Identifying ligands for the PHD1 finger of KDM5A through high-throughput screening.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

Recognition of Histone H3 Methylation States by the PHD1 Domain of Histone Demethylase KDM5A.

PHD reader domains are chromatin binding modules often responsible for the recruitment of large protein complexes that contain histone modifying enzymes, chromatin remodelers, and DNA repair machinery. A majority of PHD domains recognize N-terminal residues of histone H3 and are sensitive to the methylation state of Lys4 in histone H3 (H3K4). Histone demethylase KDM5A, an epigenetic eraser enzyme that contains three PHD domains, is often overexpressed in various cancers, and its demethylation activity is allosterically enhanced when its PHD1 domain is bound to the H3 tail. The allosteric regulatory function of PHD1 expands roles of reader domains, suggesting unique features of this chromatin interacting module. Our previous studies determined the H3 binding site of PHD1, although it remains unclear how the H3 tail interacts with the N-terminal residues of PHD1 and how PHD1 discriminates against H3 tails with varying degrees of H3K4 methylation. Here, we have determined the solution structure of apo and H3 bound PHD1. We observe conformational changes occurring in PHD1 in order to accommodate H3, which interestingly binds in a helical conformation. We also observe differential interactions of binding residues with differently methylated H3K4 peptides (me0, me1, me2, or me3), providing a rationale for PHD1's preference for lower methylation states of H3K4. We further assessed the contributions of various H3 interacting residues in the PHD1 domain to the binding of H3 peptides. The structural details of the H3 binding site could provide useful information to aid the development of allosteric small molecule modulators of KDM5A.

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors of KRasG12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D. These differences in selectivity, combined with the high resolution of 19F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.

A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain.

TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.

Allosteric N-WASP activation by an inter-SH3 domain linker in Nck.

Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott-Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP-driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes.

Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism.

The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism--enabled by the functional coupling between a reader and a catalytic domain in KDM5A--suggests a model for the spread of demethylation on chromatin.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.

Interventions to promote informed consent for patients undergoing surgical and other invasive healthcare procedures.

BACKGROUND: Achieving informed consent is a core clinical procedure and is required before any surgical or invasive procedure is undertaken.  However, it is a complex process which requires patients be provided with information which they can understand and retain, opportunity to consider their options, and to be able to express their opinions and ask questions.  There is evidence that at present some patients undergo procedures without informed consent being achieved. OBJECTIVES: To assess the effects on patients, clinicians and the healthcare system of interventions to promote informed consent for patients undergoing surgical and other invasive healthcare treatments and procedures. SEARCH METHODS: We searched the following databases using keywords and medical subject headings: Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 5, 2012), MEDLINE (OvidSP) (1950 to July 2011), EMBASE (OvidSP) (1980 to July 2011) and PsycINFO (OvidSP) (1806 to July 2011). We applied no language or date restrictions within the search. We also searched reference lists of included studies. SELECTION CRITERIA: Randomised controlled trials and cluster randomised trials of interventions to promote informed consent for patients undergoing surgical and other invasive healthcare procedures. We considered an intervention to be intended to promote informed consent when information delivery about the procedure was enhanced (either by providing more information or through, for example, using new written materials), or if more opportunity to consider or deliberate on the information was provided. DATA COLLECTION AND ANALYSIS: Two authors assessed the search output independently to identify potentially-relevant studies, selected studies for inclusion, and extracted data. We conducted a narrative synthesis of the included trials, and meta-analyses of outcomes where there were sufficient data. MAIN RESULTS: We included 65 randomised controlled trials from 12 countries involving patients undergoing a variety of procedures in hospitals. Nine thousand and twenty one patients were randomised and entered into these studies. Interventions used various designs and formats but the main data for results were from studies using written materials, audio-visual materials and decision aids. Some interventions were delivered before admission to hospital for the procedure while others were delivered on admission.Only one study attempted to measure the primary outcome, which was informed consent as a unified concept, but this study was at high risk of bias.  More commonly, studies measured secondary outcomes which were individual components of informed consent such as knowledge, anxiety, and satisfaction with the consent process.  Important but less commonly-measured outcomes were deliberation, decisional conflict, uptake of procedures and length of consultation.Meta-analyses showed statistically-significant improvements in knowledge when measured immediately after interventions (SMD 0.53 (95% CI 0.37 to 0.69) I(2) 73%), shortly afterwards (between 24 hours and 14 days) (SMD 0.68 (95% CI 0.42 to 0.93) I(2) 85%) and at a later date (15 days or more) (SMD 0.78 (95% CI 0.50 to 1.06) I(2) 82%). Satisfaction with decision making was also increased (SMD 2.25 (95% CI 1.36 to 3.15) I(2) 99%) and decisional conflict was reduced (SMD -1.80 (95% CI -3.46 to -0.14) I(2) 99%). No statistically-significant differences were found for generalised anxiety (SMD -0.11 (95% CI -0.35 to 0.13) I(2) 82%), anxiety with the consent process (SMD 0.01 (95% CI -0.21 to 0.23) I(2) 70%) and satisfaction with the consent process (SMD 0.12 (95% CI -0.09 to 0.32) I(2) 76%). Consultation length was increased in those studies with continuous data (mean increase 1.66 minutes (95% CI 0.82 to 2.50) I(2) 0%) and in the one study with non-parametric data (control 8.0 minutes versus intervention 11.9 minutes, interquartile range (IQR) of 4 to 11.9 and 7.2 to 15.0 respectively). There were limited data for other outcomes.In general, sensitivity analyses removing studies at high risk of bias made little difference to the overall results.  AUTHORS' CONCLUSIONS: Informed consent is an important ethical and practical part of patient care.  We have identified efforts by researchers to investigate interventions which seek to improve information delivery and consideration of information to enhance informed consent.  The interventions used consistently improve patient knowledge, an important prerequisite for informed consent.  This is encouraging and these measures could be widely employed although we are not able to say with confidence which types of interventions are preferable. Our results should be interpreted with caution due to the high levels of heterogeneity associated with many of the main analyses although we believe there is broad evidence of beneficial outcomes for patients with the pragmatic application of interventions. Only one study attempted to measure informed consent as a unified concept.

13C NMR metabolomic evaluation of immediate and delayed mild hypothermia in cerebrocortical slices after oxygen-glucose deprivation.

BACKGROUND: Mild brain hypothermia (32°-34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C]glucose and [1,2-C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase. METHODS: Neonatal rat cerebrocortical slices receiving a C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N=3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen-glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator. RESULTS: The most significant metabolite difference (P<0.0056) was [2-C]glutamine's higher final/control ratio for the hypothermia group (1.75±0.12) compared with ratios for the delayed (1.12±0.12) and normothermia group (0.94±0.06), implying a higher pyruvate carboxylase/pyruvate dehydrogenase ratio for glutamine formation. Least Absolute Shrinkage and Selection Operator found the most important metabolites associated with adenosine triphosphate preservation: [3,4-C]glutamate-produced via pyruvate dehydrogenase entry, [2-C]taurine-an important osmolyte and antioxidant, and phosphocreatine. Final principal component analyses scores plots suggested separate cluster formation for the hypothermia group, but with insufficient data for statistical significance. CONCLUSIONS: Starting mild hypothermia simultaneously with oxygen-glucose deprivation, compared with delayed starting or no hypothermia, has higher pyruvate carboxylase throughput, suggesting that better glial integrity is one important neuroprotection mechanism of earlier hypothermia.

Variation in family physicians' recording of auscultation abnormalities in patients with acute cough is not explained by case mix. A study from 12 European networks.

BACKGROUND: Conflicting data on the diagnostic and prognostic value of auscultation abnormalities may be partly explained by inconsistent use of terminology. OBJECTIVES: To describe general practitioners use of chest auscultation abnormality terms for patients presenting with acute cough across Europe, and to explore the influence of geographic location and case mix on use of these terms. METHODS: Clinicians recorded whether 'diminished vesicular breathing', 'wheezes', 'crackles' and 'rhonchi' were present in an observational study of adults with acute cough in 13 networks in 12 European countries. We describe the use of these terms overall and by network, and used multilevel logistic regression to explore variation by network, controlling for patients' gender, age, comorbidities, smoking status and symptoms. RESULTS: 2345 patients were included. Wheeze was the auscultation abnormality most frequently recorded (20.6% overall) with wide variation by network (range: 8.3-30.8%). There was similar variation for other auscultation abnormalities. After controlling for patient characteristics, network was a significant predictor of auscultation abnormalities with odds ratios for location effects ranging from 0.37 to 4.46 for any recorded auscultation abnormality, and from 0.25 to 3.14 for rhonchi. CONCLUSION: There is important variation in recording chest auscultation abnormalities by general practitioners across Europe, which cannot be explained by differences in patient characteristics. There is a need and opportunity for standardization in the detection and classification of lung sounds.

Design of a phosphorylatable PDZ domain with peptide-specific affinity changes.

Phosphorylation is one of the most common posttranslational modifications controlling cellular protein activity. Here, we describe a combined computational and experimental strategy to design new phosphorylation sites into globular proteins to regulate their functions. We target a peptide recognition protein, the Erbin PDZ domain, to be phosphorylated by cAMP-dependent protein kinase. Comparing the five successful designs to the unsuccessful cases, we find a trade-off between protein stability and the ability to be modified by phosphorylation. In two designs, Erbin's peptide binding function is modified by phosphorylation, where the presence of the phosphate group destabilizes peptide binding. One of these showed an additional switch in specificity by introducing favorable interactions between a designed arginine in the peptide and phosphoserine on the PDZ domain. Because of the diversity of PDZ domains, this opens avenues for the design of related phosphoswitchable domains to create a repertoire of regulatable interaction parts for synthetic biology.

Metabolomics of oxidative stress in recent studies of endogenous and exogenously administered intermediate metabolites.

Aerobic metabolism occurs in a background of oxygen radicals and reactive oxygen species (ROS) that originate from the incomplete reduction of molecular oxygen in electron transfer reactions. The essential role of aerobic metabolism, the generation and consumption of ATP and other high energy phosphates, sustains a balance of approximately 3000 essential human metabolites that serve not only as nutrients, but also as antioxidants, neurotransmitters, osmolytes, and participants in ligand-based and other cellular signaling. In hypoxia, ischemia, and oxidative stress, where pathological circumstances cause oxygen radicals to form at a rate greater than is possible for their consumption, changes in the composition of metabolite ensembles, or metabolomes, can be associated with physiological changes. Metabolomics and metabonomics are a scientific disciplines that focuse on quantifying dynamic metabolome responses, using multivariate analytical approaches derived from methods within genomics, a discipline that consolidated innovative analysis techniques for situations where the number of biomarkers (metabolites in our case) greatly exceeds the number of subjects. This review focuses on the behavior of cytosolic, mitochondrial, and redox metabolites in ameliorating or exacerbating oxidative stress. After reviewing work regarding a small number of metabolites-pyruvate, ethyl pyruvate, and fructose-1,6-bisphosphate-whose exogenous administration was found to ameliorate oxidative stress, a subsequent section reviews basic multivariate statistical methods common in metabolomics research, and their application in human and preclinical studies emphasizing oxidative stress. Particular attention is paid to new NMR spectroscopy methods in metabolomics and metabonomics. Because complex relationships connect oxidative stress to so many physiological processes, studies from different disciplines were reviewed. All, however, shared the common goal of ultimately developing "omics"-based, diagnostic tests to help influence therapies.

Outcome-related metabolomic patterns from 1H/31P NMR after mild hypothermia treatments of oxygen-glucose deprivation in a neonatal brain slice model of asphyxia.

Human clinical trials using 72 hours of mild hypothermia (32°C-34°C) after neonatal asphyxia have found substantially improved neurologic outcomes. As temperature changes differently modulate numerous metabolite fluxes and concentrations, we hypothesized that (1)H/(31)P nuclear magnetic resonance (NMR) spectroscopy of intracellular metabolites can distinguish different insults, treatments, and recovery stages. Three groups of superfused neonatal rat brain slices underwent 45 minutes oxygen-glucose deprivation (OGD) and then were: treated for 3 hours with mild hypothermia (32°C) that began with OGD, or similarly treated with hypothermia after a 15-minute delay, or not treated (normothermic control group, 37°C). Hypothermia was followed by 3 hours of normothermic recovery. Slices collected at different predetermined times were processed, respectively, for 14.1 Tesla NMR analysis, enzyme-linked immunosorbent assay (ELISA) cell-death quantification, and superoxide production. Forty-nine NMR-observable metabolites underwent a multivariate analysis. Separated clustering in scores plots was found for treatment and outcome groups. Final ATP (adenosine triphosphate) levels, severely decreased at normothermia, were restored equally by immediate and delayed hypothermia. Cell death was decreased by immediate hypothermia, but was equally substantially greater with normothermia and delayed hypothermia. Potentially important biomarkers in the (1)H spectra included PCr-(1)H (phosphocreatine in the (1)H spectrum), ATP-(1)H (adenosine triphosphate in the (1)H spectrum), and ADP-(1)H (adenosine diphosphate in the (1)H spectrum). The findings suggest a potential role for metabolomic monitoring during therapeutic hypothermia.

Treatment of acute cough/lower respiratory tract infection by antibiotic class and associated outcomes: a 13 European country observational study in primary care.

OBJECTIVES: Acute cough/lower respiratory tract infection (LRTI) is one of the commonest reasons for consulting and antibiotic prescribing. There are theoretical reasons why treatment with particular antibiotic classes may aid recovery more than others, but empirical, pragmatic evidence is lacking. We investigated whether treatment with a particular antibiotic class (amoxicillin) was more strongly associated with symptom score resolution and time to patients reporting recovery than each of eight other antibiotic classes or no antibiotic treatment for acute cough/LRTI. METHODS: Clinicians recorded history, examination findings, symptom severity and antibiotic treatment for 3402 patients in a 13 country prospective observational study of adults presenting in 14 primary care research networks with acute cough/LRTI. 2714 patients completed a symptom score daily for up to 28 days and recorded the day on which they felt recovered. A three-level autoregressive moving average model (1,1) model investigated logged daily symptom scores to analyse symptom resolution. A two-level survival model analysed time to reported recovery. Clinical presentation was controlled for using clinician-recorded symptoms, sputum colour, temperature, age, co-morbidities, smoking status and duration of illness prior to consultation. RESULTS: Compared with amoxicillin, no antibiotic class (and no antibiotic treatment) was associated with clinically relevant improved symptom resolution (all coefficients in the range -0.02 to 0.01 and all P values greater than 0.12). No antibiotic class (and no antibiotic treatment) was associated with faster time to recovery than amoxicillin. CONCLUSIONS: Treatment by antibiotic class was not associated with symptom resolution or time to recovery in adults presenting to primary care with acute cough/LRTI.

Exogenous ethyl pyruvate versus pyruvate during metabolic recovery after oxidative stress in neonatal rat cerebrocortical slices.

BACKGROUND: Exogenous pyruvate and ethyl pyruvate (EP), the key ingredient in a new Ringer's solution in clinical trials, are antioxidants as well as metabolic substrates. In vivo studies show both to be protective in oxidative stress, with EP being better. The authors used an acute rat brain slice preparation to compare EP and pyruvate rescue after H(2)O(2) oxidative stress, asking whether EP was again better and whether its actions were exclusively metabolic. METHODS: Oxygenated neonatal P7 cerebrocortical slices were exposed for 1 h to 2 mM H(2)O(2), and recovered for 4 h with artificial cerebrospinal fluid having 2 mM glucose and (1) 20 mM EP, (2) 20 mM pyruvate, or (3) 1 mM of the nonmetabolizable radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN). Perchloric acid extracts were studied with 31P/1H nuclear magnetic resonance at 14.1 T. Acute cell injury was assessed by counting terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL)-stained cells. RESULTS: At the end of recovery, preservation of adenosine triphosphate and N-acetylaspartate was better with EP than with pyruvate. Adenosine triphosphate preservation was best when PBN and EP were coadministered. 1H nuclear magnetic resonance revealed changes in lactate, alanine, gamma-aminobutyric acid, glutamate, glutamine, succinate, taurine, and myoinositol. Two-dimensional [1H-13C] heteronuclear single quantum coherence spectroscopy found that 13C-EP administration produced the same tricarboxylic acid metabolites as C-pyruvate. TUNEL-positive cell percentages with EP were less than half of those for PBN or pyruvate rescue (P < 0.05). CONCLUSION: EP enters cells, provides pyruvate as a tricarboxylic acid substrate, and is more protective. Although EP provides metabolic protection of adenosine triphosphate levels, it does not maximize antioxidant protection.