Discovery of BI-9787, a potent zwitterionic ketohexokinase inhibitor with oral bioavailability.

Fructose metabolism by ketohexokinase (KHK) is implicated in a variety of metabolic disorders. KHK inhibition is a potential therapeutic strategy for the treatment of diseases including diabetes, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis. The first small-molecule KHK-inhibitors have entered clinical trials, but it remains unclear if systemic inhibition of KHK by small-molecules will eventually benefit patients. Here we report the discovery of BI-9787, a potent, zwitterionic KHK inhibitor characterized by high permeability and favorable oral rat pharmacokinetics. BI-9787 was identified by optimizing chemical starting points generated via a ligand-based virtual screening of Boehringer's virtual library of synthetically accessible compounds (BICLAIM). It serves as a high-quality in vitro and in vivo tool compound for investigating the role of fructose metabolism in disease.

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.

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.

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.

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.

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.

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.

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.

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.

In vivo characterization of the mechanics of human uterine cervices.

The uterine cervix has to provide mechanical resistance to ensure a normal development of the fetus. This is guaranteed by the composition of its extracellular matrix, which functions as a fiber-reinforced composite. At term a complex remodeling process allows the cervical canal to open for birth. This remodeling is achieved by changes in the quality and quantity of collagen fibers and ground substance and their interplay, which influences the biomechanical behavior of the cervix but also contributes to pathologic conditions such as cervical incompetence (CI). We start by reviewing the anatomy and histological composition of the human cervix, and discuss its physiologic function and pathologic condition in pregnancy including biomechanical aspects. Established diagnostic methods on the cervix (palpation, endovaginal ultrasound) used in clinics as well as methods for assessment of cervical consistency (light-induced fluorescence, electrical current, and impedance) are discussed. We show the first clinical application of an aspiration device, which allows in vivo testing of the biomechanical properties of the cervix with the aim to establish the physiological biomechanical changes throughout gestation and to detect pregnant women at risk for CI. In a pilot study on nonpregnant cervices before and after hysterectomy we found no considerable difference in the biomechanical response between in vivo and ex vivo. An outlook on further clinical applications during pregnancy is presented.

Menarcheal onset is associated with body composition parameters but not with socioeconomic status.

In the present study the menarcheal status (pre-or postmenarcheal), body composition, weight status, and socioeconomic parameters such as type of school and parental educational level as of 1752 female adolescents ageing between 12 and 18 years (x = 14.6) from rural areas of Eastern Austria were documented. Furthermore the association patterns between body composition, socioeconomic parameters and menarcheal status were analyzed. It turned out, that body composition parameters such as BMI, lean body mass and absolute and relative fat mass were significantly associated with menarcheal status. Postmenarcheal girls were taller and exhibited a higher weight status, a higher absolute and relative amount of fat mass and a higher amount of lean body mass than their premenarcheal counterparts of the same age. In contrast to the significant association between body composition and menarcheal status, no significant impact of educational level on menarcheal status, indicating socioeconomic status could be documented.

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.

Mechanical properties of the human uterine cervix: an in vivo study.

Experimental results of in vivo measurements to characterize the mechanical behaviour of human uterine cervices are documented. Aspiration experiments were performed on eight uteri in vivo, before vaginal/abdominal hysterectomy, and four uteri were also tested ex vivo, approximately 1.5h after extraction. The reproducibility of the mechanical data from the in vivo aspiration experiments has been analysed. For an introduced "stiffness parameter" the organ specific SD is 22%, so that the proposed experimental procedure allows detections of 30% changes with respect to a reference value of the stiffness parameter. A comparison of in vivo and ex vivo data from the same organ has shown that: (i) the ex vivo mechanical response of the uterine cervix tissue does not differ considerably from that observed in vivo; (ii) some differences can be identified in tissue pre-conditioning with ex vivo showing a stronger history dependence with respect to in vivo; (iii) the differences in the time dependence of the mechanical response are not significant and might be masked by the variability of the measured data. This study represents a first step of a clinical application aiming at analysing the mechanical response of normal cervical tissue at different gestational ages, and identifying the mechanical properties that characterize pathologic conditions such as cervical insufficiency leading to preterm delivery.

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.

Clinicopathologic profile of gestational trophoblastic disease.

Much debate exists on factors predicting the development of persistent gestational trophoblastic disease (pGTD). Diagnosis is still limited by following persistently elevated or rising postevacutation beta-human chorionic gonadotropin (beta-hCG) titers. The aim of the present work was to evaluate the hypothesis that the presence of c-erbB-2 oncogene amplification and expression, in combination with parameters such as DNA-content and karyotype of the sex chromosomes, confer an increased risk of developing pGTD. Clinicopathological characteristics were evaluated in 36 cases of gestational trophoblastic diseases (GTD) and analyzed for c-erbB-2 amplification and protein p185 expression using differential polymerase chain reaction (DPCR) and immunohistochemical (IHC) techniques. The DNA-content was determined by image analysis on Feulgen stained nuclear cell preparations and karyotyping for XY chromosomes was performed by fluorescence in situ hybridization (FISH). The data was correlated with histopathological characteristics of GTD. Seventy-five percent (n = 27) of the examined cases showed spontaneous regression after evacuation, including 2 patients who received additional chemotherapy. Twenty-five percent (n = 9) resulted in a persistent or metastatic disease. The median time between antecedent pregnancy and GTD was 45.4 months. Complete remission was achieved in all patients with pGTD after administration of chemotherapeutic agents or adjuvant surgical procedures. Cases with cerbB-2 amplification and expression in combination with DNA hyperploidy showed higher proliferation and more aggressive behavior (2 complete hydatidiform moles with lung and liver metastases, 2 invasive moles and 1 choriocarcinoma). XY karyotype was evident in the choriocarcinoma and in 2 complete hydatidiform moles with advanced stage and DNA hyperploidy. From these results we conclude that c-erbB-2 amplification and/or protein expression in combination with DNA-content show a significant correlation with the proliferative and aggressive potential of GTD, suggesting their combined use as a possible marker for pGTD.

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.