Crystal structures of human and mouse ketohexokinase provide a structural basis for species- and isoform-selective inhibitor design.

A molecular understanding of the proteins involved in fructose metabolism is essential for controlling the current spread of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism starts with the phosphorylation of D-fructose to fructose 1-phosphate by ketohexokinase (KHK). KHK exists in two alternatively spliced isoforms: the hepatic and intestinal isoform KHK-C and the peripheral isoform KHK-A. Here, the structure of apo murine KHK (mKHK), which differs from structures of human KHK in overall conformation, is reported. An isoform-selective ligand, which offers a 50-fold higher potency on mKHK and human KHK-A compared with KHK-C, is further characterized. In mKHK, large-scale conformational changes are observed upon ligand binding. The structures suggest a combined strategy for the design of species- and isoform-selective KHK inhibitors.

Differential analyte derivatization enables unbiased MALDI-TOF-based high-throughput screening: A proof-of-concept study for the discovery of catechol-o-methyltransferase inhibitors.

Recent advances in label-free high-throughput screening via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offer unprecedented opportunities for the identification of novel chemical starting points in target-based drug discovery. A clear advantage of the technology is the possibility for label-free, direct quantification of analytes with high precision and robustness. Here we have expanded the range of analytes and biology that can be addressed via MALDI-TOF HTS, by developing a method based on post-reaction pyrylium-based derivatization to detect 3-methoxytyramine, the physiological enzyme product of the catechol-O-methyltransferase (COMT) enzyme. The introduction of pyrylium-type reagents as universal derivatization strategy under aqueous conditions for molecules containing primary amines represents a valuable addition to the toolbox of MALDI-TOF assay development. Characterization of COMT's enzymatic activity and inhibition by reference inhibitors, and comparison of the results obtained in our assay with data from previous mechanistic studies validated the performance of this new method. To address the problem of isobaric interference, a source of false results in MALDI-TOF assays measuring low molecular weight analytes, we devised a differential derivatization workflow which can potentially replace other counter- or orthogonal assays in future screening campaigns. Finally, we report on the first label-free HTS campaign for the identification of COMT inhibitors performed in miniaturized 1536-well microtiter plate format via MALDI-TOF MS analysis.

Discovery and Structure-Based Optimization of Fragments Binding the Mixed Lineage Kinase Domain-like Protein Executioner Domain.

Necroptosis is a form of programmed cell death that in case of misregulation can lead to inflammatory diseases. Mixed lineage kinase domain-like protein (MLKL), the effector protein in the canonical necroptosis signaling pathway, becomes activated by phosphorylation. Here, we report the identification of novel reversible binders of the MLKL executioner domain by a protein NMR-detected fragment-based screen. Determination of protein fragment costructures using NMR spectroscopy revealed a small molecule binding site that is distinct from the previously identified binding site of covalent MLKL inhibitors. Affinity optimization of the initially prioritized hit with millimolar affinity was achieved by NMR-guided structure-based design and yielded fragment-like molecules with a KD of 50 µM. Furthermore, we demonstrate that the improved fragment competes for the same binding site as nonyl-maltoside, a detergent that in conjunction with phytic acid activates the MLKL executioner domain.

Biophysical and structural investigation of the regulation of human GTP cyclohydrolase I by its regulatory protein GFRP.

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Hybrid Screening Approach for Very Small Fragments: X-ray and Computational Screening on FKBP51.

Fragment-based drug discovery (FBDD) permits efficient sampling of the vast chemical space for hit identification. Libraries are screened biophysically and fragment:protein co-structures are determined by X-ray crystallography. In parallel, computational methods can derive pharmacophore models or screen virtual libraries. We screened 15 very small fragments (VSFs) (HA ≤ 11) computationally, using site identification by ligand competitive saturation (SILCS), and experimentally, by X-ray crystallography, to map potential interaction sites on the FKBP51 FK1 domain. We identified three hot spots and obtained 6 X-ray co-structures, giving a hit rate of 40%. SILCS FragMaps overlapped with X-ray structures. The compounds had millimolar affinities as determined by 15N HSQC NMR. VSFs identified the same interactions as known FK1 binder and provide new chemical starting points. We propose a hybrid screening strategy starting with SILCS, followed by a pharmacophore-derived X-ray screen and 15N HSQC NMR based KD determination to rapidly identify hits and their binding poses.

Prognostic impact of nodal status and therapeutic implications.

The prognostic impact of lymph node (LN) metastases in gastric cancer is generally accepted. In primarily resected patients the pN-category and LN ratio are independent prognostic factors. Number of involved LNs, number of resected LNs, lymphangiosis and micrometastases also influence the prognosis significantly. To guarantee a proper D2 lymphadenectomy (LAD) at least 25 LNs according to the German S3 guidelines for the treatment of gastric cancer should be removed. Also in neoadjuvantly treated patients the ypN-category and LN ratio play an important prognostic role, despite the fact that UICC staging system was development based only on primarily resected patients. The role of response of LNs in neoadjuvantly treated patients is still unclear and needs further investigation. It seems to be less important than the response of the primary tumor. Limited data exists, suggesting that preoperative treatment might reduce the number of LNs involved and improve ypN-category. Due to further development in gastric cancer like laparoscopic resection and effective perioperative treatment in locally advanced tumor the role and the prognostic impact of LAD is again in the focus of discussion.

Endoluminal perforation of a magnetic antireflux device.

BACKGROUND: The history of surgical antireflux treatment is coined by the search for better alternatives to Nissen fundoplication. Implantable devices are one option, beginning with the "Angelchik" prosthesis 30 years ago. However, this procedure was left soon because of the high rate of foreign body connected problems (migration, perforation). A modern approach is a magnetic sphincter augmentation device (LINX Reflux Management System, Torax Medical, Shoreview, MN, USA), a magnetic chain which is implanted laparoscopically. Advantages reported are simplicity to apply and good results in reflux control, with up to now only rare complication rates as reported in the literature (Lipham et al. in Dis Esophagus, 2014). METHODS: We report one case of erosion of the esophagus by a LINX system resulting in severe dysphagia. RESULTS: A complete endoluminal removal could be achieved by a prototype OTSC-clip remover. Complete remission could be achieved. The technique is presented in detail (video). CONCLUSIONS: In principle, total endoscopic removal of the LINX device is feasible in case of major erosion.

Developments in flexible endoscopic surgery: a review.

Flexible endoscopy is increasingly developing into a therapeutic instead of a purely diagnostic discipline. Improved visualization makes early lesions easily detectable and allows us to decide ad hoc on the required treatment. Deep enteroscopy allows the exploration of even the small bowel - for long a "white spot" for gastrointestinal endoscopy - and to perform direct treatment. Endoscopic submucosal dissection is a considerable step forward in oncologically correct endoscopic treatment of (early) malignant lesions. Though still technically challenging, it is increasingly facilitated by new manipulation techniques and tools that are being steadily optimized. Closure of wall defects and hemostasis could be improved significantly. Even the anatomy beyond the gastrointestinal wall is being explored by the therapeutic use of endoluminal ultrasound. Endosonographic-guided surgery is not only a suitable fallback solution if conventional endoscopic retrograde cholangiopancreatography fails, but even makes necrosectomy procedures, abscess drainage, and neurolysis feasible for the endoscopist. Newly developed endoscopic approaches aim at formerly distinctive surgical domains like gastroesophageal reflux disease, appendicitis, and cholecystitis. Combined endoscopic/laparoscopic interventional techniques could become the harbingers of natural orifice transluminal endoscopic surgery, whereas pure natural orifice transluminal endoscopic surgery is currently still in its beginnings.

A probe-based electromagnetic navigation system to integrate computed tomography during upper gastrointestinal endoscopy.

BACKGROUND AND STUDY AIMS: For preoperative work-up, an examination tool that visualizes separately compiled diagnostics in augmented reality would be desirable. We developed a probe-based electromagnetic navigation system, which can be passed through the working channel of an endoscope, to integrate computed tomography (CT) information during upper gastrointestinal endoscopy. PATIENTS AND METHODS: The target registration error (TRE) of the system was evaluated experimentally and clinically. A total of 24 study patients with upper gastrointestinal cancer were included in the study. The cancerous lesion was endoscopically located (mean duration 8.4 minutes, range 7.1 - 23.2) and the TRE (coronal, transverse, sagittal layer) was measured by comparing the distance between the navigation probe (at the tip of the endoscope) and the target lesion shown on the corresponding CT cross section. RESULTS: Experimental evaluations showed an accuracy in line with the system's inherent failure rate, with a median TRE of 2.8 mm (IQR 1.8 - 4.3), 2.2 mm (0.4 - 3.7), and 2.8 mm (1.1 - 5.9) in the coronal, transverse, and sagittal planes, respectively. Clinical evaluation revealed a median TRE of 4.8 mm (1.9 - 10.1), 3.9 mm (0.7 - 7.1), and 4.2 mm (0.9 - 8.9), respectively. No complications occurred during navigated endoscopy. CONCLUSIONS: The probe-based electromagnetic navigation system revealed high accuracy (TRE < 5 mm), facilitating improved interpretation of endoluminal imaging.

The role of morphology in combination with ploidy analysis in characterizing early gestational abortion.

Morphology in combination with flow cytometry is an inexpensive and fast tool to characterize important reasons for early gestational loss. Early partial hydatidiform moles are especially difficult to identify as not all have developed the typical histological features of central cistern formation and trophoblastic hyperplasia of the chorionic villi. Angiomatoid formation of the blood vessels, irregular chorionic villi, diffuse villous stromal fibrosis, trophoblastic pseudoinclusions, and pleomorphic trophoblast at the implantation site have therefore been proposed as additional morphological criteria. In our study, we investigated the correlation between morphological features and results of ploidy analysis to assess the additional criteria for practical use. Morphological features of the placentas of up to 13 weeks of gestation were evaluated on hematoxylin and eosin sections. Ploidy analysis was performed by flow cytometry on formalin-fixed, paraffin-embedded tissue. Statistical analysis was performed by binary logistic regression. One hundred fifty samples were included in the study. Of the samples, 23 were triploid, 19 were tetraploid, and the remaining ones were diploid. Statistical analysis showed a poor predictive power based on all morphological criteria alone. The most important result of this study was the identification of five triploid placentas that showed diffuse fibrosis of the chorionic villi, angiomatoid changes of the blood vessels, trophoblastic pseudoinclusions, and focal pleomorphic changes at the implantation site, but none of the classical morphological features of triploid pregnancies were identified. Our study demonstrates that the use of additional morphological criteria increases the number of missed abortions with identified causes for pregnancy loss.

One target-two different binding modes: structural insights into gevokizumab and canakinumab interactions to interleukin-1ß.

Interleukin-1ß (IL-1ß) is a key orchestrator in inflammatory and several immune responses. IL-1ß exerts its effects through interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor accessory protein (IL-1RAcP), which together form a heterotrimeric signaling-competent complex. Canakinumab and gevokizumab are highly specific IL-1ß monoclonal antibodies. Canakinumab is known to neutralize IL-1ß by competing for binding to IL-1R and therefore blocking signaling by the antigen:antibody complex. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1ß bioactivity by reducing the affinity for its IL-1RI:IL-1RAcP signaling complex. How IL-1ß signaling is affected by both canakinumab and gevokizumab was not yet experimentally determined. We have analyzed the crystal structures of canakinumab and gevokizumab antibody binding fragment (Fab) as well as of their binary complexes with IL-1ß. Furthermore, we characterized the epitopes on IL-1ß employed by the antibodies by NMR epitope mapping studies. The direct comparison of NMR and X-ray data shows that the epitope defined by the crystal structure encompasses predominantly those residues whose NMR resonances are severely perturbed upon complex formation. The antigen:Fab co-structures confirm the previously identified key contact residues on IL-1ß and provide insight into the mechanisms leading to their distinct modulation of IL-1ß signaling. A significant steric overlap of the binding interfaces of IL-1R and canakinumab on IL-1ß causes competitive inhibition of the association of IL-1ß and its receptor. In contrast, gevokizumab occupies an allosteric site on IL-1ß and complex formation results in a minor reduction of binding affinity to IL-1RI. This suggests two different mechanisms of IL-1ß pathway attenuation.

Molecular structure of human GM-CSF in complex with a disease-associated anti-human GM-CSF autoantibody and its potential biological implications.

Polyclonal autoantibodies against human GM-CSF (granulocyte/macrophage colony-stimulating factor) are a hallmark of PAP (pulmonary alveolar proteinosis) and several other reported autoimmune diseases. MB007 is a high-affinity anti-(human GM-CSF) autoantibody isolated from a patient suffering from PAP which shows only modest neutralization of GM-CSF bioactivity. We describe the first crystal structure of a cytokine-directed human IgG1λ autoantibody-binding fragment (Fab) at 1.9 Å (1 Å=0.1 nm) resolution. Its CDR3-H substantially differs from all VH7 germline IgG1 structures reported previously. We derive a reliable model of the antigen-autoantibody complex by using NMR chemical shift perturbation data in combination with computational methods. Superposition of the modelled complex structure with the human GM-CSF-GM-CSF ternary receptor complex reveals only little overlap between receptor and Fab when bound to GM-CSF. Our model provides a structural basis for understanding the mode of action of the MB007 autoantibody.

Assessment of the in vivo biomechanical properties of the human uterine cervix in pregnancy using the aspiration test: a feasibility study.

OBJECTIVE: To date no diagnostic tool is yet available to objectively assess the in vivo biomechanical properties of the uterine cervix during gestation. METHODS: We show the first clinical application of an aspiration device to assess the in vivo biomechanical properties of the cervix in pregnancy with the aim to describe the physiological biomechanical changes throughout gestation in order to eventually detect pregnant women at risk for cervical insufficiency (CI). RESULTS: Out of 15 aspiration measurements, 12 produced valid results. The stiffness values were in the range between 0.013 and 0.068 bar/mm. The results showed a good reproducibility of the aspiration test. In our previous test series on non-pregnant cervices our repetitive measurements showed a standard deviation of >20% compared to <+/-10% to our data on pregnant cervices. Stiffness values are decreasing with gestational age which indicates a progressive softening of cervical tissue towards the end of pregnancy. Three pregnant women had two subsequent measurements within a time interval of four weeks. Decreasing stiffness values in the range of 20% were recorded. DISCUSSION: This preliminary study on the clinical practicability of aspiration tests showed promising results in terms of reproducibility (reliability) and clinical use (feasibility). Ongoing studies will provide further insights on its usefulness in clinical practice and in the detection of substantial changes of the cervix in pregnancy indicative for threatened preterm birth or cervical insufficiency.

[Amniotic fluid insulin levels versus mean maternal blood glucose levels in gestational diabetes mellitus: an analysis of the neonatal outcome]. / Insulinbestimmung aus dem Fruchtwasser versus mittlere Blutglukose zur Therapieentscheidung beim Gestationsdiabetes: Eine Analyse des kindlichen Outcome.

PURPOSE: To compare the obstetric and neonatal outcome in pregnancies complicated by gestational diabetes mellitus after amniocentesis for amniotic fluid insulin measurement or maternal blood glucose monitoring or both as selection criterion for therapy. MATERIAL AND METHODS: In a retrospective study, 408 diabetic pregnancies were analyzed; 307 were treated with diet alone (group 1) and 101 with diet and insulin (group 2). Pearson's chi2 or Fisher's exact test was used to assess obstetric and anthropometric data within these groups, and p values <0.05 were considered statistically significant. RESULTS: Group 1 - According to the method used, no significant differences were found for gestational age at birth (p = 0.396), mode of delivery (p = 0.79) and neonatal outcome parameters determined as mean birth weight (p = 0.348), birth weight above the 75th percentile (p = 0.473), mean ponderal index (PI; p = 0.434), pH of umbilical artery (p = 0.065) and of umbilical-vein blood (p = 0.052), mean Apgar scores at 1 (p = 0.56) and 5 min (p = 0.072), insulin (p = 0.25) and glucose (p = 0.535) in cord blood. Significant differences were found for birth weight above the 90th percentile (p = 0.005) and Apgar score <7 at 1 min (p = 0.019). Group 2 - Again, no significant differences were observed in terms of gestational age at birth (p = 0.219), mode of delivery (p = 0.386), mean birth weight (p = 0.59), birth weight above the 75th (p = 0.701) and 90th percentiles (p = 0.487), mean PI (p = 0.156), pH of umbilical-artery (p = 0.197) and umbilical vein blood (p = 0.056), Apgar scores at 1 (p = 0.58) and 5 min (p = 0.52), insulin (p = 0.67) and glucose (p = 0.11) in cord blood. CONCLUSION: In retrospective analysis there was no significant difference in outcome parameters in pregnancies complicated by gestational diabetes dependent on the method used as selection criterion for therapy.

Cystic periventricular leukomalacia in preterm infants: an analysis of obstetric risk factors.

OBJECTIVE: To identify obstetric risk factors and to elucidate the effect of prolonged rupture of the membranes on the development of cystic periventricular leukomalacia (PVL) in preterm infants. METHODS: A retrospective case-control study of 95 preterm infants with the diagnosis of PVL and 245 healthy controls matched for gestational age. A total of 52 antenatal, intrapartum and neonatal characteristics were studied by univariate methods and logistic regression. RESULTS: Preterm premature rupture of membranes (PPROM) (odds ratio 2.1 [95% CI 1.3-3.4], P=.003), gestational age at PPROM (P=.025), prolonged rupture of membranes (P<.0001), administration of tocolytic agents (1.8 [1.1-3.0], P=.019) and antibiotics (1.9 [1.2-3.1], P=.008) were associated with PVL. The use of tocolytic agents >24 h (P=.008), prolonged latency between the increase in maternal leukocyte count and birth (P=.034), spontaneous onset of labor (1.8 [1.0-2.9], P=.026), vaginal delivery (1.7 [1.1-2.8], P=.029) and male gender (1.5 [1.0-2.0], P=.04) were found more frequently in PVL cases. Preeclampsia (0.4 [0.1-0.9], P=.034), hypertension at booking (P=.009), sonographic IUGR (P=.020), abnormal blood flow of the umbilical artery (P=.032) and cesarean section without labor (0.5 [0.3-0.8], P=.006) were found less frequently. In logistic regression analysis, prolonged rupture of the membranes (P=.748), preeclampsia (P=.973), the use of antibiotics (P=.617) and beta-sympathomimetic tocolytic agents (P=.563) lost statistical significance, whereas birth weight (P=.036) became significant. CONCLUSION: PPROM and prolonged rupture of the membranes may provoke adverse effects on the neurodevelopmental outcome of the preterm fetus. These findings may have implications on the obstetric management of PPROM beyond 30 weeks of gestation. Cesarean section without labor was less likely associated with the diagnosis of PVL.

Neonatal outcome and two-year follow-up after expectant management of second trimester rupture of membranes.

OBJECTIVE: To assess neonatal outcome and 2-year follow-up of pregnancies complicated by second trimester preterm premature rupture of membranes (PPROM). METHODS: A retrospective review of obstetric and neonatal records for 87 pregnancies (56 singletons, 6 twins, 1 triplet) with PPROM between 14+0 and 24+6 weeks of gestation. Patients received antibiotics and steroids for fetal lung maturity once they reached 24 weeks of gestation. Placentas were examined histopathologically. Surviving infants were followed-up at 2 years of age. RESULTS: Median latency from PPROM to delivery was 4 days. Survival rate of 56 singletons was 45% (25/56); and 13 died in hospital. Survival rate of infants discharged from hospital was 23% (12/56). Chorioamnionitis was seen histologically in 42% (5/12) of surviving infants compared with 92% (12/13) of those that died in hospital. Of the 12 surviving infants, 50% had a normal neurological and developmental outcome at 2 years of age. CONCLUSION: Gestational age, birth weight, and histologic chorioamnionitis have prognostic importance in pregnancies complicated by PPROM. Surviving infants have a 50% chance of achieving an adequate health status at 2 years of age.