator . ChIP was performed using 72 ng of chromatin and 1.5 µg of an anti-BCD polyclonal antibody described previously (D. pseudoobscura (Flybase release 1.0) and D. melanogaster (Flybase release 5) genome using Bowtie with options set at -5 5 -3 5 -n 2 -x 2000. The aligned reads were converted to WIG files using custom scripts available on request. WIG files were normalized to 10 million mapped reads. The data have been deposited in the NCBI Gene Expression Omnibus (GEO) database with accession number GSE103695.Embryos were collected from a population cage for 90 min and then aged for 2 h in order to enrich for embryos at developmental stage 5. Embryos were then fixed with formaldehyde as described previously  and sorteviously . The BCDThe posterior embryo ChIP-seq data were compared with published data on whole embryos from the same developmental stage  download


CAM4066, a specific CK2α kinase inhibitor, is anchored in the cryptic αD pocket outside the active site and inserts a “warhead” into the active site, blocking ATP binding and thereby inhibiting the kinase. Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a  Despite many clinical success stories, the strong conservation of the ATP binding site across the kinase family makes it challenging to achieve selectivity.2 For example, imatinib inhibits not only its original target, Abl kinase, but eight other kinases with low nanomolar inhibitory constants.3 Therefore, inhibitors must derive selectivity from less conserved features of the kinase.4Protein kinases are key regulators of many cellular processes and are thus one of the main protein classes targeted in modern drug discovery.9 CK2α provides pro-survival and anti-apoptotic effects on the cells, and is often overexpressed in cancer cells, promoting their proliferation by multiple mechanisms such as potentiation of the Akt pathway and activation of NF-Kβ.11 Cancer cells therefore rely on CK2α's activity to survive, becoming addicted to high levels of its non-oncogenic activity. This is particularly true for cells that are challenged with chemotherapeutic agents and the most promising uses for CK2α stem from these observations. Indeed, there have been many successful studies where CK2α inhibitors have been used in combination with established drugs and synergy has been demonstrated between them.17 In several recent examples, it has been shown that CK2α inhibitors can re-sensitize cells to treatments that they have become resistant to.18 For these reasons, selective inhibition of CK2α appears to be a promising strategy for cancer therapy.19 A number of potent ATP-competitive CK2α inhibitors21 have been shown to inhibit the growth of cancer cell lines and one of these, CX4945, has progressed to phase II clinical trials ,26 a more open αD pocket can be observed, but no ligand is bound to it and no comment was made in the paper describing it. Nevertheless, the real size and potential of this pocket has only been revealed upon binding of our fragment , however, 6 was chosen as the fragment to link to 4 as it showed no inhibition of phosphorylation at 500 μM or detectable binding by ITC, but was clearly observed to bind in the crystal structure. The idea being that the binding to CK2α should not be dominated by the interaction with the conserved ATP site but instead by the interaction with the αD site. Several fragments were found that interacted with the hinge region, however, these were later found to be unsuitable for progression as the movement of Met168 (4 and 6 was investigated by the extension of 4 into the αD site with the synthesis of a series of compounds extending towards the ATP site (details to be published elsewhere) and eventually linking with 6. The structure of the resulting compound CAM4066  is positioned in the same place as CX4945 would bind , neither of which showed any inhibition at 500 μM.The s 320 nM , correspf 370 nM . This is50. At this concentration CK2α was inhibited by 75% (s.d. 19%), whereas no other kinases in the panel showed significant inhibition by CAM4066 .30 Indeed, the IC50 of CX4945 against Clk2 was reported to be 3.8 nM which is more potent than that reported against CK2α itself, in that study .30 D11 inhibits 61 other kinases by 70% or greater at 10 μM (354 kinases)31 and TF inhibits 7 other kinases by 70% or greater at 10 μM (61 kinases).32 HIPK2, HIPK3 and PIM1 are noteworthy as they are significantly inhibited by four (HIPK2-3) or 3 (PIM1) of these inhibitors,32 but show only 0% ± 4, and 3% ± 4 and –5% ± 5 inhibition, respectively, by CAM4066 at 2 μM. The off-target activities of these CK2α inhibitors are more significant when the drop-off in activity against cancer cell lines is considered. For all these inhibitors there is at least a 1000 fold reduction in activity against cell lines,32 compared to the inhibition/affinity against the isolated protein. The inhibitors are therefore being administered at concentrations higher than those that are known to cause significant inhibition of a large number of off target kinases. Thus, our initial data validates the novel strategy of targeting the newly discovered αD site. Furthermore, this data reveals the promise offered by further development of compounds targeting the αD site rather than the traditional method of targeting the ATP site.Although Clk2 itself is not included within our panel, several closely related kinases in the CMGC family are . None of these related kinases are inhibited by CAM4066. Added to this information, analysis of the crystal structure of Clk2 indicates that it is unlikely to have an αD pocket that could accommodate CAM4066, especially with the side chain of Tyr374 blocking the bottom of that site  in contr8), was used for the cellular assays, as esters are the most common form of pro-drug.34 This approach proved to be successful, and pro-CAM4066 showed good dose responses in HCT116, Jurkat and A549 cells, with a GI50 of 9, 6 and 20 μM, respectively  inhibition of the phosphorylation of AKT1 Ser129 after 24 hours. However, at later time points of 48 and 72 hours, pro-CAM4066 inhibited the phosphorylation of this targets at a similar level to CX4945 (35 It is possible that this delay in inhibition, when compared to CX4945, is because the hydrolysis of pro-CAM4066 to release the active compound CAM4066 delays the achievement of pharmacologically active concentrations within cells which delays the onset of inhibition.This compares favorably with the clinical trial compound CX4945, which shows GIo CX4945 . In contIn conclusion, we have used high concentration fragment soaks to probe the surface of CK2α leading to the identification of a cryptic pocket close to the ATP site. Fragments that bound in this cryptic pocket were linked successfully to a low affinity fragment in the ATP site. This quickly and efficiently generated a potent and highly selective inhibitor of CK2α, and validated the use of this cryptic pocket for the further development of CK2α inhibitors. Most significantly, we have fulfilled our initial aim and shown that it is possible to achieve higher levels of selectivity towards CK2α than previously reported for any inhibitor by interacting with features outside the active site. This can be rationalized by the low level of conservation around the αD pocket in kinases, compared to significantly higher similarity seen in the ATP site. The strategy of using such cryptic pockets outside of the active site to gain selectivity for inhibitors could be applied to other protein classes where selectivity is hard to achieve because of the high level of sequence conservation in the active site. This strategy could also be used to target orthologous proteins from pathogenic organisms to gain selectivity over host proteins.


ECL1, R80ECL1, and Y893.29) are important in the hTGR5–nomilin interaction. Based on these results, an hTGR5–nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5–nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5–nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse–human chimeric TGR5, we also found that three amino acid residues (Q77 TGR5, also known as G protein-coupled bile acid receptor 1 (GPBAR1), is a class A G protein-coupled receptor (GPCR) comprising seven transmembrane helices (TM), three extracellular loops (ECL), and three intracellular loops (ICL). TGR5 recognizes bile acids (BA) as its endogenous ligands , and ligTGR5 is expressed in different tissues and organs, including brown adipose tissue (BAT), small intestine and skeletal muscle . WatanabRecently, a number of novel TGR5 agonists were described , 14–18. ECL1, R80ECL1, and Y893.29. On the basis of these results, we constructed the hTGR5–nomilin binding model, which suggests that the molecular structure of nomilin is important to its being an hTGR5 agonist. Obacunone, a limonoid with a molecular structure similar to nomilin, also exhibited semi-specific hTGR5 ligand activity depending on the three amino acid residues mentioned earlier. Notably, the binding pattern between hTGR5 and these limonoids was different from that between hTGR5 and taurolithocholic acid (TLCA) and INT-777 [In the present study, we planned to determine key amino acid residues in the TGR5–nomilin interaction and identify the molecular features of nomilin necessary for it to be recognized as a TGR5 agonist. We found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5) regardless of their high homology. Considering this, several mouse–human TGR5 chimeras were developed to ascertain the critical region for nomilin response. Point mutation studies on the identified region revealed three key hTGR5 residues essential for hTGR5–nomilin interaction: Q77 INT-777 , 20. TheNomilin was purchased from Tokyo Chemical Industry Co., obacunone was purchased from ChromaDex, and TLCA and limonin was purchased from Sigma.The pCRE-Luc reporter plasmid, which contains four copies of the consensus sites of CRE, was purchased from Agilent Technologies. Mouse–human TGR5 chimeras (1 and 2) were constructed by using the primer set shown in Table A in HEK293 cells obtained from ATCC were cultured at 37°C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) fetal bovine serum (FBS).5 cells/well. Twenty hours later, the cells were transfected with pCRE-Luc, p3×FLAG-CMV-TGR5, and pEF-β-galactosidase (100 ng/well each) using the calcium phosphate method. After 4 h of incubation, the medium was replaced with DMEM, which contained 10% dextran charcoal-stripped FBS. Twenty-four hours after transfection, the cells were treated with the previously mentioned compounds and control vehicle (DMSO). After 5 h, the cells were then incubated with lysis buffer  on ice, and the luciferase and β-galactosidase activity were measured. The luciferase activity values were divided by those of β-galactosidase to obtain normalized luciferase values.HEK293 cells were plated in 12-well plates at a density of 1.0 × 10A 3D molecular model of hTGR5 was constructed based on metarhodopsin structure , an actiThe initial complex structures were energy-minimized and then optimized using molecular mechanics and dynamics calculations, tethering the α-carbon of the main chain in the TM regions at the initial positions. The final complex structures were then structure-optimized in the lipid bilayer model installed in AMBER software.All data are presented as mean ± standard deviation (SD). Two-tailed unpaired Student’s t-tests and one-way ANOVA (Tukey’s post hoc test) were used to determine p-values. Statistical significance was set at p < 0.05.We previously reported that nomilin, which is a limonoid frequently found in citrus plants , has theTo identify candidate amino acid residues that are crucial for nomilin recognition, various human–mouse TGR5 chimeras were developed and analyzed for their responsiveness to TLCA and nomilin. Initially, we constructed two versions of chimeric TGR5: chimera-1  and chimera-2  . BecauseECL1, Q77ECL1, S78ECL1, R79ECL1, R80ECL1, V883.28, Y893.29, A913.31, and S953.35 in hTGR5—were identified as candidate residues for playing a key role in hTGR5–nomilin interaction, which satisfies the following conditions: (1) they are evolutionarily unconserved between hTGR5 and mTGR5, and (2) they exist in ECL1 or TM3. To assess the contribution of these nine residues to nomilin response, nine mouse-to-human one-point mutants were constructed: mTGR5 S75NECL1, R76QECL1, N77SECL1, H78RECL1, Q79RECL1, L87V3.28, H88Y3.29, T90A3.31, and C94S3.35. First, the effects of one-point mutation in ECL1 on nomilin recognition were evaluated using CRE-luciferase reporter assay . Notably, mTGR5-R76QECL1/Q79RECL1/H88Y3.29 exhibited strong nomilin response equal to that of hTGR5-WT . As expected, alanine-mutated hTGR5 shows a very weak nomilin response relative to WT-hTGR5  values from dose-response curves for nomilin and TLCA to measure the affinity of these agonists for human-to mouse and mouse-to human triple mutated TGR5    . The EC55.99 μM) . Taken tECL1, R80ECL1, and Y893.29 (shown in red-dashed line), resulting in the activation of hTGR5. The binding of nomilin in this study showed that it binds in the mode vertical to the membrane. This mode is different from that in the complexes reported by Macchiarulo et al in which rhodopsin, an inactive GPCR, was used as the template for the TGR5 receptor [On the basis of these findings, we constructed an hTGR5–nomilin binding model in silico. Because the structural model of nomilin-bound hTGR5 should be constructed from its activated form, we selected metarhodopsin II , one of receptor . This maECL1, R80ECL1, and Y893.29 in hTGR5   . These rECL1 and S78ECL1  were identified as key residues for nomilin response . The values represent the mean ± SD.Transient transfection assays using HEK293 cells with a CRE-luciferase reporter plasmid and an expression vector for TGR5 with indicated point mutation. Twenty-four hours after transfection, cells were treated with TLCA or nomilin (100 μM each) for another 5 h. Then, luciferase reporter activity was quantified (hTGR5/DMSO was set at 1) (n = 3). Significant differences between the nomilin responses were analyzed using one-way ANOVA (Tukey’s post hoc test) (*(PDF)Click here for additional data file.S2 Figp < 0.05; **p < 0.01 for hTGR5). The values represent the mean ± SD.Transient transfection assays using HEK293 cells with a CRE-luciferase reporter plasmid and an expression vector for triple alanine mutated TGR5. Twenty-four hours after transfection, cells were treated with TLCA or nomilin (100 μM each) for another 5 h. Then, luciferase reporter activity was quantified (hTGR5/DMSO was set at 1) (n = 3). Significant differences in nomilin treated group were analyzed using one-way ANOVA (Tukey’s post hoc test) (*(PDF)Click here for additional data file.S3 Figp < 0.01. The values represent the mean ± SD.(A) Comparison of binding pattern of hTGR5 and limonin (cyan). Steric repulsion formed between limonin and hTGR5 is indicated by red double-headed arrows. (B) HEK293 cells were transfected with the CRE-driven luciferase reporter plasmid and the hTGR5 expression plasmid. After transfection for 24 h, the cells were treated with TLCA (positive control) and limonin (100μM each) for another 5 h. Then, luciferase reporter activity was quantified (hTGR5 (-)/DMSO was set at 1) (n = 3). Significant differences were analyzed using one-way ANOVA (Tukey’s post hoc test); **(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen . Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species ( Acetylcholinesterase  is a serine hydrolase enzyme found predominantly in muscle tissue and the nervous system, and controls the propagation of neuronal action potential through the hydrolysis of the neurotransmitter acetylcholine. This enzyme has a high affinity for choline esters, but is also able to hydrolyze other esters, such as organophosphate compounds (OP), at lower hydrolysis rate  (%)  (%) .-1.cm-1 at 401 nm /[E0]) and incubation time, indicating a first-order reaction between the enzyme and inhibitor.Inhibition kinetic constants (IKC) were obtained using the methods described by Kemp and Wallace  and CarrMugil liza, from 7 to 34 μM for Genidens genidens, from 7 to 41 μM for Lagocephalus laevigatus and from 0.069 to 0.339 μM for Gallus domesticus), with 0.45 mL of the enzyme solution containing around 5–6 mU of AChE in sodium phosphate buffer, pH 7.5, plus 0.1 g% Triton X-100. After 1, 2, 3, 4 and 5 min of incubation, enzyme inhibition was stopped by adding 0.5 mL of 0.64 mM DTNB-3.75 mM acetylthiocholine  in sodium phosphate buffer 0.1 M, pH 7.5, plus 0.1 g% of Triton X-100 prepared prior to use. The residual enzyme activities [Er] were evaluated. Control enzyme activities [E0] were measured by adding the pesticide after the substrate. Regressions were calculated with MP concentrations that resulted in 10%-90% enzyme inhibitions, as indicated by Kemp and Wallace /[E0]) over time (plot) and linear regressions of double-reciprocals plots (replots) were considered only when R2 was higher than 0.95. As a measure of enzyme sensitivity to MP, the IC50 was calculated for each species for 30 min of incubation using the following equation One Unit of AChE (U) is the amount of enzyme that forms one μmol of products per minute. The Coefficient of Determination (Resticide . The lin)(ki*30) .Gallus domesticus brain AChE, an example of a highly sensitive enzyme, was compared to brain AChE from fish (Mugil liza), less sensitive. Curves were fitted as proposed by Radic et al. [Inhibitions by excess substrate  and the c et al.  for inhiEnzyme activity towards acetylthiocholine with progressive inhibition in excess of substrate is described as characteristic AChE property , 23 indiThese results demonstrated the predicted activity inhibition in high substrate concentration , and theDifferent MP concentrations were evaluated for the IKC calculations in the AChE inhibition assay and for the animal comparisons .kapp linearity (R2 > 0.95) was observed, indicating adequate MP concentrations for IKC calculations, according to Kemp and Wallace [For all replots, a minimum of 10 and a maximum of 90% AChE inhibition was observed. Higher inhibitions led to results out of the range of the plotted straight line. For all inhibitor concentrations tested herein, with the exception of one hen assay, kapp were observed for all investigated species, both fish and hen. Linear regressions presented different slopes, with the exception of L. laevigatus and M. liza, that showed similar slopes , which is only possible with proportional increments or decrements in kp and Kd values . As displayed in G. genidens) AChE presented higher sensitivity to MP inhibition when compared to the other evaluated fish species. This was confirmed by the IKC values and the calculated IC50 for this species  AChE to inhibition by MP is revealed by the ki value, three times higher than for the other evaluated fishes. However, puffer fish (L. laevigatus), and catfish (G. genidens) showed similar kp values. These differences must, therefore, be related to differences in AChE affinity to the inhibitor MP. Indeed, the catfish enzyme dissociation constant (Kd) was about three times lower than puffer fish (L. laevigatus), and 8 times lower than mullet (M. liza), confirming the fact that catfish AChE sensitivity to MP is higher as a consequence of a higher enzyme affinity for the inhibitor, expressed as Kd differences [The greater sensitivity of Catfish , puffer fish (L. laevigatus), and hen , with a slightly higher value for mullet (M. liza). However, ki and Kd values differed significantly between animals. Hen AChE presented higher sensitivity to MP, as indicated by a lower IC50 value.Similar AChE enzyme M. liza) and puffer fish (L. laevigatus) showed about the same sensitivity (ki) to MP, as indicated by the parallel lines displayed in kp and Kd values for brain mullet and puffer fish AChE (ki is the slope of these contants (kp/Kd) this leads to similar ki and, hence, similar AChE sensitivity. Hen  AChE IKC showed a much higher ki value (Kd value and, therefore, hen AChE presented a much higher affinity to MP when compared to fish brain AChE.Brain AChE from mullet (sitivity ki to MPish AChE  were difki value , and thuAChE sensitivity to MP, expressed as IC50, and substrate specificity, expressed as P% , confirmKd observed for brain AChE in some fish and vertebrates such as hen and rat, with similar kp values [The low variability of the catalytic center residues indicate that the enzyme mechanism was subjected to a strong selection under natural conditions, suggesting that sensitivity variability is due to affinity differences based on changes in peripheral residues of aromatic gorge or anionic center, and not based on catalytic center residues. This is further supported by the higher variability of the p values . In lighKd values. This may be due to a reduced access to the catalytic center, providing greater substrate selectivity for fish brain AChE.The results of the present study demonstrate that the insoluble fraction of brain AChE with detergents provides suitable enzyme preparations with a simple method for accurate enzyme assays eliminating complex purification steps. Furthermore, this study demonstrated that terrestrial vertebrate brain AChE enzymes appear to significantly more sensitive to inhibitors and much less specific to artificial substrates than fish brain AChE that demonstrated low affinity, as expressed by the high


Orthologues of AMP-activated protein kinase (AMPK) occur in essentially all eukaryotes as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. The canonical role of AMPK is as an energy sensor, monitoring levels of the nucleotides AMP, ADP, and ATP that bind competitively to the γ subunit. Once activated, AMPK acts to restore energy homeostasis by switching on alternate ATP-generating catabolic pathways while switching off ATP-consuming anabolic pathways. However, its ancestral role in unicellular eukaryotes may have been in sensing of glucose rather than energy. In this article, we discuss a few interesting recent developments in the AMPK field. Firstly, we review recent findings on the canonical pathway by which AMPK is regulated by adenine nucleotides. Secondly, AMPK is now known to be activated in mammalian cells by glucose starvation by a mechanism that occurs in the absence of changes in adenine nucleotides, involving the formation of complexes with Axin and LKB1 on the surface of the lysosome. Thirdly, in addition to containing the nucleotide-binding sites on the γ subunits, AMPK heterotrimers contain a site for binding of allosteric activators termed the allosteric drug and metabolite (ADaM) site. A large number of synthetic activators, some of which show promise as hypoglycaemic agents in pre-clinical studies, have now been shown to bind there. Fourthly, some kinase inhibitors paradoxically activate AMPK, including one (SU6656) that binds in the catalytic site. Finally, although downstream targets originally identified for AMPK were mainly concerned with metabolism, recently identified targets have roles in such diverse areas as mitochondrial fission, integrity of epithelial cell layers, and angiogenesis. The catalytic subunits  contain kinase domains (α-KDs) at their N-termini, and the small N-lobe and larger C-lobe . Thr172 phosphorylation is brought about by one of two principal upstream kinases: the tumour suppressor LKB1 or the Ca2+/calmodulin-activated kinase CaMKK2 (CaMKK-β). The α-KD is followed by an auto-inhibitory domain  that maintains the α-KD in an inactive conformation in the absence of AMP5. The α-AID is connected to the globular C-terminal domain  by a region of more extended polypeptide termed the α-linker8 (in dark blue). The β subunits (β1 or β2) contain myristoylated N-terminal regions9 . The latter forms the core of the complex by cross-linking the α-CTD to the N-terminal region of the γ subunit12. The γ subunits  contain N-terminal regions of variable length, followed by four tandem repeats of sequence motifs known as cystathionine-beta-synthase (CBS) repeats that generate the binding sites for the regulatory nucleotides AMP, ADP, and ATP13. The two pairs of repeats (CBS1:CBS2 and CBS3:CBS4) assemble head-to-head to form a disc-like structure with one repeat in each quadrant14  completely abolish activation by AMP, while in the active AMP-bound conformation the α-linker makes close contacts with residues that bind AMP in this site12  is best known as a sensor of cellular energy status in vertebrate cellssite12 . Moreove18. Under these circumstances, AMPK is activated by three complementary mechanisms: (i) binding of AMP or ADP promotes Thr172 phosphorylation by LKB1 , (ii) binding of AMP or ADP inhibits Thr172 dephosphorylation by protein phosphatases19, and (iii) binding of AMP but not ADP causes an allosteric activation of up to 10-fold19. The structural data suggest plausible mechanisms to explain mechanisms (ii) and (iii) since there is evidence that displacement of AMP by ATP at site 3 triggers dissociation of the α-linker from the γ subunit, causing the catalytic and nucleotide-binding modules to move apart6 (see previous section). This may not only allow the α-AID to rotate back into its inhibitory position behind the kinase domain5 but also expose Thr172, which in the compact conformation is partly buried in a deep cleft between the two modules6  or that stimulate ATP consumption  increase the cellular ADP:ATP ratio, analogous to the “battery” of the cell running flat. This is invariably accompanied by even larger increases in the AMP:ATP ratio, which are due to displacement of the adenylate kinase reaction (2ADP ↔ ATP + AMP)dules6 , thus al19, suggesting that complexes at different locations could be tuned to respond differently to changes in adenine nucleotides, depending on which γ subunit isoform is present.Interestingly, AMPK complexes with different γ subunit isoforms  display subtle variations in their responses to increases in AMP and ADP21) normally appears to be constitutively active22, and the regulation of AMPK phosphorylation appears to be modulated instead by binding of adenine nucleotides to AMPK. However, AMPK activation by energy stress does not occur in many tumour cells that lack LKB1  because the basal activity of CaMKK2 is too low to trigger significant Thr172 phosphorylation23. The CaMKK2-AMPK pathway represents instead an alternate Ca2+-activated pathway that mediates AMPK activation by hormones that release Ca2+ from intracellular stores, such as thrombin24 or ghrelin25.The LKB1 complex  almost exclusively to generate ATP. When glucose runs low, yeast needs to switch on (i) genes required for metabolism of other fermentable carbon sources such as sucrose or (ii) genes of mitochondrial oxidative metabolism, which are required for growth on low concentrations of glucose or on non-fermentable carbon sources such as ethanol. None of these metabolic adaptations occurs in the absence of genes encoding the α, β, or γ subunits of the AMPK orthologue, termed the SNF1 complex26. The SNF1 complex is activated upon glucose starvation28 by phosphorylation of the threonine residue equivalent to Thr172 (Thr210)29. Although glucose starvation is accompanied by large changes in the cellular ratios of AMP:ATP and ADP:ATP, the yeast SNF1 complex is not allosterically activated by AMP28. Neither phosphorylation nor dephosphorylation of Thr210 appears to be sensitive to AMP either, although dephosphorylation may be inhibited by ADP30. Thus, the ancestral role of the AMPK orthologue in unicellular fungi appears to have been in response to glucose starvation, but it remains unclear whether changes in adenine nucleotides are the crucial signals or whether there is instead some mechanism by which the SNF1 complex responds more directly to the availability of glucose.When the yeast31, but it had generally been assumed that this effect was mediated by the canonical energy stress mechanism . However, recent studies suggest that the mammalian kinase may be able to sense glucose by a non-canonical mechanism independently of changes in adenine nucleotides. The first clue came with unexpected findings that Axin  forms a ternary complex with LKB1 and AMPK in response to glucose starvation, thus bringing the upstream and downstream kinases together and promoting Thr172 phosphorylation32. AMPK activation, both in mouse embryo fibroblasts (MEFs) starved of glucosein vitro and in livers of mice starvedin vivo, was subsequently found to require not only Axin32 but also Lamtor133, a resident lysosomal protein that associates with the vacuolar ATPase (v-ATPase). Lamtor1 is a component of the pentameric Ragulator complex, which acts as a guanine nucleotide exchange factor (GEF) for RagA or RagB, whose GTP-bound forms trigger translocation of mTORC1 to the lysosome where it is activated34. Axin, along with bound LKB1, was found to translocate to the surface of the lysosome upon glucose starvation, and these results suggested a model in which glucose deprivation led to the formation of a lysosomal complex involving v-ATPase, the Ragulator, Axin, LKB1, and AMPK, thus triggering AMPK activation33. AMPK is known to phosphorylate Raptor (a key component of mTORC1) as well as the upstream regulator TSC2, thus causing rapid inhibition of mTORC136. However, glucose starvation still suppresses mTORC1 even in TSC2- and AMPK-null MEFs by causing dissociation of mTORC1 from the lysosome in a Rag GTPase-dependent manner38. Importantly, knockout of Axin in MEFs led to prolonged activation and much slower dissociation of mTORC1 from the lysosome after glucose starvation, most likely due to the ability of Axin to inhibit the GEF activity of the Ragulator complex33. The ability of Axin to negatively regulate mTORC1 may also account for some of the beneficial roles of metformin39. Taken together, these new findings suggest that the regulation of the AMPK and mTORC1 signalling pathways is much more closely intertwined than previously realised.It has been known for many years that glucose deprivation activates AMPK in mammalian cells32, it now appears that AMPK can sense glucose starvation independently of changes in adenine nucleotides40. In MEFs, removing glucose from the medium (as long as glutamine and pyruvate were still present) caused rapid AMPK activation without any changes in AMP:ATP or ADP:ATP ratios. The AMPK activation that occurred upon glucose removal, but not the larger activation that occurred following energy stress , was dependent on Axin, Lamtor1, and also N-terminal myristoylation of the AMPK-β subunits, and the last of these was required for lysosomal localisation of AMPK. The ability of glucose to repress AMPK activation required its metabolism by glycolysis as far as fructose-1,6-bisphosphate (FBP), and the sensor for glucose availability appears to be the glycolytic enzyme that metabolises FBP, i.e. aldolase40. Intriguingly, aldolase has previously been shown to associate with the lysosomal v-ATPase in both yeast and mammalian cells43. These findings led to a model in which the lack of availability of glucose, and hence FBP, causes changes in the interaction between aldolase and the v-ATPase, promoting the formation of a complex between the Ragulator, Axin, LKB1, and AMPK  and may exert some of the therapeutic effects of that drug. Salicylate is currently the only natural product known to bind this site on AMPK, but there is much speculation in the field that there may be a naturally occurring metabolite from animal cells that binds there, which is why it has been termed the allosteric drug and metabolite (ADaM) site55.The β-CBM is a member of the CBM20 family of carbohydrate-binding modules, which are non-catalytic domains that usually occur in proteins that metabolise starch or glycogen. The β-CBM causes a proportion of mammalian AMPK to associate with glycogen particles in intact cells56 and sorafenib58. SU6656 was developed as an inhibitor of Src family kinases such as Src, Yes, and Fyn and was proposed to activate AMPK by inhibiting phosphorylation of tyrosine residues on LKB156 or AMPK59 by Fyn. However, activation of AMPK by SU6656 does not require phosphorylation of these tyrosine residues or even the presence of an Src family kinase in the cells60. SU6656 is in fact a potent inhibitor of AMPK that binds at the catalytic site in competition with ATP, but this paradoxically causes a conformational change that promotes Thr172 phosphorylation by LKB1. This mechanism would still promote phosphorylation of downstream targets of AMPK as long as the lifetime of Thr172 phosphorylation was sufficient for SU6656 to dissociate and for one or more catalytic events to occur prior to Thr172 dephosphorylation. Indeed, SU6656 promotes the phosphorylation of the downstream target acetyl-CoA carboxylase in intact cells60.It has recently been reported that AMPK is paradoxically activated by two kinase inhibitors: SU665661. Sorafenib also paradoxically activates AMPK58, but this is because sorafenib, in contrast to SU6656, is an inhibitor of the mitochondrial respiratory chain, so that it activates AMPK indirectly by increasing cellular AMP:ATP ratios60.Sorafenib was originally developed as an inhibitor of tyrosine kinases and is used clinically for the treatment of hepatocellular carcinoma as well as advanced renal cell or thyroid carcinoma62 XXS/TXXXΦ62 , where Φ64. Thus, respiratory chain inhibitors cause mitochondrial fission, possibly as a means of segregating regions of mitochondria that have undergone oxidative damage for subsequent autophagy and recycling. Mitochondria also become more fragmented in mitotic cells, perhaps to ensure even distribution to daughter cells. On the other hand, mitochondrial fusion into larger networks tends to occur in quiescent cells, which are more reliant on oxidative metabolism for ATP generation. Since mitochondria are the major suppliers of ATP in most quiescent cells and since AMPK knockout cells are known to accumulate abnormal mitochondria67, it is perhaps not surprising that AMPK should have a key role in mitochondrial dynamics. Indeed, in either U2OS cells or MEFs that lack both catalytic subunits of AMPK, the effects of mitochondrial inhibitors to trigger mitochondrial fission were attenuated68. Moreover, compounds such as A-769662 (which activate AMPK by binding at the ADaM site) caused mitochondrial fission in the absence of energy stress. Fission is triggered by the GTPase dynamin-related protein-1 (DRP1), which is recruited to mitochondria in part by the mitochondrial fission factor (MFF). The latter is phosphorylated at two sites—Ser155 and Ser172—by AMPK  epithelial cells and that AMPK activators also protected tight junctions from disassembly induced by Ca2+ depletion71. However, the direct target(s) of AMPK responsible for these effects remained unclear. One candidate to explain these effects is Gα-interacting vesicle-associated protein (GIV), also known as Girdin. AMPK phosphorylates Ser245, a good fit to the AMPK consensus motif that is located in the junction between the N-terminal and coiled-coil domains of Girdin , when tight junctions are known to be turning over. Evidence was obtained by expressing non-phosphorylatable (S245A) and phosphomimetic (S245D) mutants in type II MDCK cells (which have low Girdin expression), suggesting that Ser245 phosphorylation was responsible for maintaining tight-junction integrity during glucose starvation. Ser245 phosphorylation also appeared to be responsible for interaction of Girdin with microtubules that are associated with tight junctions.The integrity and polarity of epithelial cell layers are other factors that are crucial to survival in vertebrates. Tight junctions between epithelial cells maintain a permeability barrier that helps to ensure that solutes and other materials such as pathogens cannot pass between cells but instead have to pass through them, where their uptake and onward transport can be monitored and regulated. Several years ago, it was shown that AMPK was activated during Ca Girdin . This wa73  promotes angiogenesis73. Thus, AMPK appears to be critical in enhancing angiogenesis, a process that would be beneficial in nutrient-deprived cells.A recent phosphoproteomic screen in wild-type and AMPK-null MEFs treated with the ADaM site ligand A-769662 identified Ser-243 on glutamine:fructose-6-phosphate amidotransferase-1 (GFAT1) as an AMPK target73 . GFAT1 cAlthough important questions remain, good progress has been made in obtaining structural data that provide insights into the molecular mechanisms by which the AMPK heterotrimer is activated by the canonical energy-sensing mechanism involving changes in AMP, ADP, and ATP. At the same time, it has become clear that AMPK is activated by glucose starvation by a lysosomal mechanism that is independent of changes in adenine nucleotides; this may even have evolved before the energy-sensing mechanism and may represent the ancestral role of the AMPK system. The number of pharmacological agents that activate AMPK has continued to increase, including several that bind in the so-called “ADaM” site and at least one (SU6656) that binds in the catalytic site yet causes paradoxical activation. Finally, the number of well-validated downstream targets for AMPK has continued to increase, including targets involved in mitochondrial fission, in the maintenance of tight junctions, and in the N-acetylglucosamine synthesis pathway.α-AID, alpha subunit auto-inhibitory domain; α-CTD, alpha C-terminal domain; α-KD, alpha subunit kinase domain; β-CBM, beta carbohydrate-binding module; β-SID, beta subunit interaction domain; ADaM, allosteric drug and metabolite; AMPK, AMP-activated protein kinase; CBS, cystathionine-beta-synthase; DRP1, dynamin-related protein-1; FBP, fructose-1,6-bisphosphate; GEF, guanine nucleotide exchange factor; MDCK, Madin-Darby canine kidney; MEF, mouse embryo fibroblast; MFF, mitochondrial fission factor; v-ATPase, vacuolar ATPase.