UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Calibration and thermal test results of prototype bolometer sensors for ITER fusion reactor.

For over 10 years, several bolometer sensors with different properties have been tested in the IBOVAC facility. The aim has been to develop a bolometer sensor that can be operated in ITER and can withstand harsh operating conditions. For this purpose, important physical properties of the sensors, i.e., cooling time constant τ, normalized heat capacity κ, and normalized sensitivity sn, have been characterized in a vacuum condition and at various temperatures up to 300 °C. The calibration is achieved by ohmic heating of the sensor absorbers by applying a DC voltage and recording exponential current fall during heating. Recently, a Python program was developed to analyze the data and extract the above mentioned parameters including the uncertainties from recorded currents. In the present series of experiments, the latest prototype sensors developed for ITER are tested and evaluated. These include three different sensor types: two with Au absorbers on ZrO2 membranes (self-supporting substrate sensors) and one with Au absorbers on Si3N4 membranes supported by a Si frame (supported membrane sensors). Tests revealed that the sensor with ZrO2 substrate can only be operated up to 150 °C, while the supported membrane sensors passed the tests up to 300 °C successfully. These results will be used, together with other upcoming tests, such as irradiation testing, to select the most suitable sensors to be employed in ITER.

Screening of Chemical Libraries Using Xenopus Embryos and Tadpoles for Phenotypic Drug Discovery.

Phenotypic drug discovery assesses the effect of small molecules on the phenotype of cells, tissues, or whole organisms without a priori knowledge of the target or pathway. Using vertebrate embryos instead of cell-based assays has the advantage that the screening of small molecules occurs in the context of the complex biology and physiology of the whole organism. Fish and amphibians are the only classes of vertebrates with free-living larvae amenable to high-throughput drug screening in multiwell dishes. For both animal classes, particularly zebrafish and Xenopus, husbandry requirements are straightforward, embryos can be obtained in large numbers, and they develop ex utero so their development can be monitored easily with a dissecting microscope. At 350 million years, the evolutionary distance between amphibians and humans is significantly shorter than that between fish and humans, which is estimated at 450 million years. This increases the likelihood that drugs discovered by screening in amphibian embryos will be active in humans. Here, we describe the basic protocol for the medium- to high-throughput screening of chemical libraries using embryos of the African clawed frog Xenopus laevis Bioactive compounds are identified by observing phenotypic changes in whole embryos and tadpoles. In addition to the discovery of compounds with novel bioactivities, the phenotypic screening protocol also allows for the identification of compounds with in vivo toxicity, eliminating early hits that are poor drug candidates. We also highlight important considerations for designing chemical screens, choosing chemical libraries, and performing secondary screens using whole mount in situ hybridization or immunostaining.

Functional characterization of two 20ß-hydroxysteroid dehydrogenase type 2 homeologs from Xenopus laevis reveals multispecificity.

The African clawed frog, Xenopus laevis, is a versatile model for biomedical research and is largely similar to mammals in terms of organ development, anatomy, physiology, and hormonal signaling mechanisms. Steroid hormones control a variety of processes and their levels are regulated by hydroxysteroid dehydrogenases (HSDs). The subfamily of 20ß-HSD type 2 enzymes currently comprises eight members from teleost fish and mammals. Here, we report the identification of three 20ß-HSD type 2 genes in X. tropicalis and X. laevis and the functional characterization of the two homeologs from X. laevis. X. laevis Hsd20b2.L and Hsd20b2.S showed high sequence identity with known 20ß-HSD type 2 enzymes and mapped to the two subgenomes of the allotetraploid frog genome. Both homeologs are expressed during embryonic development and in adult tissues, with strongest signals in liver, kidney, intestine, and skin. After recombinant expression in human cell lines, both enzymes co-localized with the endoplasmic reticulum and catalyzed the conversion of cortisone to 20ß-dihydrocortisone. Both Hsd20b2.L and Hsd20b2.S catalyzed the 20ß-reduction of further C21 steroids (17α-hydroxyprogesterone, progesterone, 11-deoxycortisol, 11-deoxycorticosterone), while only Hsd20b2.S was able to convert corticosterone and cortisol to their 20ß-reduced metabolites. Estrone was only a poor and androstenedione no substrate for both enzymes. Our results demonstrate multispecificity of 20ß-HSD type 2 enzymes from X. laevis similar to other teleost 20ß-HSD type 2 enzymes. X. laevis 20ß-HSD type 2 enzymes are probably involved in steroid catabolism and in the generation of pheromones for intraspecies communication. A role in oocyte maturation is unlikely.

Engineering Xenopus embryos for phenotypic drug discovery screening.

Many rare human inherited diseases remain untreatable despite the fact that the disease causing genes are known and adequate mouse disease models have been developed. In vivo phenotypic drug screening relies on isolating drug candidates by their ability to produce a desired therapeutic phenotype in whole organisms. Embryos of zebrafish and Xenopus frogs are abundant, small and free-living. They can be easily arrayed in multi-well dishes and treated with small organic molecules. With the development of novel genome modification tools, such a zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas, it is now possible to efficiently engineer non-mammalian models of inherited human diseases. Here, we will review the rapid progress made in adapting these novel genome editing tools to Xenopus. The advantages of Xenopus embryos as in vivo models to study human inherited diseases will be presented and their utility for drug discovery screening will be discussed. Being a tetrapod, Xenopus complements zebrafish as an indispensable non-mammalian animal model for the study of human disease pathologies and the discovery of novel therapeutics for inherited diseases.

Stratified high-throughput screening sets enable flexible screening strategies from a single plated collection.

Customized compound picking and plating of very large corporate screening decks (many 100,000s) for high-throughput screening is generally restricted, both from a time and cost perspective. Here we present a stratified screening deck with accompanying plating design for use with very large corporate compound collections. The deck is plated as a whole, but copies for screening can be downsized flexibly and quickly on the fly, without the need for repicking of physical samples. We show that such downsized sets maximize returns and yield results superior to randomly picked subsets of the same size. For the proposed stratified plating design, structurally diverse subsets that cover the full collection in terms of compound diversity and favorable compound properties can be produced economically and quickly from the full set of master plates. The design was implemented globally at AstraZeneca in 2009 and has enabled substantial cost-saving in screening campaigns, as set size requirements can be met on a per-screen basis, using a single, preplated master deck.

High-temperature scanning tunneling microscopy study of the ordering transition of an amorphous carbon layer into graphene on ruthenium(0001).

The ordering transition of an amorphous carbon layer into graphene was investigated by high-temperature scanning tunneling microscopy. A disordered C layer was prepared on a Ru(0001) surface by chemical vapor deposition of ethylene molecules at ~660 K. The carbon layer grows in the form of dendritic islands that have almost the same density as graphene. Upon annealing of the fully covered surface, residual hydrogen desorbs and a coherent but still disordered carbon layer forms, with almost the same carbon coverage as in graphene. The ordering of this layer into graphene at 920 to 950 K was monitored as a function of time. A unique mechanism was observed that involves small topographic holes in the carbon layer. The holes are mobile, and on the trajectories of the holes the disordered carbon layer is transformed into graphene. The transport of C atoms across the holes or along the hole edges provides a low-energy pathway for the ordering transition. This mechanism is prohibited in a dense graphene layer, which offers an explanation for the difficulty of removing defects from graphene synthesized by chemical methods.

DrugPred: a structure-based approach to predict protein druggability developed using an extensive nonredundant data set.

Judging if a protein is able to bind orally available molecules with high affinity, i.e. if a protein is druggable, is an important step in target assessment. In order to derive a structure-based method to predict protein druggability, a comprehensive, nonredundant data set containing crystal structures of 71 druggable and 44 less druggable proteins was compiled by literature search and data mining. This data set was subsequently used to train a structure-based druggability predictor (DrugPred) using partial least-squares projection to latent structures discriminant analysis (PLS-DA). DrugPred performed well in discriminating druggable from less druggable binding sites for both internal and external predictions. The method is robust against conformational changes in the binding site and outperforms previously published methods. The superior performance of DrugPred is likely due to the size and composition of the training set which, in contrast to most previously developed methods, only contains cavities that have evolved to bind a natural ligand.

Inflammatory autoimmune neuropathy, presumably induced by bortezomib, in a patient suffering from multiple myeloma.

Bortezomib is a proteasome inhibitor demonstrating substantial activity in multiple myeloma. One of its key toxicities is peripheral neuropathy, which is reversible in most patients. The possibility that bortezomib might in rare cases induce severe neuropathies by auto-inflammatory mechanisms remains controversial. We report here the case of a 65-year-old female myeloma patient who was initially treated with bortezomib, doxorubicin, and dexamethasone (PAD). At the end of the second cycle of PAD, the patient presented with a rapid and severe onset of paresis of the left arm, accompanied by progressive sensory neuropathy and increasing neuropathic pain. After an extensive neurological work-up, including electrophysiological and laboratory evaluations as well as magnet resonance tomography imaging, we diagnosed an inflammatory autoimmune neuropathy, presumably induced by bortezomib, with accentuation of the left arm nerve plexus. We subsequently initiated regular treatment with polyvalent immunoglobulins, which gradually improved the neurological symptoms. In conclusion, the identification of an inflammatory autoimmune neuropathy, presumably associated with bortezomib, is a rare but important complication. An extensive neurological examination should be performed in patients who develop severe or unusual sensory or motor deficits under therapy with bortezomib, so as to differentiate autoimmune from toxic neuropathies, as therapeutic strategies differ for each.

Timing of the high-dose therapy in the area of new drugs.

The treatment approach in patients with multiple myeloma (MM) has been essentially changed with introduction of novel agents such as thalidomide, bortezomib and lenalidomide. In patients eligible for autologous stem cell transplant, combinations of novel agents with chemotherapy have been recognized as induction regimens. New induction regimens have significantly increased the rate of complete remission before and after autologous stem cell transplant with positive impact on the length of progression-free survival followed by the possibility for further improvement with the application of consolidation or use of thalidomide and lenalidomide as maintenance therapy. These results offer new perspectives in the treatment of MM with a reasonable hope of cure.

A combined ecological and epidemiologic investigation of metal exposures amongst Indigenous peoples near the Marlin Mine in Western Guatemala.

In August 2009 a combined epidemiological and ecological pilot study was conducted to investigate allegations of human rights abuses in the form of exposures to toxic metals experienced by mine workers and Indigenous Mam Mayan near the Marlin Mine in Guatemala. In the human study there were no differences in blood and urine metals when comparing five mine workers with eighteen non-mine workers, and there were no discernible relationships between metal exposures and self-reported health measures in any study group. On the other hand, individuals residing closest to the mine had significantly higher levels of certain metals (urinary mercury, copper, arsenic, and zinc) when compared to those living further away. The levels of blood aluminum, manganese, and cobalt were elevated in comparison to established normal ranges in many individuals; however, there was no apparent relationship to proximity to the mine or occupation, and thus are of unclear significance. In the ecological study, several metals (aluminum, manganese, and cobalt) were found significantly elevated in the river water and sediment sites directly below the mine when compared to sites elsewhere. When the human and ecological results are combined, they suggest that exposures to certain metals may be elevated in sites near the mine but it is not clear if the current magnitude of these elevations poses a significant threat to health. The authors conclude that more robust studies are needed while parallel efforts to minimize the ecological and human impacts of mining proceed. This is critical particularly as the impact of the exposures found could be greatly magnified by expected increases in mining activity over time, synergistic toxicity between metals, and susceptibility for the young and those with pre-existing disease.

The oral histone deacetylase inhibitor LBH589 is a potential and promising therapeutic agent in multiple myeloma after at least two lines of chemotherapy including bortezomib or lenalidomide.

BACKGROUND: Multiple myeloma as the second most common hematological malignancy is characterized by proliferation of monoclonal plasma cells. This entity still remains a non-curable disorder leading, amongst others, to complications as myeloma bone disease, bleeding events, kidney failure and neurological impairment. LBH589 is a histone deacetylase inhibitor with an epigenetic mechanism of action and the potential for treatment in myeloma. CASE REPORT: We report here about the successful treatment of a 44-year-old woman suffering from progressive myeloma with LBH589 after five different chemotherapies. During the 9 years after first diagnosis of myeloma in April 2000, our patient twice underwent an autologous stem cell transplantation and was also treated with the new substances bortezomib, thalidomide and lenalidomide. RESULTS: A rapid decline of myeloma activity parameters could be reached, with an approximately exponential decrease of kappa light chains in the 24-h urine. As a consequence, a near-complete remission was determined after about 6 months of LBH589 treatment. Additionally, the adverse event profile was acceptable, and the patient's quality of life showed a considerable advancement of well-being. CONCLUSIONS: LBH589 may be very effective in multiple myeloma after a multitude of preceding treatments that could not induce a long-term anti-myeloma effect. Future trials with LBH589 should search for the specific characteristics of responding patients.

Effective prophylaxis of thromboembolic complications with low molecular weight heparin in relapsed multiple myeloma patients treated with lenalidomide and dexamethasone.

The immunomodulatory drugs thalidomide and lenalidomide have enhanced activity in patients with multiple myeloma (MM). Their efficacy is increased with the addition of dexamethasone, but significant rates of venous thromboembolism (VTE) are a severe side effect. Based on this evidence, it is recommended that VTE prophylaxis be prescribed in these patients. However, the optimal prophylaxis remains controversial. We analyzed 45 patients with relapsed MM who were treated with lenalidomide and dexamethasone at our center. The 45 patients received a total number of 192 cycles, respectively a median of three cycles; the median dosage of dexamethasone was 240 mg per cycle. All patients received prophylactic anticoagulation with low molecular weight heparin (LMWH). Moreover, 86.6% of patients had at least one additional VTE risk factor beside the myeloma-related risk. One out of 45 patients developed a deep vein thrombosis and pulmonary embolism. None of the other 44 patients had clinical signs of thrombosis or embolism and none of all patients experienced complications or side effects due to anticoagulation. Our results indicate that prophylactic anticoagulation with LMWH is safe and effective. Therefore, we propose LMWH should be used in patients being treated with lenalidomide and dexamethasone at least for the first 3 months of treatment until randomized trials have proven the equality of other pharmacological prophylaxis.

An alternative method for the evaluation of docking performance: RSR vs RMSD.

A new assessment criterion for docking poses is proposed in which experimental electron density is taken into account when evaluating the ability of docking programs to reproduce experimentally observed binding modes. Three docking programs (Gold, Glide, and Fred) were used to generate poses for a set of 88 protein-ligand complexes for which the crystal structure is known. The new criterion is based on the real space R-factor (RSR), which measures how well a group of atoms-in our case the ligand-fits the experimental electron density by comparing that density to the expected density, calculated from the model (i.e., the predicted ligand pose). The RSR-based measure is compared to the traditional criterion, the root-mean-square distance (RMSD) between the docking pose and the binding configuration in the crystallographic model. The results highlight several shortcomings of the RMSD criterion that do not affect the RSR-based measure. Examples illustrate that the RSR-derived approach allows a more meaningful a posteriori assessment of docking methods and results. Practical implications for docking evaluations and for methodological development work in this field are discussed.

Do structurally similar ligands bind in a similar fashion?

The scope of the current work is to investigate whether structurally similar ligands bind in a similar fashion by exhaustively analyzing experimental data from the protein database (PDB). The complete PDB was searched for pairs of structurally similar ligands binding to the same biological target. The binding sites of the pairs of proteins complexing structurally similar ligands were found to differ in 83% of the cases. The most recurrent structural change among the pairs involves different water molecule architecture. Side-chain movements are observed in half of the pairs, whereas backbone movements rarely occurred. However, two structurally similar ligands generally confirm a high degree of structural conservation. That is, a majority of the ligand pairs occupy the same region in the binding sites, providing support for the use of shape matching in the drug design process. We allow ourselves to draw general conclusions because our data set consists of ligands with drug-like physicochemical properties complexed to a broad spectrum of different protein classes.

From the similarity analysis of protein cavities to the functional classification of protein families using cavbase.

In this contribution, the classification of protein binding sites using the physicochemical properties exposed to their pockets is presented. We recently introduced Cavbase, a method for describing and comparing protein binding pockets on the basis of the geometrical and physicochemical properties of their active sites. Here, we present algorithmic and methodological enhancements in the Cavbase property description and in the cavity comparison step. We give examples of the Cavbase similarity analysis detecting pronounced similarities in the binding sites of proteins unrelated in sequence. A similarity search using SARS M(pro) protease subpockets as queries retrieved ligands and ligand fragments accommodated in a physicochemical environment similar to that of the query. This allowed the characterization of the protease recognition pockets and the identification of molecular building blocks that can be incorporated into novel antiviral compounds. A cluster analysis procedure for the functional classification of binding pockets was implemented and calibrated using a diverse set of enzyme binding sites. Two relevant protein families, the alpha-carbonic anhydrases and the protein kinases, are used to demonstrate the scope of our cluster approach. We propose a relevant classification of both protein families, on the basis of the binding motifs in their active sites. The classification provides a new perspective on functional properties across a protein family and is able to highlight features important for potency and selectivity. Furthermore, this information can be used to identify possible cross-reactivities among proteins due to similarities in their binding sites.

Inhibition of angiogenesis by non-toxic doses of temozolomide.

It is well established that certain chemotherapeutic agents have potent antiangiogenic properties which may be part of their antitumor activity. Temozolomide (TMZ) is a lipophilic methylating agent used in the therapy of malignant melanoma and other tumors. We sought to determine whether TMZ is capable of inhibiting angiogenesis or influencing endothelial function. We used the in vivo chorioallantoic membrane (CAM) assay, and HUVEC-based in vitro Matrigel, adhesion and proliferation assays to determine the antiangiogenic effects of different doses of TMZ. In the CAM assay, angiogenesis was significantly inhibited by 5 microM TMZ, a concentration also found to be effective in interfering with in vitro angiogenesis as measured by the Matrigel assay. For the inhibition of basic fibroblast growth factor (bFGF)-, vascular endothelial growth factor (VEGF)- or beta-phorbol 12-myristate-13-acetate (PMA)-induced endothelial cell proliferation or endothelial cell adhesion to fibronectin, TMZ concentrations of at least 25 microM were necessary, indicating that bFGF-, VEGF- or protein kinase C-mediated pathways may not primarily be involved in the observed antiangiogenic effect. Thus, we could demonstrate that TMZ inhibits angiogenesis at low, non-toxic doses that correspond to the plasma concentrations achieved by an oral application of 20 mg/m2 every 8 h. This 'metronomic' scheduling has already been used in phase I studies and has produced antitumor effects. Therefore, the antitumor activity of TMZ may, at least in part, be due to its antiangiogenic properties. The precise mechanism of its antiangiogenic action remains to be elucidated.

A new method to detect related function among proteins independent of sequence and fold homology.

A new method has been developed to detect functional relationships among proteins independent of a given sequence or fold homology. It is based on the idea that protein function is intimately related to the recognition and subsequent response to the binding of a substrate or an endogenous ligand in a well-characterized binding pocket. Thus, recognition of similar ligands, supposedly linked to similar function, requires conserved recognition features exposed in terms of common physicochemical interaction properties via the functional groups of the residues flanking a particular binding cavity. Following a technique commonly used in the comparison of small molecule ligands, generic pseudocenters coding for possible interaction properties were assigned for a large sample set of cavities extracted from the entire PDB and stored in the database Cavbase. Using a particular query cavity a series of related cavities of decreasing similarity is detected based on a clique detection algorithm. The detected similarity is ranked according to property-based surface patches shared in common by the different clique solutions. The approach either retrieves protein cavities accommodating the same (e.g. co-factors) or closely related ligands or it extracts proteins exhibiting similar function in terms of a related catalytic mechanism. Finally the new method has strong potential to suggest alternative molecular skeletons in de novo design. The retrieval of molecular building blocks accommodated in a particular sub-pocket that shares similarity with the pocket in a protein studied by drug design can inspire the discovery of novel ligands.