Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to proline binding in the GSAL site.Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of  The intermediate P5C is in equilibrium with its hydrolysis product, l-glutamate-γ-semialdehyde (GSAL). The latter species is the substrate for the GSAL dehydrogenase (GSALDH) active site of PutA, which catalyzes the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of GSAL to glutamate. The two active sites are separated by a linear distance of ~40 Å and connected by a tunnel through which the intermediate P5C/GSAL is transferred via substrate channeling.Proline utilization A (PutA) proteins are bifunctional enzymes that catalyze the oxidation of lutamate  1,2]. P,2. Pl-pr+, and an electron acceptor for the FAD coenzyme, and the production of NADH is monitored. The reaction rate displays non-Michaelis–Menten behavior in that, above a certain proline concentration, the rate decreases with increasing proline. This result is consistent with substrate inhibition, and fitting of the reaction velocity data to a substrate inhibition model has yielded Ki values in the range of 24–263 mM for the various PutAs examined. In particular, the enzyme used in the present work, PutA from Bradyrhizobium japonicum (BjPutA), has a Ki of 24 mM  is the varied P5C concentration, Km is the Michaelis–Menten constant for P5C, [I] is the proline concentration, and Ki is the competitive inhibition constant for proline.The BjPutA R456M mutant was expressed and purified as described ,6. DL-P5escribed ,30. The Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a hallmark of PutAs. Inhibition of PutA by proline may be advantageous during osmotic stress, when bacteria need to accumulate high levels of proline rather than catabolizing it. The inhibition of the PutA GSALDH site by proline would also lead to a build-up of P5C, which is a competitive inhibitor of the PutA PRODH activity . Thus, u


Therefore it is possible that estrogen levels can dictate the balance of kynurenic acid in the brain. Inhibition assay results and modelling suggests that the 17-sulfate moiety in estradiol disulfate is very important in improving its potency as an inhibitor, increasing the inhibition by approximately 10–100 fold compared to estradiol.The kynurenine aminotransferase (KAT) enzymes are pyridoxal 5′-phosphate-dependent homodimers that catalyse the irreversible transamination of kynurenine into kynurenic acid (KYNA) in the tryptophan metabolic pathway. Kynurenic acid is implicated in cognitive diseases such as schizophrenia, and several inhibitors have been reported that selectively target KAT-II as it is primarily responsible for kynurenic acid production in the human brain. Not only is schizophrenia a sexually dimorphic condition, but women that have schizophrenia have reduced estrogen levels in their serum. Estrogens are also known to interact in the kynurenine pathway therefore exploring these interactions can yield a better understanding of the condition and improve approaches in ameliorating its effects. Enzyme inhibitory assays and binding studies showed that estradiol disulfate is a strong inhibitor of KAT-I and KAT-II (IC Sexual dimorphism has been described for the age of onset of schizophrenia in several studies6. Males typically have been shown to have an earlier onset, with a peak in those aged 15–25 years6. In comparison, the onset for females peak in the ages of 20–29 years6. The relatively lower incidence of schizophrenia in females during adolescence corresponds to a time of major hormonal changes, including that of increasing estrogen levels7. A smaller secondary peak for late onset schizophrenia has also been observed in females aged 45–49 years6 which again coincides with a period of estrogen change in women, with this time it being a drop in estrogen levels during menopausal transition9. The association of estrogen deficits in schizophrenia has been supported by molecular, animal and clinical studies. Several studies have identified increased severity of schizophrenia or surrogate measures of schizophrenia associated with low circulating estrogen levels11. In women with schizophrenia, reduced levels of serum estradiol has been reported in all phases of their menstrual cycle and although some reduction in estrogen is known to be associated with some antipsychotic medications, for which mechanisms leading to hypoestrogenism are known, it is thought that the reduction in women with schizophrenia exists independently of medication11.Schizophrenia has a prevalence of approximately 1% worldwide12 where it is a precursor for the serotonin pathway and the kynurenine pathway -dependent enzymes called kynurenine aminotransferase (KAT)14, of which there are four KAT isoforms in mammals. Between them, they are responsible for the irreversible transamination of kynurenine (KYN) to kynurenic acid (KYNA), using PLP as a cofactor 29, and often simultaneously increase the levels of the key neurotransmitters, glutamate30, acetylcholine31, dopamine32, and GABA33, which have recognized roles in cognitive function18. Supplementing this, the use of S-ESBA, BFF-816 and PF-04859989 on rats and, in the case of the latter inhibitor, also on non-human primates, have shown that these inhibitors may improve memory and spatial learning34.S-ESBA, BFF-122, PF-04859989, BFF-816 48, although other evidence refutes TDO2 induction49. This discrepancy might arise from inhibition of enzymes such as kynureninase and KAT by estrogens, thereby causing a build-up of kynurenine. As kynurenine is the measured, downstream product of TDO2 activity, a build-up of kynurenine can potentially give an appearance of TDO2 induction in a tissue homogenate.Steroid compounds, such as estrogens, and their sulfate and phosphate esters forms, have activity on various PLP-dependent enzymes54. These changes are caused by the estrogen component in the oral contraceptives, as women administered progestogen alone do not display increased excretion of these metabolites56. Similarly, elevated metabolite excretory patterns were displayed during pregnancy57 and in ovulation in the menstrual cycle58, both correlating with rising estrogen levels.The effects of estrogen on the kynurenine pathway metabolites have been observed in women. Relative to controls, young women taking oral contraceptives have increased excretion of kynurenine pathway metabolites, including kynurenine, xanthurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid, after they were given a tryptophan loadOwing to the sexual dimorphism in schizophrenia and the observed links between the kynurenine pathway and schizophrenia, exploring the relationship between estrogen and the kynurenine pathway will aid in the molecular understanding of this pathway and its associated conditions in which variations occur. In our current study, our goal was to accurately establish the inhibitory capacity of estradiol, estradiol 3-sulfate, estradiol disulfate and estrone sulfate Fig.  directly50: >2 mM). Estradiol disulfate displayed relatively good inhibitory capacity and had an IC50 of 291.5 (±19.6) μM . Estrone sulfate also could not be fully profiled and hence its IC50 is approximately ~2 mM. Estradiol disulfate however was found to inhibit KAT-II comparatively well, with an IC50 of 26.3 (±1.4) μM , and irreversible inhibitors BFF-122 (IC50: 15–20 μM) and PF-04859989 (1–3 μM) on KAT-II have been measured35. Our IC50 for the reversible estradiol disulfate suggests that it inhibits KAT-II strongly by the standards of the previously reported inhibitors, as it compares in magnitude to the irreversible inhibitor, BFF-122.Previously the inhibition profiles of the known reversible inhibitor NS-1502  35. The KD suggests that estradiol disulfate binds with affinity as strong as that would be expected from a compound that is neither irreversible nor very tightly binding.SPR was employed to evaluate the binding profile of estradiol disulfate on KAT-II. KAT-II was bound to a CM5 chip by amine coupling (achieving approximately 8000 RU). Estradiol disulfate was injected at increasing concentrations (170 nM–43750 nM) and the corrected, reference subtracted data displayed that estradiol disulfate interacted with KAT-II in a dose-dependent manner, and it had rapid association and dissociation with KAT-II. Due to the nature of this interaction, kinetic analyses could not be performed to directly evaluate the association and dissociation rate constants. Instead, a steady state affinity model was fitted with this data which showed that estradiol disulfate binds with a KD of 5.05 × 1015. By binding similarly, estradiol is likely to directly compete in the active site with the substrate and cause some competitive inhibition. This was seen in the inhibition assay results, but ultimately the inhibition by estradiol was relatively weak.Computational docking showed that estradiol disulfate displays the best potential for affinity with KAT-II at the active site, with a G score of −8.0 Kcal/mol . Methanol and dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific . PBST  was purchased from Sigma, and SPR reagents  were purchased from Bio-Rad Laboratories .SPR binding affinity measurements were performed using the Biacore T200 with a CM5 sensor chip . The experiment was prepared and obtained with the Biacore T200 Control Software, and analysed with the Biacore T200 Evaluation Software.59 was used to develop a nonlinear regression fit of the data collected in the inhibition assays. The IC50 is reported with the standard error.GraphPad Prism v7.02 software61.The expression and purification of recombinant KAT-I and KAT-II protein was completed as previously described35. 0.7 µg of KAT-I or KAT-II was incubated at 37 °C for 10 minutes in a 50 µL reaction mixture containing PLP (50 µM), α-ketoglutarate (5 mM), L-KYN (5 mM) in PBS, pH 7.4, and the inhibitor studied (1–2000 µM). Following incubation, the reaction was terminated by adding formic acid (0.8 M) in a 1:1 ratio. 50 µL of the mixture was transferred into a vial with 950 µL of water for HPLC analysis.The KAT inhibition assay was based on that described previouslyThe production of KYNA was measured using HPLC with UV detection at a wavelength of 330 nM, using a C18 reverse-phase column, and 50% (v/v) water and 50% (v/v) methanol mobile phase. The data was collected in triplicate.Prior to performing the SPR experiments, the solutions were filter sterilised for 10 min at 25 °C. KAT-II was diluted to a concentration of 350 µg/mL using sodium acetate (pH 4.5). The Biacore T200 compartment temperature was controlled at 25 °C and the flow rate was set to 10 µL/min. Using a 1:1 mixture of EDC (400 mM) and sulfo-NHS (100 mM), two flow cells on a CM5 sensor chip (containing a carboxymethylated dextran surface) were activated. Flow cell one contained a blank immobilisation (PBST buffer injection) and flow cell two had the prepared KAT-II sample injected. The two flow cells were deactivated using an injection of 1 M ethanolamine.The analyte, estradiol disulfate, was prepared in running buffer (PBST with 5% DMSO) in a concentration series from 170 nM-43750 nM. The analytes were injected in a multicycle kinetics mode using a flow rate set to 30 µL/min, contact time of 90 seconds and a dissociation time of 300 seconds, with an extra 50% DMSO wash performed after each injection. Solvent correction was performed before and after the estradiol disulfate injections, and also every thirty cycles, to account for the use of DMSO in the analyte sample preparation and the running buffer. The data was collected in triplicate.62) was downloaded from the Protein Data Bank. The PLP cofactor was removed as it is thought to compete with the ligands in the active site rather than being present simultaneously. The protein preparation wizard was used in Maestro (version 10.4.017) to optimise and minimise the protein, and hydrogen atoms were added and water molecules not participating in the reaction were removed. The ligands were prepared using LigPrep63, using the OPLS-2005 force field64. The active site was defined by the location of the existing  ligand, kynurenine, in the PDB file. The ligands were then docked using the Glide docking program65, with XP (extra precision) docking66.The human KAT-II crystal structure (PDB ID: 2R2Nhttps://figshare.com/projects/Inhibition_of_human_kynurenine_aminotransferase_isozymes_by_estrogen_and_its_derivatives/24823).The data generated and analysed during the current study is available in the Figshare repository (KYNA, a metabolite in the tryptophan metabolic pathway, is produced by the KAT enzymes, of which KAT-II is thought to be the isoform that produces the majority of KYNA in the human brain. KYNA is found elevated in the brain of patients with schizophrenia, and is therefore implicated in the condition. Low circulating estrogen levels are also associated with the onset and severity of patients with schizophrenia. The activity of the KAT enzymes has been previously studied with estrogens and estrogen conjugates to determine their inhibitory ability, but in rat homogenates only.We have found estradiol disulfate to be the most potent amongst the group of derivatives inhibiting KAT activity, with some activity also displayed by estrone sulfate. Despite showing inhibition, estradiol and estradiol 3-sulfate were, relatively, the weaker inhibitors. In combination with molecular docking studies, this shows that increased potency for KAT-II inhibition for estrogens is mainly derived from the 17-sulfate moiety. This can also be exploited in the design of novel KAT-II inhibitors, and also contribute to the improvement of existing inhibitors.Changes in estrogen levels induced by oral contraceptives, pregnancy and the menstrual cycle can influence the tryptophan metabolic pathway. Estrogens have the ability to be formed in the brain analogous to that of peripheral tissues, and have a similar capacity to being metabolised as a consequence of enzymatic activity in the brain. Owing to the inhibitory capability of the steroids, and particularly their sulfate derivatives, the distribution of the metabolic enzymes in the brain may dictate the potential for these compounds to inhibit the KAT enzymes. Consequently, this will affect the production of tryptophan metabolites, including that of KYNA, in the brain.Ultimately, steroid dysfunction in schizophrenia may not be confined to estrogen dysfunction alone, and is it possible that many steroid hormones and their metabolites are involved. There are multiple pathways in which estrogen may be affecting the brain but a role involving the NMDA receptor antagonist KYNA is potentially a significant one, which may be consistent with the kynurenic acid hypothesis of schizophrenia as a lack of KAT inhibition by estrogen derivatives in the brain can cause an imbalanced oversupply of KYNA.Supplementary Information


Compensation postchronic myocardial infarction (CMI) in rats is characterized in trabeculae as increased force and power production during physiological shortening, which occurs alongside classical hypertrophy. Sarcomeric contractile gain is influenced by mechanisms involving reduced myosin binding protein C (MyBP-C) and raised regulatory light chain (RLC) phosphorylation. Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic “compensation” and congestive “decompensation.” Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae. Compensation postchronic myocardial infarction (CMI) in rats is characterized in trabeculae as increased force and power production during physiological shortening, which occurs alongside classical hypertrophy. Sarcomeric contractile gain is influenced by mechanisms involving reduced myosin binding protein C (MyBP-C) and raised regulatory light chain (RLC) phosphorylation.chronic myocardial infarction (CMI) is typified by the obstruction of blood flow in the coronary arteries, which creates cardiac remodeling in surrounding tissue regions not directly affected by the infarct  permeabilized cardiac trabeculae, extracted from sites distant from the infarct zone, are “compensated”  in a CMI model, which is not accounted for by the hypertrophic response (2) that contractile characteristics parallel RLC phosphorylation level enrichment in disease progression. We measure the phosphorylation levels of myosin binding protein C (MyBP-C) and troponin I (TnI), which influence muscle mechanics, to test the hypothesis that sarcomeric protein alteration is a phenomenon of sarcomeric level contractile compensation  ensation , 51, 63.Vmax) are altered by compensation and decompensation. We used full activation (32 μM [Ca2+]), and partial activation (1 μM [Ca2+]) to ascertain whether the mechanics are altered by MI in a consistent manner, independently of [Ca2+]. In addition we interrogated changes in the morphological features of the exact same trabecula preparations using electron microscopy (EM) and confocal microscopy to assess type I collagen abundance. Confocal two-photon excitation (TPE) and s harmonic generation (SHG) microscopy was used to assess relative collagen abundance and infiltration (Force-velocity (FV) measurements provide a robust methodology for exploring changes in cross-bridge mechanics during physiological shortening  in permeltration . The proltration . EM was This study couples mechanical-morphological observations with in-vivo clinical measures of heart function. Bringing these data together highlights that contractile protein phosphorylation is important in the compensatory mechanism of increased mechanical output, defined by physiological shortening under load. We hypothesize that posttranslational modification alters muscle function at the cross-bridge level and propose how sarcomeric protein alterations affect trabecular force, shortening, and power in this disease.Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health under assurance number A5634-01 and also conformed to the UK Animals (Scientific Procedures) Act 1986. Imperial College Ethical Review Committee and the Project License authorized these studies in accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. Adult male Sprague-Dawley rats (250–300 g) (n = 18) underwent proximal left coronary artery ligation to induce myocardial infarction (MI) as described previously  were immediately rinsed with oxygenated ice-cold Krebs-Henseleit solution. Cylindrical trabecular preparations (50–200 μm in diameter and 0.8–2 mm in length) were excised from the inner wall of the left ventricle superior to the left ventricular papillary muscle distant from the anterior apical infarct region. T-clips were crimped onto the ends of the trabeculae and the tissue was permeabilized by immersion in a 2% Triton solution. The samples were attached to the experimental rig by hooks and shellac was applied to the ends of the trabecular segments to minimize compliance of the damaged ends of the preparations. Sarcomere length was set to 2.1 μm by laser light diffraction assessed both before and after activation cycles. Preparations were activated and held at isometric tension for 1 s to make sure there was no slippage of the preparation. Tension was measured at 20°C in an activating solution with either 1 or 32 μmol/l free free), affected the contractile properties. Isometric force, Vmax, and peak power were lower in the submaximally activated trabeculae, from both MI4W, and AMC4W hearts. Curvature of the FV relationships (a/P0) was reduced in submaximal activations, and this effect was accompanied by overall reduced shortening velocity at which peak power is observed (Vpp). Key signs that MI4W hearts were mechanically “compensated” were 1) isometric force was increased significantly ; and 2) peak power was elevated significantly ; and 3) force at peak power (FPP) was raised by MI in both maximal and submaximal activation The features of FV and power-velocity relationships in maximally (32 μmol/l CaVmax (P = 0.015), which was manifest as the relatively higher (2-fold) Vmax in the compensated MI4W compared with control AMC4W, at submaximal activation (see a/Po showed a similar interaction effect (P = 0.011), where the curvature in the MI4W submaximal activations  was found to be intermediate to the mean values at full activation (ranging 0.05 to 0.13) and the value for AMC4W at submaximal activation . Vpp was 30% higher in MI4W compared with AMC4W, but the difference was not significant.Interestingly, there was a significant interaction effect between MI and Ca level on average tion see . The ave2+]free). Similarly, the approximate twofold higher average power and Vmax seen in the partially activated compensated hearts, when compared with controls, had reversed in trabeculae from MI20W hearts. In the fully activated decompensated hearts, Vmax was reduced by one-third, while power was 53% of the mean of AMC20W. The main effect of CMI on Vmax and power was observed at full activation, where the control values remained relatively similar to the full activation values for AMC4W. FPP was reduced at MI20W by 20 and 30% in maximal and submaximal activation compared with AMC20W (P < 005). Mechanical decompensation was even evident in the fully activated MI20W group. There was a significant interaction effect between MI and Ca level on Vmax and Vpp; both of these mean velocity measures were relatively low in the fully activated MI20W group, compared with the respective AMC20W values.At MI20W, most mechanical adaptions observed in the MI4W cohort were reversed ; Table 2B and D). In both cases collagen had become more central in the trabeculae disrupting the myocardial network (B and D).TPE and SHG microscopy were used to measure collagen infiltration in the trabeculae used for mechanical experiments. Trabeculae were mounted on a custom-made device for three-dimensional simultaneous recording of SHG signal (at sarcomere length of 2.1 μm), as a collagen marker, and autofluorescence for tissue volume evaluation  4). Qua. Qua4).  network , B and DMyocardial tissue sections, also taken from noninfarct regions of the hearts, were imaged in the electron microscope to confirm the effect of MI on sarcomeric content, structure, and tissue morphology at higher resolution. Control trabeculae, had well ordered sarcomeres, intercalated discs (ICDs) and normal mitochondria A. Age dii/mol MyBP-C (n = 5) vs. AMC4W 2.9 ± 0.1 mol Pi/mol MyBP-C (n = 5), a 17% reduction (P < 0.05) (P < 0.05) at MI20W to 3.5 ± 0.4 mol Pi/mol MyBP-C (n = 5) from 2.5 ± 0.4 mol Pi/mol MyBP-C (n = 5) in AMC20W, and RLC phosphorylation increased at MI4W to 0.45 ± 0.02 mol Pi/mol RLC (n = 5) from 0.30 ± 0.04 mol Pi/mol RLC (n = 5) in AMC4W (P < 0.05). This was a 50% increase in RLC phosphorylation that was maintained at MI20W 0.52 ± 0.04 mol Pi/mol RLC (n = 5) compared with control at 0.31 ± 0.02 mol Pi/mol RLC .Adaptation during disease progression SDS-PAGE incorporating Phos-tag was used to quantify protein phosphorylation of tTnI, MyBP-C, andRLC  8, 35, , 35, 58. < 0.05) . PhosphoEnhanced power output, isometric force, and force at peak power were consistent features of cardiac compensation in this CMI model. However, hypertrophy and activation level seemed to be only relatively minor determinants of power and force. Cardiac hypertrophy largely reflected the increased collagen deposition in the noninfarcted area of a compensated heart. Mechanical compensation was not observed at the level of the intact organ, likely due to tissue damage in the infarct zone. This highlights the impact of the mechanical compliance and the morphological effect of the scar tissue created by CMI . In the A and B, and PP and not VPP. Therefore, the ability of each contractile unit to produce force during shortening has been fine-tuned post-CMI, independently of hypertrophy.The contractile apparatus of the MI4W myocardium outperformed AMC4W at both physiological (1 μM) and maximally activating (32 μM) calcium, in spite of the extensive damage in the infarcted region and loss of functional tissue A and 5B.N); the force produced by each individual cross-bridge (F); and the fraction of attached cross-bridges (f/f + g), where f is the rate constant of cross-bridge attachment and g is the rate constant of detachment. Alterations in both the kinetics and mechanics of myosin have previously been observed by altering RLC and MyBP-C phosphorylation.A straightforward two-state model describes how force production may be altered in muscle . This mof, by moving myosin heads closer to actin and thereby increasing the probability of cross-bridge formation  leading to increased isometric force, which we observe at MI4W. Power is also affected at MI4W. Power can be increased either by an increase in force, in velocity, or by both simultaneously. RLC phosphorylation has been previously found to alter Vmax . This accompanied severe disruption of the sarcomeric environment, ICDs, and mitochondrial structure . E. E17). Ein force , B–D. Thtructure C. Contratructure . TheoretWe have observed novel mechanical adaptation post-CMI during physiological muscle shortening, which accompanies RLC, and MyBP-C phosphorylation change. These effects are evident at saturating calcium concentrations and maximal thin filament activation and therefore are at least partially independent of calcium sensitive regulation of the thin filament. Thus we identify a mechanism of sarcomeric compensation, which is mediated by changes in myosin function, created by sarcomeric protein phosphorylation change. These changes alter cross-bridge binding to actin and unbinding in a way that promotes increases in sarcomeric force, and power production. With these data we show the effect of CMI on myocardial contractile output during physiological shortening; providing a starting point to quantitatively assess the efficacy of interventions targeted to sarcomeric protein modification .Wellcome Trust Grants 091460/Z/10/Z and 092852/Z/10/Z and Biotechnology and Biological Sciences Research Council Grant BB/I019448/1.The work presented herein was supported by No conflicts of interest, financial or otherwise, are declared by the author(s).C.N.T., V.C., K.T.M., T.G.W., and M.A.F. conception and design of research; C.N.T., M.B.S., V.C., A.V., I.T., and O.C. performed experiments; C.N.T., M.B.S., V.C., A.V., I.T., O.C., and T.G.W. analyzed data; C.N.T., M.B.S., V.C., A.V., I.T., O.C., T.G.W., and M.A.F. interpreted results of experiments; C.N.T., V.C., A.V., and I.T. prepared figures; C.N.T., M.B.S., V.C., A.R.L., S.B.M., P.K.L., K.T.M., T.G.W., and M.A.F. drafted manuscript; C.N.T., M.B.S., V.C., A.R.L., S.B.M., P.K.L., K.T.M., T.G.W., and M.A.F. edited and revised manuscript; C.N.T., T.G.W., and M.A.F. approved final version of manuscript.


Lycopodium alkaloids, lyconadins G (1) and H (2), together with four known ones (3–6), were isolated from Lycopodium complanatum. The structures were determined on the basis of their spectroscopic analyses, and the absolute configuration of 1 was established by an X-ray crystallographic analysis. It is the first time to establish the absolute configuration of lyconadin-type Lycopodium alkaloid by an X-ray diffraction experiment. In addition, these findings may provide more information for the biosynthesis of lyconadins.Two rare lyconadin-type The online version of this article (doi:10.1007/s13659-016-0111-9) contains supplementary material, which is available to authorized users. Lycopodium alkaloids are a unique family of complex natural products that have garnered long-standing interest from chemists as challenging targets for total synthesis [Qian Ceng Ta [whole plant of Huperzia serrata (Thunb.ex Murray) Trev.], is a promising agent to treat Alzheimer’s disease with highly specific and potent inhibitory activity against acetylcholinesterase (AChE) , four methylenes, and one methyl. Based on the above evidence, the structure of 1 was determined to be similar to that of lyconadin C (4) + in the ESI–MS, and the molecular formula, C16H22N2O, was established by HR-EI-MS [m/z 258.1732 Calc. 258.1732]. The IR absorption implied the presence of amide carbonyl (1680 cm−1) functionality. The 13C NMR , five methines (including one olefinic (δC 123.1) with corresponding proton as a singlet signal at δH 5.85 in the 1H NMR spectrum), six methylenes, and one methyl, implying that the structure of 2 was similar to that of lyconadin G (1). The sole difference was the loss of a 1,2-disubstituted double bond between C-2 and C-3, which was confirmed by the HMBC correlations of H2-2 and H2-3 with C-4, and of H2-3 with C-5, as well as the 1H-1H COSY correlation between H2-2 and H2-3.Lyconadin H  was inferred from the coupling constant of 3JH-13/H-14b value (4.3 Hz) and ROESY correlation of H-12/H-13 .6 c = 0., MeOH; U1) and H (2) was assayed by the spectrophotometric method developed by Ellman with slightly modification. S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′-dithio-bis-(2-nitrobenzoic) acid , AChE derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture  containing phosphate buffer (pH 8.0), test compound (50 μM), and AChE (0.02 U/mL), was incubated for 20 min (30 °C). Then, the reaction was initiated by the addition of 40 μL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 3 min for 1 h. Tacrine was used as positive control with final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).AChE inhibitory activity of lyconadins G Below is the link to the electronic supplementary material.


ANGPTL4 and PDK4, which are involved in glucose and lipid metabolism. CDK5-mediated phosphorylation at serine 273 (S273) is a unique regulatory mechanism reserved for PPARγ, and this event is linked to insulin resistance in type 2 diabetes mellitus. Our data demonstrates that chiglitazar modulates gene expression differently from two TZDs, rosiglitazone and pioglitazone, via its configuration-restricted binding and phosphorylation inhibition of PPARγ. Chiglitazar induced significantly greater expression of ANGPTL4 and PDK4 than rosiglitazone and pioglitazone in different cell models. These increased expressions were dependent on the phosphorylation status of PPARγ at S273. Furthermore, ChIP and AlphaScreen assays showed that phosphorylation at S273 inhibited promoter binding and cofactor recruitment by PPARγ. Based on these results, activities from pan agonist chiglitazar can be an effective part of a long-term therapeutic strategy for treating type 2 diabetes in a more balanced action among its targeted organs. Type 2 diabetes mellitus is often treated with insulin-sensitizing drugs called thiazolidinediones (TZD), which improve insulin resistance and glycemic control. Despite their effectiveness in treating diabetes, these drugs provide little protection from eminent cardiovascular disease associated with diabetes. Here we demonstrate how chiglitazar, a configuration-restricted non-TZD peroxisome proliferator-activated receptor (PPAR) pan agonist with moderate transcription activity, preferentially regulates These drugs also exhibit a more preventive effect and provide more durable HbA1c control than other diabetes treatments [Metabolic syndromes, including type 2 diabetes mellitus (T2DM) and its associated complications , have significant worldwide impact. Current antidiabetic treatments, especially the insulin-sensitizing class of drugs called thiazolidinediones (TZD)  and pioglitazone (Pio)), improve insulin resistance and glycemic control with benefit of improvement in rental complication. However, they offer ambiguous protection from eminent cardiovascular risks associated with the diseases. Moreover, the related side effects, such as water retention and body weight gain, rather impair their extended uses in long-term management of diabetic patients. Nevertheless, the TZD class of peroxisome proliferator-activated receptor eatments . Therefoγ, a primary target of TZDs along with PPARα and PPARδ, is a ligand-activated member of the nuclear hormone receptor superfamily which is expressed in various tissues with overlapping or distinct biological activity in controlling glucose, lipid, and energy homeostasis. In brief, PPARγ is predominantly present in adipose tissue and macrophages and functions to repartition fatty acids (FAs) to adipose tissue from muscle, liver, and circulation, thus improving insulin resistance [α is mainly expressed in liver, heart, muscle, and kidney, where it stimulates FA oxidation and improves lipoprotein metabolism [δ is expressed ubiquitously, its function is less defined and PPARδ is mainly considered as an important regulator of FA metabolism and thermogenesis [α and PPARδ promote osteoblast activity in bone, while PPARγ promotes osteoclast activity [γ agonists in T2DM patients [PPARsistance . PPARα itabolism . Althougogenesis . All throgenesis . Controvactivity . Considepatients , 10. Thuγ phosphorylation, induced by “pathological stimuli” such as a high-fat diet, obesity, and inflammation, at serine 273 (S273) by TZD and synthetic ligands, resulted in alterations in gene expression profile which led to insulin resistance in animals and humans. Therefore, this event may play a unique role in discriminating insulin-sensitizing from other adverse effects, such as weight gain and edema, used to be linked to PPARγ activation by TZDs [Recently, several studies reported that specific inhibition of PPAR by TZDs –14.μM in CV-1 cells for PPARα, PPARγ, and PPARδ, respectively, which is currently in phase III clinical development in China. This compound is a moderate transcriptional activator of all three PPAR subtypes and induces different patterns of gene regulation compared to the TZD class of drugs in vitro and in vivo [ KKAy and db/db diabetic mouse models [Chiglitazar (Chi) is a configuration-restricted non-TZD PPAR pan agonist, with AC50 of 1.2, 0.08, and 1.7  in vivo , 16. In e models . ANGPTL4 (angiopoietin-like 4) and PDK4 , which are involved in lipid metabolism and insulin sensitization, relative to Ros and Pio. These genes are regulated mainly by PPARδ and to a lesser extent by other PPAR subtypes [γ at S273, which affects cofactor recruitment by the receptor and binding of a PPARγ-containing transcription complex to PDK4 and ANGPTL4 promoters. The unique structure of Chi allows its distinct binding and interactions with the receptor, which results in significantly stronger inhibition of site-specific phosphorylation leading to higher induction of ANGPTL4 and PDK4 expression. Since the phosphorylation status of PPARγ is directly linked to “pathological stimuli” such as a high-fat diet, obesity, and inflammation commonly seen in T2DM patients, the prominent activity of Chi in this regard in addition to its pan agonist activity can thus act cooperatively to rebalance glucose, FA uptake, and substrate utilization in energy production upon insulin resistance and obesity, which may improve the clinical prognosis of T2DM patients over time.In this article, we report that Chi preferentially induced two important PPAR target genes,subtypes , 18. TheChiglitazar was discovered and synthesized by Chipscreen Biosciences Ltd. . Rosigliμg/ml streptomycin, and 50 units/ml penicillin at 37°C in a humidified incubator with 5% CO2.Human preadipocyte-visceral (HPA-v) cells were purchased from ScienCell  and cultured in preadipocyte medium  containing 10% fetal bovine serum. The human normal liver cell line L-02 was purchased from the Shanghai cell bank of the Chinese Academy of Sciences  and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 50 γ binding to its ligands were downloaded from the Protein Data Bank  [The published crystal structures of PPARG, 2XKW) . Images G, 2XKW) . MDS wasG, 2XKW) . The conG, 2XKW) , 19. TheG, 2XKW) . vol/vol dimethyl sulfoxide (DMSO)), cells were collected and total RNA was extracted using TRIzol reagent  followed by purification using the RNeasy Mini Kit . The concentration of RNA was assessed using the UV spectrometer DU520 . First-strand cDNA was synthesized by SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed with StepOnePlus™  using the FastStart High Fidelity PCR kit . Primer sequences used were as follows: CD36, F: 5′ CATCGCTGGGGCTGTCATT 3′, R: 5′ GCGTCCTGGGTTACATTTTCC 3′; ANGPTL4, F: 5′ GCAGCCATTCCAACCTCAA 3′, R: 5′ CAAGAGTCACCGTCTTTCGTG 3′; PDK4, F: 5′ ATGTCATTGGCAAGAGGAAGAA 3′, R: 5′ ATTACCAGAAGCACCACAACACT 3′; LIPE , F: 5′ CCCTGCTCCTCCGAGACTT 3′, R: 5′ GGACTTGCGCCCACTTAACT 3′; β-actin, F: 5′ AGTTGCGTTACACCCTTTC 3′, R: 5′ TGTCACCTTCACCGTTCC 3′. The relative gene expression level was normalized against β-actin. The fold change of expression induced by the various treatments was calculated as the relative expression level in treated samples divided by the vehicle sample or treatment control.HPA-v cells were cultured overnight in 6-well plates with the appropriate medium before use. After a 24-hour incubation with the tested compounds at varying concentrations or vehicle control (0.1% (vol/vol) DMSO) for 48 hours. Proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit and quantitated with Pierce Micro BCA Protein Assay kit according to the manufacturer's instructions . The appropriate quantity of protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membrane . Blots were blocked for 1 hour at room temperature in TBST buffer  with 5% nonfat dry milk and subsequently incubated with individual primary antibodies. Primary antibodies for PPARγ , Danvers, MA; 81B8), phospho-(Ser) CDKs substrates , β-Actin , LIPE , CD36 , PDK4 , and ANGPTL4  were used according to the manufacturer's recommendation. After three washes with TBST buffer, membranes were incubated at room temperature for 1 hour with the respective secondary antibodies according to the manufacturer's recommendation. The bands were visualized with Super ECL Plus Detection Reagent  and exposed to Kodak X-OMAT film. The film was scanned and transformed to gray-scale graphs with the Bio-Print gel scanner and accessory software .HPA-v cells were cultured in 60 mm plates overnight and then incubated with the indicated compounds at varying concentrations or vehicle control (0.1% (vol/vol) DMSO) for 24 hours. Luciferase activity was measured using a luciferase assay kit from Promega . GFP fluorescence and luciferase activity were sequentially detected using the Fluoroskan Ascent FL reader . Reporter gene expression as represented by luciferase activity was normalized against GFP fluorescence in the same well. Reporter induction by the tested compounds was compared with the vehicle control .Cell transfection was performed as described previously using the same constructs for the luciferase reporter assay , 13. Bri (vol/vol) DMSO) for 24 hours. Total RNA was extracted and then real-time RT-PCR was performed as described above.To detect changes in gene expression, L-02 cells were seeded into 6-well plates and cotransfected with plasmids expressing hRXR and different PPAR subtypes. At 48 hours after transfection, cells were incubated with the tested compounds at the indicated concentrations or vehicle control  at 4°C overnight prior to mixing with protein G-Sepharose beads (Thermo Fisher Scientific) for 2 hours. The beads were washed three times with lysis buffer, and the immunoprecipitates were recovered and dissolved in SDS loading buffer. Equal amounts of immunoprecipitates were loaded and separated by SDS-PAGE prior to immunoblot analysis using primary antibodies against PPARγ  and phospho-(Ser) CDKs substrates .HPA-v cells were cultured in 60 mm plates overnight and then incubated with the indicated compounds or DMSO. Cell lysates were dissolved in Pierce IP Lysis Buffer (Thermo Fisher Scientific) and then incubated with anti-PPARγ2  [ PPARγ2 into the pcDNA3.1 vector using the QuikChange site-directed mutagenesis kit  as recommended by the manufacturer. The following primer pairs were used for site-directed mutagenesis: PPARγ S289A, F: 5′ GCTGCCAGTTTCGCGCCGTGGAGGCTGT 3′, R: 5′ ACAGCCTCCACGGCGCGAAACTGGCAGC 3′, where TCC (serine) was substituted with GCC (Alanine); Y473D, F: 5′ CTCCTGCAGGAGATCGACAAGGACTTGTACTAG 3′, R: 5′ CTAGTACAAGTCCTTGTCGATCTCCTGCAGGAG 3′, where TAC (tyrosine) was substituted with GAC (aspartic acid); E343A, F: 5′ GGGTTCTCATATCCGCGGGCCAAGGCTTCA 3′, R: 5′ TGAAGCCTTGGCCCGCGGATATGAGAACCC 3′, where GAG (glutamate acid) was substituted with GCG . The primer pair and its endonuclease site for subcloning were as follows: NheI F: 5′ CTGGCTAGCGTTATGGGTGAAACTCTGG 3′, XhoI R: 5′ GGCCTCGAGCTAGTACAAGTCCTTGTA 3′. All constructs were confirmed by full-length sequencing.Site-directed mutagenesis of amino acid sites in human PPARine 473)  was condγ antibody  as described above. The DNA fragments were subsequently recovered using a QIAquick column (Qiagen). Real-time PCR was performed as described above using the following sequence-specific primer pairs against target gene promoters: ANGPTL4, F: 5′ TGAGCTCTTCTCCGTTCATCTCGAACCAC 3′, R: 5′ GAGTCTAGACATCTCAGAGGCTCTGCCTG 3′; PDK4, F: 5′ GGATTTCAACAGCCAGTGCT 3′, R: 5′ ATAGTGCTGCCCAGTGTGTG 3′; CD36, F: 5′ ATTTGTGGTTGGTTGCCAAG 3′, R: 5′ AGGTGATGGGTCTTCACCAG 3′; LIPE, F: 5′ CAAGTGATTGGGATGAAGCA 3′, R: 5′ CTAGCCAGCCCAGTCTTCAG 3′; insulin, F: 5′ CTTCAGCCCAGTTGACCAAT 3′, R: 5′ AGGGAGGAGGAAAGCAGAAC 3′. Real-time PCR of the insulin promoter was used as a negative control. The respective input samples were taken as internal (loading) controls. Real-time PCR was performed as described above. Promoter binding was evaluated by normalizing the Ct (threshold cycle of PCR) value of samples immunoprecipitated with anti-PPARγ antibody against the Ct of respective Input controls.The Chromatin Immunoprecipitation (ChIP) assay kit  was used according to the manufacturer's instructions. Briefly, after incubation with the indicated treatments, HPA-v cells were fixed with 1% formaldehyde at 37°C for 15 minutes and then sonicated using the XO-900D Ultrasonic cell disruption apparatus . After removing debris with protein A-agarose beads, an aliquot of supernatant was taken as the input control. The same sample volume was subjected to immunoprecipitation with anti-PPARγ-LBD fragment was expressed and purified as described previously [ In vitro phosphorylation of PPARγ-LBD (ligand binding domain) was performed in a 50 μl reaction volume consisting of the individual tested agonists or vehicle control (DMSO) at the indicated concentration, 1 μg of His-PPARγ-LBD, 30–50 ng of active CDK5/p25, and 100 mM ATP in 1x kinase buffer. The reaction was incubated at 30°C for 30 minutes and then subjected to immunoblot analysis or the AlphaScreen assay of cofactor binding.Active heterodimer of CDK5/p25 was purchased from Millipore (cat. number 14-516). Recombinant human His-PPAReviously . In vitrγ-LBD was determined using the AlphaScreen assay (AlphaScreen® Histidine (Nickel Chelate) Detection Kit, Perkin-Elmer, Waltham, MA, USA) as described previously [γ-LBD and 40 nM of the respective biotinylated peptides in the presence of 5 μg/ml donor and acceptor beads in a buffer containing 50 nM MOPS, 50 mM NaF, 50 mM CHAPS, and 0.1 mg/ml bovine serum albumin, pH 7.4. For the cofactor binding assay with phosphorylated PPARγ-LBD, in vitro phosphorylation was first performed as described above followed by the conventional AlphaScreen procedure. The phosphorylation status of PPARγ-LBD was varied depending on the presence of CDK5/p25, ATP, or tested agonists or their order of addition into the in vitro phosphorylation reaction. For example, “LBD + CDK5 − ATP + agonist” represents no ATP in the phosphorylation reaction; “LBD + CDK5 + ATP + agonist” represents that all components were present in the phosphorylation reaction. Furthermore, “(LBD + CDK5 + ATP) + agonist” denotes that in vitro phosphorylation was completed before agonist was added. The representative biotinylated peptide motifs used were as follows: NCOR2 (nuclear receptor corepressor 2): Biotin-GHSFADPASNLGLEDIIRKALMGSF; PGC1a : Biotin-QEAEEPSLLKKLLLAPANTQ; SRC1-2 (nuclear receptor coactivator 1): Biotin-SPSSHSSLTERHKILHRLLQEGSP; SRC2-3 (nuclear receptor coactivator 2): Biotin-SPKKKENALLRYLLDKDDT; SRC3-3 (nuclear receptor coactivator 3): Biotin-SPKKKENNALLRYLLDRDD; TRAP1b : Biotin-FSKVSQNPILTSLLQITGN.The representative LXXLL peptide motifs of different cofactors were synthesized according to published literature. The binding of various peptide motifs to PPAReviously . The conThe fold change of baseline cofactor recruitment induction is represented as the ratio of the relative luciferase unit (RLU) value from the ligand-free binding reaction with cofactor divided by the value with no peptide in presence. The fold change of cofactor recruitment induction by the tested agonists was calculated as the ratio of the RLU value from the binding reaction with agonist divided by vehicle control (DMSO).t-test was performed to determine statistical significance as needed. Statistical significance between the different comparisons was defined as p < 0.01.Student's γ targets , ANGPTL4 and PDK4, which were preferentially induced by Chi. As shown in  ANGPTL4 and PDK4 were significantly induced by Chi compared to Ros or Pio. Consistent with previous findings [ CD36 and LIPE, two well-established PPARγ target genes, were similar following the treatments with each compound . As shown in  ANGPTL4 and PDK4 significantly in PPARδ-transfected cells, this PPAR pan agonist also upregulated both genes even more significantly in PPARγ- but not PPARα-transfected cells compared to Ros and Pio in this cell model. These results suggest that ANGPTL4 and PDK4 expressions are upregulated not only in response to PPARδ activation but also by PPARγ. However, this induction of gene expression following agonist treatments does not appear to be correlated with PPARγ transactivity, since Chi consistently produces less potent PPARγ transactivation relative to Ros in different cell model systems [50 for PPARγ transitivity of 0.120 ± 0.047, 0.035 ± 0.037, and 0.288 ± 0.514 μM, respectively .subtypes , 18. Is  systems . Consistγ crystal structure [γ via Tyr-473 of helix 12 and His-323 of helix 5, which is consistent with a typical full agonist binding model [β-sheet. To verify the different binding modes between them, we performed serial site-directed mutations of the referred residues on the receptor including Tyr-473Asp (Y473D), Ser-289Ala (S289A), and Glu-343Ala (E343A), respectively. Unexpectedly, the Y473D mutation significantly diminished the transactivity of Chi as well as Ros and Pio in reporter gene assay  [γ activity can be modulated by CDK5-mediated phosphorylation at S273 [γ by preferentially inhibiting the receptor phosphorylation and, thus, producing a differential pattern of the PPARγ-targeted gene regulation.Chi possesses a non-TZD structure with a relatively restricted configuration. Simulated molecular docking with the published PPARtructure  revealedng model , 26. How Figures , despiteigure 4) . Among oα, an inducer of CDK5-mediated PPARγ phosphorylation [γ and then the immunoprecipitates were visualized by immunoblotting using anti-phospho-(Ser) CDKs substrate antibody. While Chi and the two TZDs all exhibited dose-dependent inhibition in TNFα-enhanced phosphorylation of PPARγ in HPA-v cells, Chi produced a stronger inhibitory effect even at 0.05 and 0.2 μM ( in vitro phosphorylation reaction with purified recombinant human His-PPARγ ligand binding domain (LBD) [μM Chi inhibits CDK5-mediated phosphorylation of the PPARγ-LBD significantly greater than the two TZDs tested   and the s tested . These eγ phosphorylation in regulating target gene expression, we examined PPAR target gene expression in TNFα-treated HPA-v cells incubated with a panel of kinase inhibitors . Expressions of ANGPTL4 and PDK4 were only induced by roscovitine treatment, indicating that this event is CDK5 kinase-dependent. Meanwhile, induction of the classic PPARγ target genes, CD36 and LIPE, was not changed in HPA-v cells by the treatment with above-mentioned kinase inhibitors  [γ at S273 may affect its interaction with RXR and subsequent promoter binding. We performed ChIP assays to test the promoter binding of PPARγ-containing transcription complex induced by Chi, Ros, and Pio in the presence and absence of S273 phosphorylation. In absence of TNFα, it is clear that Chi induced greater recruitment of PPARγ-containing complexes to the ANGPTL4 and PDK4 promoters than CD36 and LIPE  to recruit cofactors  upon specific ligand binding or to induce protein-protein interactions  that result in formation of a transcriptome that regulates downstream target genes , 28. Stuin (DBD) , 30. Thuand LIPE . Most stromoters . These rignaling .γ-LBD to see whether specific cofactor recruitment is affected by S273 phosphorylation during target gene regulation. Our data show, for the first time, that in vitro phosphorylation of PPARγ by CDK5 inhibited cofactor recruitment , were significantly inhibited by CDK5-mediated phosphorylation of the receptor. The inhibition was partially and differentially restored upon additions of the three ligands, consistent with their inhibition potency on in vitro phosphorylation at S273   . Insteadof PPARγ . Differeof PPARγ –42. Ros -v cells  and in vn assays  than the ANGPTL4 and PDK4. In the human preadipocyte HPA-v cell line, Chi differentially regulated a different set of target genes compared to Ros and Pio. Among them, expression of ANGPTL4 and PDK4 was dramatically higher following Chi treatment  but not others  inhibited by CDK5-mediated S273 phosphorylation  was specifically involved in the recruitment or stabilization of cAMP response element-binding protein and p300-containing cofactor complexes to a subset of target genes [ ANGPTL4 and PDK4 expressions promoted by Chi compared with the other two TZDs. In contrast, we found that Chi more significantly dissociates corepressor NCOR2 from PPARγ-LBD than Ros in AlphaScreen assay; knockout NCOR in adipocyte was recently reported to repress PPARγ S273 phosphorylation and increase insulin sensitivity in mouse model [γ by Chi partially contributes to its phosphorylation inhibition. In contrast, another report demonstrated that short chain FA butyrate could induce a unique set of PPAR target genes, including ANGPTL4, even though no significant activity in cofactor recruitment (except for induced binding of two corepressors NCOR1 and NCOR2 binding) was observed by PamChip® arrays [γ transcriptome; it is possible to induce ANGPTL4 expression via interfering in the interaction of HDAC and NCOR2. Considering the pathophysiological and therapeutic relevance of PPARγ S273 phosphorylation in T2DM and its associated complications, the impact of this biological process on specific target gene regulation and cofactor recruitment is worthy of further investigation.Our ChIP and AlphaScreen assays revealed that TNFrylation . These ding site . Althouget genes , our undse model ; the mor® arrays . Butyratα and PPARδ could contribute additional effects to lipid metabolism over PPARγ. In fact, Pio brings cardiovascular benefits to patients most likely due to weaker PPARγ and PPARα activation compared with Ros [γ and other PPAR subtypes may not only explain the drastic differences in clinical outcomes, such as CV events, resulting from the same chemical class of TZDs  but also potentially enable the development of configuration-restricted non-TZD molecules with fewer side effects associated with PPARγ activation.Insulin resistance, T2DM, and its cardiovascular complications reflect a very complex outcome due to dysfunction in the mutually compensatory interactions among glucose, lipid, and energy utilization in relevant tissues. One consequence, namely, lipid overload in adipose tissue, liver, muscle, and heart tissue due to a metabolic syndrome often seen in T2D patients, is a critical factor leading to body weight gain, heart failure, and other cardiovascular conditions. Therefore, treatments that restore glucose, lipid, and energy homeostasis would potentially provide the most clinical benefit. Among the three subtypes, PPARwith Ros , 46. Suc ANGPTL4 and PDK4, involved in glucose and lipid metabolism via inhibition of PPARγ phosphorylation. Dietary saturated fatty acids (SFAs) can be presented to cells in the form of circulating free fatty acids (FFAs) or FAs hydrolyzed by lipoprotein lipase (LDL) from chylomicrons or VLDL in the lymph nodes and blood. SFAs are strong proinflammatory nutrients that can trigger activation of intracellular inflammatory pathways in innate immune cells such as macrophages, as well as the main insulin target cells, adipocytes, myocytes, and hepatocytes, leading to insulin resistance, obesity, and cardiovascular disease [ ANGPTL4 is an endogenous inhibitor of LDL activity and plays a crucial role in preventing fat-induced inflammation elicited by SFAs, diet-induced obesity, and myocardial infarction through preservation of vascular integrity [ ANGPTL4 decreases circulating FFAs derived from triglyceride- (TG-) rich lipoproteins to enhance insulin sensitivity of target organs. The positive relationship of ANGPTL4 activity with increased plasma level of TG-rich VLDL lipoproteins is seen in human with less-of-function variance ANGPTL4 E40K as well as ANGPTL4-deficient or liver-specific overexpression transgenic mice [ ANGPTL4 suppresses foam cell formation to reduce atherosclerosis development in atherosclerosis-prone E3L mice [ ANGPTL4 [ ANGPTL4 may potentially contribute to increased lipid accumulation  in the liver, muscle, and heart [β-oxidation in these tissues through the additional activation of PPARα and PPARδ activity [ ANGPTL4 by Ros and a weak PPARα transactivity of Pio [ UCP-1 in fat and skeletal tissues [ db/db mice were noted after treatment by Chi [Beyond its moderate pan agonist activity, Chi greatly induces two target genes, disease , 48. ANGntegrity –51. ANGPnic mice –54; howeE3L mice . Similar ANGPTL4 , highlignd heart , which iactivity . Indeed,y of Pio , 59. In  tissues . Furthert by Chi . Much let by Chi . PDK4 inactivates the pyruvate dehydrogenase complex, which leads to reliance on β-oxidization of FFA as a primary energy source rather than glucose. The glycerol-3-phosphate catalyzed from glucose thus serves to immobilize circulating FFA in adipose tissue, which further enhances the insulin sensitivity of other glucose-utilizing organs. PDK4 is upregulated in skeletal muscle in insulin-resistant states and proposed to be involved in the etiology of insulin resistance [ PDK4 knockout in mice has no obvious phenotype change and no effect on blood glucose levels and insulin sensitivity in the fed state and only leads to hypoglycemia after the prolonged starvation [ PDK4 for glucose homoeostasis. Although cardiac-specific overexpression of PDK4 exacerbates hypertrophic cardiomyopathy caused by calcineurin stress-activated pathway in PDK4/CnA double transgenic mice [ PDK4, distinct metabolic modulation profile of PPARδ from two other PPAR subtypes and cardiac protection in ischemia/reperfusion mice as previously reported [Increased insulin sensitivity occurs following enhanced FA reesterification in adipose tissue, reduced FA efflux, and limited ectopic lipid deposition.sistance . Howeverarvation , which inic mice , it exernic mice . Myocardreported  may endo ANGPTL4 and PDK4, which are important regulators of glucose and lipid metabolism and insulin resistance, is directly linked to S273 phosphorylation of PPARγ in response to common T2DM factors such as a high-fat diet, obesity, and inflammation. Chi preferentially induced ANGPTL4 and PDK4 expressions by inhibiting these cascades most likely via its unique configuration-restricted binding to the receptor, which influenced cofactor recruitment and promoter binding of the PPARγ-containing transcription complex. Further investigation is required to elucidate the detailed mechanism for gene-specific regulation by the phosphorylated receptor. Nevertheless, the effect of Chi on the regulation of insulin resistance-related gene expression by PPARγ, together with its moderate activity on PPARα and δ, could rebalance glucose and FA uptake and substrate utilization in adipose tissue, muscle, liver, and heart upon insulin resistance and obesity, which may provide comprehensive clinical benefits to T2DM patients in the future.In summary, we demonstrate that regulation of the expression ofSupplementary Figure 1: The expression induction of target genes by three agonists in human pre-adipocyte HPA-v cells. Supplementary Figure 2: The expression induction of target genes by three agonists in L-02 cells. Supplementary Figure 3: PPARγ transactivation of three agonists in L-02 cells. Supplementary Figure 4: The transactivation of SR1664 on different PPARγ constructs in L-02 cells. Supplementary Figure 5: In vitro cofactors recruitment by PPARγ LBD independent of ligand.


Procyon lotor) are sentinels of local exposure to trace element contaminants at a coal fly ash site and whether exposure resulted in health impairment or changes in the intestinal helminth communities. We compared trace element accumulation and the impact on health responses and intestinal helminth communities of raccoons inhabiting contaminated and reference sites of the U.S. Department of Energy’s Savannah River Site . Data on morphometry, hematology, histopathology, helminth community and abundance, and liver trace element burdens were collected from 15 raccoons captured adjacent to a coal fly ash basin and 11 raccoons from a comparable uncontaminated site nearby. Of eight trace elements analyzed, Cu, As, Se, and Pb were elevated in raccoons from the contaminated site. Raccoons from the contaminated site harbored higher helminth abundance than animals from the reference site and that abundance was positively associated with increased Cu concentrations. While we found changes in hematology associated with increased Se exposure, we did not find physiological or histological changes associated with higher levels of contaminants. Our results suggest that raccoons and their intestinal helminths act as sentinels of trace elements in the environment associated with coal fly ash contamination.Anthropogenic pollutants disrupt global biodiversity, and terrestrial sentinels of pollution can provide a warning system for ecosystem-wide contamination. This study sought to assess whether raccoons (The online version of this article (doi:10.1007/s00244-016-0340-2) contains supplementary material, which is available to authorized users. Human-driven environmental factors have rapidly altered ecosystem composition and function on a global scale. Drivers of change include deforestation, hunting, introduction of invasive species, pathogen spread, and pollution  are considered a sentinel species of environmental hazards . SRS is a former nuclear weapons production facility characterized by the occurrence of industrial areas, including a coal-powered electricity generation facility that released industrial contaminants into the surrounding environment (White and Gaines 2) and has had restricted public access since 1951  was selected as the control site for this study and is a natural creek originating from a spring-fed aquifer system that has not been impacted by industrial activities on the SRS Bowers . Beaver To evaluate whether raccoons were sentinels of trace element contamination, we tested individual parameters  as predictors of elevated concentration of eight trace elements in the liver of raccoons collected in contaminated versus reference sites. For trace elements that exhibited differences among contaminated and reference sites, we considered them as predictor variables to assess changes in morphometry, hematology, and parasite burden of individual raccoons across contaminated and reference sites.In August and December 2013, raccoons were captured using steel wire box live traps , baited with whole corn, fish meat, and/or plaster scent tabs. Traps were placed strategically along roads, water bodies, or in a grid within 500 m from the centroid of the reference site or the fly ash containment basins to target animals utilizing resources known to be uncontaminated or contaminated, respectively. After capture, animals were transported from the field to the Savannah River Ecology Lab animal care facility, where they were immobilized using Telazol (2.2 mg/kg IM) and subsequently euthanized via intracardiac injection of potassium chloride administered while the animal was under general anesthesia. All animal handling practices and euthanasia were performed in accordance with University of Georgia Animal Care and Use guidelines under protocol A2012 12-010-Y2-A3.n = 4 ; reference site n = 7 ] and 15 raccoons in December . Seven raccoons were aged at 1–2 years , 12 aged at 3–4 years , and 7 aged at 5–6 years .Twenty-six raccoons were trapped throughout the two trapping seasons, and sex and estimated age classes were recorded. Raccoons were grouped into three age classes: I (1–2 years), II (3–4 years), and III (5–6 years) based on tooth wear. We captured 11 raccoons in August  analysis was conducted on all liver samples. Wet weights of liver samples were recorded and tissues subsequently freeze-dried and homogenized. For trace element analysis, approximately 250 mg of dry sample was used for microwave digestion  with 10.0 ml trace metal-grade nitric acid  were analyzed using a DMA 80 Dual Cell Mercury Analyzer  according to U.S. Environmental Protection Agency (EPA) method 7473, which has an instrument detection limit (IDL) for this method of 0.01 nanograms (ng) of total mercury. For quality assurance, we included with each set of 10 samples a replicate, a blank, and two standard reference materials . Solid SRMs were used to calibrate the instrument. Method detection limits  averaged 0.0233 parts per billion (ppb) dry mass. Mean percent recoveries of THg for the SRMs TORT-2 and PACS-2 were 102.1 ± 1.6 and 103.8 ± 3.9, respectively. All THg concentrations were converted to parts per million (ppm) presented on a dry mass basis to be comparable to the other trace element concentrations.Complete blood cell counts were conducted for 24 of 26 raccoons, excluding the other two animals in which blood samples were clotted. Anticoagulant (EDTA) blood tubes were processed for red blood cell (RBC) and total white blood cell (WBC) counts, mean corpuscular volume (MCV), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), platelet counts, mean platelet volume (MVP), plasma protein, and reticulocyte and absolute reticulocyte counts by automation using the Advia 2120i Hematology System . Blood smears were stained with Wright’s (Romanosky) and Giemsa Stain (counter stain) procedure , and examined for cellular morphology, WBC differential cell counts, and blood parasites.Histopathologic analyses were conducted in liver, spleen, kidney, ileum, and colon samples collected from the 11 raccoons trapped in August 2013. These analyses were not conducted in raccoons from December 2013 because of logistical constraints. After fixation, sections of these tissues were processed routinely and embedded in paraffin. Sections that were 5-μm thick were stained with hematoxylin and eosin. Slides were examined by a single, blind observer (LF) for inflammation, fibrosis, degeneration/necrosis, pigment deposition, and other changes. A grading scheme was created and each tissue was graded subjectively for multiple parameters on a 0 (no changes), 1 (mild changes), 2 (moderate changes), and 3 (marked changes) scale for a total of 103 categorical variables. We included in the final analyses only 16 variables that were found to have substantial variation in the breadth of abnormalities and 3 binary categories .Upon thawing, intestines were carefully opened along their length with scissors and contents were initially screened for helminths by hand. Intestines and digested material were rinsed through a 180-µm sieve to ensure all parasites were collected; the solid material left after rinsing was sorted again under a dissecting microscope. Parasites were preserved in absolute ethanol and identified to genus or species (when possible) using a compound microscope. Total number of helminths was determined for each raccoon using a dissecting scope.p < 0.05), and all further statistical tests were conducted using log-transformed data. Based on a backward elimination procedure, stepwise regression models were used to test for differences in trace element exposure between raccoons from the contaminated and reference sites. Previous to their definitive inclusion in stepwise regression models, we tested for the univariate effect of site, age and sampling season on trace element accumulation of raccoons. We only included male data to test the univariate effect of age and sampling season because of the unbalanced sample size of sexes distributed among contaminated and reference sites. We broke down age by site and season to rule out the potential association of either site or season with animal age in the univariate analysis.Distributions of all variables were tested for normality , hematology , and parasite burden (helminth abundances) of individual raccoons, we considered the spectrum of trace elements that were at higher concentrations in livers of raccoons from the contaminated site compared to the reference site as predictor variables. We used stepwise regression models with backward elimination, including morphometry, hematology, and parasitology responses in separate models. To test the impact of trace elements on raccoon morphometry and hematology, we considered all raccoons from contaminated and reference sites. Before the inclusion of trace element concentrations in the stepwise regression models, Pearson correlation coefficients were calculated to determine collinearity between the predictor variables. To reduce the number of variables analyzed, we conducted principal component analyses (PCA) independently on the morphometric dataset. Principal components that accounted for at least 70% of the total variance and whose eigenvalues were >1 were retained for interpretation and incorporation into multivariate regression models.To determine the impact of trace element accumulation and sampling season on morphometry . To test whether binary response variables  correlated to contaminant exposure, simple logistic regression models were performed.Nonmetric multidimensional scaling (NMDS) was used to evaluate visually the variation in community composition and abundance of intestinal helminth species harbored by all raccoons from contaminated and reference sites. We used abundance data for each helminth species from individual raccoons to calculate the Sorensen (Bray–Curtis) distance metric among raccoons from both sites. Two-dimensional analysis was chosen by minimizing stress, a goodness-of-fit measure of the mismatch between the rank-order of Euclidean distance in the ordination space, and the distance among species indicated by the Bray–Curtis dissimilarity matrix. Relationships between trace element concentrations and the ordination scores were graphically explored.Stepwise regression models with backward elimination were used to test for the influence of trace element concentrations and sampling season on (non-log-transformed) parasite abundances of raccoons collected at both contaminated and reference sites. Because male and female raccoons have previously been found to have different parasite abundances ; thus, raccoons of all three age classes were pooled in stepwise regression models. As such, we only included site, sampling season and their interaction as predictors of trace element accumulation in raccoons. We found a significant influence of site and sampling season on the exposure of trace elements in raccoons. As, Se, and Pb were at higher concentrations in livers of raccoons inhabiting the contaminated site than in the reference site  on trace element accumulation in raccoons (βte Table . Only Nite Table . In addite Table . Additiote Table . The maite Table . No corrPC1) accounted for 82% of the variance ; PC loadings (eigenvectors) for each morphometric measurement were reported . PC1 was interpreted as the range of body size variation among sampled raccoons, supported by the inverse relationship of PC values to morphometric measurements. Neither exposure to trace elements nor sampling season were significantly related to PC1 in raccoons in the final stepwise regression model.Mean (±SE) values for all morphometric measurements were reported for male and female raccoons captured in the contaminated and reference sites . For the morphometric PCA, the first principal component . For binary variables, we found no significant association of As and Se to the presence or absence of abnormalities and granulomas in liver, ileum, or colon in raccoons .Relevant histological injuries detected in raccoons trapped in August 2013 were confined to 16 of the 103 variables . These injuries were defined as hepatic fibrosis, eosinophilic hepatic portal inflammation, hepatic granulomas, bile duct proliferation and pigment in hepatocytes; splenic lymphoid and reticuloendothelial hyperplasia; cellular infiltrates in intestinal lamina propria and submucosa, and intestinal granulomas. We tested nominal histological variables against the two trace elements, As and Se, which were at higher concentrations in livers of raccoons from the contaminated site compared to the reference site in August 2013. We did not find any association, however, between histological findings and As and Se , one cestode (Atriotaenia procyonis), and one acanthocephalan (Macracanthorhynchus ingens). In the contaminated site, we found one trematode but could only identify it to genus (Isthmiophora sp.); one raccoon had an unidentifiable strigeid trematode. Of the raccoons from the contaminated site, there was a mix of the four identified parasite species, with no individual having all four. M. ingens was the most abundant and prevalent helminth in both contaminated and reference sites, whereas A. procyonis was only present in raccoons from the contaminated site  in raccoons. Helminth total abundance was higher in raccoons captured in December than in August . WBC counts was not a significant predictor in any of the stepwise regression models that included helminth abundance .NMDS two-dimensional ordination showed a minimum stress of 0.21 and accounted for 74% of the variance in the helminth abundance data. The ordination pattern showed that the helminth community composition differed between raccoons from contaminated and reference sites, but there was a larger variance in the helminth abundance in males from the contaminated site than the reference site  Eisler , As in dP. rara compared with animals exposed to lower concentrations of Cu. Raccoons from the contaminated site harbored ~23% more endoparasites per raccoon than animals from the reference site due to higher abundances of A. procyonis and P. rara. The identified parasitic taxa represent a broad spectrum of transmission strategies that cannot be explained by changes in intermediate host densities alone. While A. procyonis, P. rara, and M. ingens have arthropod or millipede intermediate hosts Supplementary material 2 (PDF 73 kb)Supplementary material 3 (PDF 81 kb)Supplementary material 4 (PDF 77 kb)Below is the link to the electronic supplementary material.


This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors.The DYRK1A  gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer’s disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC DS, the most common chromosomal abnormality associated with birth defects and developmental disabilities, is caused by full or partial trisomy of chromosome 213. Almost all DS cases inevitably lead to the development of Alzheimer’s disease (AD)-type pathology4. Transgenic mice carrying an extra copy of DYRK1A have been shown to exhibit symptoms similar to DS, including brain abnormalities, neurodevelopmental delay, and memory impairments7. DYRK1A in the brain displays a distinct structure-specific distribution pattern8, and it interacts with an array of factors involved in neuronal development, proliferation, and differentiation9. The level of DYRK1A is elevated in a gene dosage-dependent manner in DS, suggesting that the protein not only plays an important role in regulating normal brain functions, but also in the etiology of DS10.The human DYRK1A gene11. DYRK1A was shown to phosphorylate microtubule-associated protein tau at T212 to prime tau for subsequent phosphorylation by GSK-3β at S30813. This inhibits tau’s ability to stimulate microtubule assembly and promotes self-aggregation, like abnormally hyperphosphorylated tau in AD brain13. DYRK1A was also found to phosphorylate amyloid precursor protein (APP) at T66814 and presenilin-1 at T35415, which correlated with increased cleavage of APP by β and γ secretases16 respectively, and leads to the formation of neurotoxic β-amyloid peptides (Aβ). Moreover, Aβ is shown to be involved in a positive feedback loop for promoting DYRK1A expression, which may further accelerate production of Aβ17.DYRK1A has been linked to neurofibrillary degeneration and β-amyloidosis of AD20. The potency of such inhibitors has mostly been analyzed using radioactive tracer methods despite the availability of non-radioactive assays22. It may be that these methods typically require multiple steps, which is undesirable for screening. Here, we describe a simple ELISA assay for DYRK1A inhibition using dynamin 1a and its phosphorylation site antibody for detection24. The ELISA assay has been verified using two known DYRK1A inhibitors and found to be consistent with radioactive tracer methods.The collected evidence suggests that DYRK1A is a potential drug target for the treatment of DS and AD. To this end, many classes of DYRK1A inhibitors, both natural and synthetic, have been testedAB_2631263, the antibody has been deposited with theDevelopmental Studies of Hybridoma Bank) was prepared as described24. The antibody was partially purified from ascites using Bakerbond ABx resins (#7269-02) before use. Anti-dynamin mAb Hudy-1 (RRID:AB_309677)25 was obtained from EMD Millipore. Alkaline phosphatase (AP) conjugated goat anti-mouse IgG secondary antibody (#115-055-146) was purchased from Jackson ImmunoResearch Laboratories, Inc.Epigallocatechin gallate  and harmine (#10010324) were obtained from Cayman Chemical. Para-nitrophenyl phosphate (PNPP) tablets and diethanolamine substrate buffer were purchased from Thermo-Fisher Scientific. EGCG and harmine were initially prepared as 50 mM stock in 100% DMSO. Working solutions of EGCG and harmine (0.01 μM – 3.2 μM) were prepared from stocks in 2% DMSO by serial dilution. Dynamin 1a pS857-specific mouse mAb 3D3  at the start codon and an in-frame termination at codon 498 plus a Xho I site (ggactcgagtcaagggctggtggacacactgtt), respectively, at 5’ and 3’ ends. PCR was performed in 50 μl mixture containing 10 ng template, 0.2 μg of each primer, 0.2 mM ATP, and PfuUltra (Agilent Technologies), as recommended by the supplier. Amplification was conducted with 20 cycles of the following steps: 94°C, 30-sec, 72°C 90-sec, and 62°C 30-sec. Custom primers were purchased from Integrated DNA Technologies. The resulting amplicon was then cloned into a modified T7 promoter-driven vector pND128 via the Cla I and Xho I sites, as described29. Proline rich domain  of dynamin 1a was also prepared as N-terminal tagged 6xHis fusion protein (HT-PRD) exactly as described above. The PRD fragment was first produced from the semi-synthetic dynamin 1a gene24 by PCR using a pair of primers, (aggatcgatgcatcatcatcatcatcataacacgaccaccgtcagcacg) and (aggctcgagtcataggtcaaaaggtggtcg) for subsequent cloning into expression vector pND1, like HT-497. It should be noted that the HT-PRD construct carries an inadvertent F862L substitution. Both pHT-497 and pHT-PRD have been deposited withAddgene.6xHis tagged rat truncated DYRK1A containing residues 1-497 (HT-497) was used for all assays. This truncation preserves activity of DYRK1A29. Proteins were quantified by Bradford method30 and stored at -80°C until use.Both HT-497 and HT-PRD were expressed and purified using TALON metal affinity resin (Clontech Laboratories) under native conditions as described2, 100 μM ATP , inhibitor if needed, and 5 ng HT-497 (unless otherwise indicated). Reactions were initiated by adding HT-497 and continued for 30 min (unless otherwise indicated) at 30°C. For time course experiments, reactions were terminated by the addition of 20 mM EDTA at the indicated time points. A set of inhibition experiments typically consists of a no-inhibitor control plus a series of eight inhibitor concentrations . Each point was run in duplicate with DMSO present in all assays at 0.2% final concentration. DMSO, up to 2%, does not affect the potency of EGCG and harmine. HT-PRD phosphorylation was subsequently determined by the sandwich antibody staining protocol, first with 100 μl mAb (60 min at room temperature) then with 100 μl AP-linked anti-mouse secondary antibody (60 min at room temperature), followed by colorimetric reaction with 100 μl PNPP solution. The extent of AP reaction was monitored at λ=405 nm. For Hudy-1 staining, wells were coated, blocked, and then stained with the antibody (1:3000 dilution) for colorimetric detection as described above.Substrate, HT-PRD, was diluted in dilution buffer  to a concentration of 2 ng/μl  and 1:2000 dilutions of commercial secondary antibody for the assay.Dilution factors for both mAb 3D3 and secondary antibody were pre-determined for each batch of antibody to ensure that neither antibody was limiting in the assay. A stock to be determined was serially diluted  and each dilution was used together with a non-limiting concentration of the other antibody to assess the level of HT-PRD phosphorylated under standard ELISA reaction conditions without inhibitor (see Results and Discussion). OD50 calculation were performed in KaleidaGraph . Data was corrected for background (readings from wells with only PNPP) before subsequent manipulations. To determine IC50, the residual DYRK1A activity was first calculated as the ratio to the no-inhibitor control in that set. The resulting residual activity was then plotted against its corresponding inhibitor concentrations in semi-log graph and the plot was fit to the sigmoidal equation, y = a+(b-a)/(1+(x/c)d), for IC50 calculation. Data throughout the article are presented as mean ± standard error of mean.Data transformation, calculation, plotting, curve fitting, and IC50 of the inhibitor. All other procedures of the assay are unchanged. Residual kinase activity with the inhibitor at each ATP concentration was first calculated as a percentage of the corresponding no-inhibitor control. The residual kinase activity was subsequently converted to inhibition potency as the difference from 1. The value for each ATP concentration was then normalized to the inhibition potency at 100 μM ATP and plotted.The standard ELISA protocol was modified to run under conditions allowing a constant inhibitor to compete against varying ATP concentrations in inhibiting DYRK1A. Briefly, a set of competition experiments had four DYRK1A assays in the presence of different ATP concentrations  with a single fixed concentration of the inhibitor to be tested. An identical set, except without inhibitor, was performed in parallel (no-inhibitor controls). The inhibitor concentration used was roughly twice the IC32 in developing our assay, by immobilizing the substrate followed by kinase phosphorylation in the wells, as this format offers the advantage of a simple, proven design versus other non-radioactive approaches33. Like many non-radioactive approaches33, our assay relies on a phospho-specific antibody to differentiate between phosphorylated from un-phosphorylated substrates. The antibody used in the assay, mAb 3D3, was raised against DYRK1A-phosphorylated Dynatide 3; a peptide derived from the DYRK1A phosphorylation site of dynamin 1a at S85724. 3D3 has been shown to recognize only pS857-dynamin 1a in rat brain extracts upon extensive phosphorylation24.We chose to follow the ELISA-based protocol35, was first tested as a substrate. Unfortunately, it failed to produce any signal upon phosphorylating coated Dynatide 3 with DYRK1A, presumably due to a lack of peptide coating. To circumvent this problem, we used a 6X histidine-tagged PRD of dynamin 1a (HT-PRD) as a DYRK1A substrate. This fragment coats wells in a concentration-dependent manner and the amount of immobilized proteins, as revealed by mAb Hudy-1 staining, are proportional to input proteins up to 200 ng/well of HT-PRD (15 pmole/well)  .in situ with excess DYRK1A (HT-497) (see Methods) for 60 min and probed with excess (non-limiting) mAb 3D3 and secondary antibodies  to identify the proper conditions. The system response to changes of HT-497 was first examined . Our ELI 75 min . Therefo 75 min .50, the amounts of antibody, both 3D3 and secondary antibody, must not be limiting. Otherwise, immunostaining will most likely under-report the actual phosphorylation level at lower concentrations of inhibitor, which could skew the IC50 calculation. Therefore, each batch of antibody was titered to determine the maximal dilution can be used. As shown for titering of 3D3, when the antibody is limiting (provided that a non-limiting concentration of secondary antibody is used), the readout will increase upon addition of 3D3 until a plateau indicating saturation Click here for additional data file.Copyright: © 2017 Liu Y et al.2017Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).22. These assays employ similar solution DYRK1A reactions at first stage and then different approaches for measuring the phosphorylated products. One used a phospho-specific antibody to capture products for subsequent immuno/colorimetric detection21, while another used a fluorescein-tagged substrate and analyzed products by high performance liquid chromatography/fluorescence detection22. The above methods have been optimized for sensitivity to measure cellular DYRK1A activity. We do not know whether our ELISA method, at the current stage, affords such level of sensitivity. Nevertheless, as we have demonstrated, our ELISA assay provides sufficient sensitivity for analyzing inhibitor activity with recombinant DYRK1A. Furthermore, because of the standard ELISA protocol, our assay is straightforward to perform with the entire process carried out in a single well. The tools and equipment for adapting this plate-based assay for high throughput automation are widely available, and if necessary, the assay can be refined to further improve the sensitivity. We believe that our assay offers a simple, rapid, and reliable non-radioactive method suitable for replacing the radioactive trace assays in quantifying and screening DYRK1A inhibitors.As noted, non-radioactive DYRK1A assays have been describedThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Liu Y et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Dataset 1.Raw data for10.5256/f1000research.10582.d15531742 Zipped file, containing the following:Data for Figure 1. Coating ELISA plate with HT-PRD. Data (OD405) for 0 – 800 ng of coated HT-PRD per well were shown. Background measurements for allData for Figure 2. Phosphorylation of coated HT-PRD by DYRK1A. Data (OD405) for 0 – 800 ng coated HT-PRD per well were shown.Data for Figure 3. DYRK1A concentration-dependent phosphorylation of coated HT-PRD. Data (OD405) for phosphorylation with 0 – 80 ng HT-497 were shown.Data for Figure 4. Time-course phosphorylation of coated HT-PRD by DYRK1A. Time-course phosphorylation data (OD405) with 0–90 min incubation time were shown.Data for Figure 5. 3D3 dilution factor determination. Data (OD405) for 3D3 dilution 1:1,000 – 256,000 were shown.Data for Figure 6A. Epigallocatechin gallate (EGCG) inhibition profile. Data (OD405) for EGCG 0 – 3.2 μM were shown.Data for Figure 6B. Harmine inhibition profile. Data (OD405) for harmine 0 – 3.2 μM were shown.Data for Figure 7. ATP competition assay. Data (OD405) for ATP 100 – 800 μM were shown.Data for Supplementary Figure. Secondary antibody dilution factor determination. Data (OD405) for secondary antibody dilution 1:1,000 – 256,000 were shown. I approve the article by Liu et al. DYRK1A has developed into an interesting pharmacological target. The assay described in this paper offers an interesting alternative to previously published assays. For assay validations the authors made however a poor choice. Epigallocatechin gallate (EGCG) has been identified as a DYRK1 inhibitor but this compound is highly reactive and not a meaningful inhibitor for assay validation . Recently the diverse problem using promiscuous, reactive and chemically unstable inhibitors (PAINS) has been highlighted in the literature. Certainly EGCG should not be used as a control compound in any assay. However, the data is well presented (antibody source should be described) and a good addition to the kinase assay repertoire that can be used studying DYRK1A inhibitors.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank Dr. Knapp for the comments. in vitro system consisting of only DYRK1A, substrate, ATP, and buffer, like our ELISA-based assay, EGCG is a well-behaved small molecule. Its property is predictable and can be reliably measured under such conditions as has been done by us and others. In addition, EGCG inhibits DYRK1A through a non-ATP-competitive mechanism. Pairing EGCG with ATP-competitive inhibitors such as harmine provides different prospects for assessing the ELISA assay.    We agree with Dr. Knapp that EGCG is known to be unstable and is reactive toward many cellular targets. Clearly, EGCG is not a suitable reagent for validating biological assays. However, in a defined The antibody used in the study, 3D3, has been deposited with the Developmental Studies Hybridoma Bank of the University of Iowa. Liu and coworkers have developed an ELISA-based assay for the protein kinase DYRK1A and show that their assay compares well with the traditional radioactive assays in the analysis of DYRK1A inhibitors. As correctly pointed out the authors, a previous ELISA-based DYRK1A assay developed in my own lab had been optimized for sensitive detection of endogenous DYRK kinases, while the new ELISA assay is more straightforward and provides a simple and rapid method for analyzing inhibitor activity with recombinant DYRK1A. The inhibitions curves with the DYRK1A inhibitors EGCG and harmine support the conclusion “that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors”. This methods article clearly represents a substantial modification of an existing procedure. The study is well designed, the methods and the analysis of the results are appropriately described and the conclusions are justified on the basis of the results. In summary, this is sound and useful study that will be valuable for other researchers in the field.Recommendationsnd paragraph the introduction, presenilin is misspelled.In the 2I appreciate the identification of the used antibodies in the method section by their accession numbers in the antibody registry. Direct links to this registry would be helpful for the reader.For the convenience of the reader, I suggest to include the harmine inhibition curve  in the main text as a second panel in Fig. 6.1).The final statement of the manuscript suggests the use their assay for the screening of DYRK1A inhibitors. It may be worth to validate this application by determining the Z’–factor of the assay  and antibody 3D3 with Addgene and the Developmental Studies Hybridoma Bank of the University of Iowa, respectively.  The error bars are SEM. This is now indicated in the revised article. The raw data in Excel format have been submitted.The term “kinase” is added to the title.We thank Dr. Becker for comments and suggestions. We have revised the article and addressed most of the questions. Here is the summary of changes.