Thermodynamic, Kinetic and Crystallographic Investigations of Benzenesulfonamides as Ligands of Human Carbonic Anhydrase II

The data presented here show that different geometries of para-alkyl substituents of a benzenesulfonamide scaffold can have a significant influence on the thermodynamic and kinetic binding parameters and suggest, that the fine-tuning of the ligand geometry to match the geometry of the active site leads to a reduced dissociation rate of the protein-ligand complex and thus to a prolonged binding in the active site. In addition, it was found that the kinetic data extracted by isothermal titration calorimetry correlate with data from experiments with surface plasmon resonance for a subset of compounds, despite a difference of one order of magnitude that could not be explained so far. Furthermore, the investigation of fluorinated benzenesulfonamide ligands revealed complex structural-thermodynamic and structural-kinetic relationships, which could not be fully elucidated herein, but suggest that fluorination of a meta-position of the aromatic part of the benzenesulfonamide backbone favors high association as well as small dissociation rates. In addition, it has been shown that a higher degree of fluorination does not necessarily have an advantage for the thermodynamic or kinetic binding profile or affnity itself. Furthermore, structurally similar ligands with only one para-substituent but different acidities indicate that the association rate of complex formation benefits from increased acidity. A new measurement protocol for microcalorimetric measurements was analyzed for its precision and compared with other possible measurement protocols and showed that it is best suited for the reliable simultaneous determination of thermodynamic and kinetic data by microcalorimetry. The microcalorimetric investigation of an already known and supposedly very potent Carbonic Anhydrase inhibitor showed an unexpected two-step binding behavior, which, however, excludes a 2 : 1 binding of inhibitor and protein. Time-dependent experiments suggest that the binding process between protein and ligand leads to a thermodynamically and kinetically preferred complex. The investigation of a chiral heterocyclic Carbonic Anhydrase inhibitor could not conclusively clarify, whether the crystallographically apparently non-binding enantiomer also shows inhibitory activity. However, microcalorimetric investigations allow the conclusion that racemization occurs under experimental conditions, which makes the clarification of the original question considerably more difficult. Soaking of Carbonic Anhydrase crystals in solutions of different concentrations of a ligand showed, that the occupancy of a second binding site was too weak to allow modeling of the ligand, which made it impossible to investigate the concentration dependence of the occupancy in this binding site. Concentration differences, however, resulted in different occupancies of the active site. A crystallographic model based on neutron diffraction data of human Carbonic Anhydrase II in complex with saccharin was not obtained. However, an attempt was made to assess the protonation state of saccharin in the active site of the enzyme by experimental phasing using high-resolution X-ray diffraction data. The continuation of a fragment screening showed the binding of further small molecules in Carbonic Anhydrase crystals. An analysis with the Pan-Dataset Density Analysis program suggests, that electron density maps are strongly dependent on the time of soaking of the crystals

The rapid synthesis of oxazolines and their heterogeneous oxidation to oxazoles under flow conditions.

A rapid flow synthesis of oxazolines and their oxidation to the corresponding oxazoles is reported. The oxazolines are prepared at room temperature in a stereospecific manner, with inversion of stereochemistry, from β-hydroxy amides using Deoxo-Fluor®. The corresponding oxazoles can then be obtained via a packed reactor containing commercial manganese dioxide.We are grateful to the Swiss National Science Foundation (DNT), the Ralph Raphael fellowship (RJI), the German Academic Exchange Service DAAD (SF), the Royal Society Newton International Fellowship (ZEW), Pfizer Worldwide Research and Development (CB) and the EPSRC (SVL, SF and ZEW, grant nº EP/K0099494/1) for financial support. The Labtrix Start was kindly loaned by Chemtrix BV and the liquid-liquid phase separator by Zaiput Flow Technologies (we are grateful to Dr Andrea Adamo for technical support with this device).This is the final version. It was first published by the Royal Society of Chemistry at http://dx.doi.org/10.1039/C4OB02105

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi

ABSTRACT: BACKGROUND: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. RESULTS: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. CONCLUSIONS: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A Fine-grained Analysis of the Jumping to Conclusions Bias in Schizophrenia: Data-Gathering, Response Confidence, and Information Integration

Impaired decision behavior of schizophrenia patients has been repeatedly observed. We investigated the aspects of the jumping to conclusions bias (JTC): biases in information-gathering, information weighting and integration, and overconfidence, using the process tracing paradigm Mouselab, which allows for an in-depth exploration of various decision-making processes in a structured information environment. Although showing less focused and systematic information search, patients practically considered all pieces of information and showed no JTC in the sense of collecting less pieces of evidence. Choices of patients and controls both approximated a rational solution quite well, but patients showed more extreme and, in view of the ambiguous nature of the task, unjustified confidence. Both groups mainly used weighted additive decision strategies for information integration and only a small proportion relied on simple heuristics. Under high stress, induced by high affective valence plus time pressure, however, schizophrenia patients showed a less appropriate weighting of information and switched to equal weighting strategies. Patients that scored higher on the schizophrenia scale PANSS showed less information search and tended to rely more often on simple heuristics.Decision Making, Schizophrenia, Jump to conclusion, Heuristics

Thermodynamic, Kinetic and Crystallographic Investigations of Benzenesulfonamides as Ligands of Human Carbonic Anhydrase II

The data presented here show that different geometries of para-alkyl substituents of a benzenesulfonamide scaffold can have a significant influence on the thermodynamic and kinetic binding parameters and suggest, that the fine-tuning of the ligand geometry to match the geometry of the active site leads to a reduced dissociation rate of the protein-ligand complex and thus to a prolonged binding in the active site. In addition, it was found that the kinetic data extracted by isothermal titration calorimetry correlate with data from experiments with surface plasmon resonance for a subset of compounds, despite a difference of one order of magnitude that could not be explained so far. Furthermore, the investigation of fluorinated benzenesulfonamide ligands revealed complex structural-thermodynamic and structural-kinetic relationships, which could not be fully elucidated herein, but suggest that fluorination of a meta-position of the aromatic part of the benzenesulfonamide backbone favors high association as well as small dissociation rates. In addition, it has been shown that a higher degree of fluorination does not necessarily have an advantage for the thermodynamic or kinetic binding profile or affnity itself. Furthermore, structurally similar ligands with only one para-substituent but different acidities indicate that the association rate of complex formation benefits from increased acidity. A new measurement protocol for microcalorimetric measurements was analyzed for its precision and compared with other possible measurement protocols and showed that it is best suited for the reliable simultaneous determination of thermodynamic and kinetic data by microcalorimetry. The microcalorimetric investigation of an already known and supposedly very potent Carbonic Anhydrase inhibitor showed an unexpected two-step binding behavior, which, however, excludes a 2 : 1 binding of inhibitor and protein. Time-dependent experiments suggest that the binding process between protein and ligand leads to a thermodynamically and kinetically preferred complex. The investigation of a chiral heterocyclic Carbonic Anhydrase inhibitor could not conclusively clarify, whether the crystallographically apparently non-binding enantiomer also shows inhibitory activity. However, microcalorimetric investigations allow the conclusion that racemization occurs under experimental conditions, which makes the clarification of the original question considerably more difficult. Soaking of Carbonic Anhydrase crystals in solutions of different concentrations of a ligand showed, that the occupancy of a second binding site was too weak to allow modeling of the ligand, which made it impossible to investigate the concentration dependence of the occupancy in this binding site. Concentration differences, however, resulted in different occupancies of the active site. A crystallographic model based on neutron diffraction data of human Carbonic Anhydrase II in complex with saccharin was not obtained. However, an attempt was made to assess the protonation state of saccharin in the active site of the enzyme by experimental phasing using high-resolution X-ray diffraction data. The continuation of a fragment screening showed the binding of further small molecules in Carbonic Anhydrase crystals. An analysis with the Pan-Dataset Density Analysis program suggests, that electron density maps are strongly dependent on the time of soaking of the crystals

Performance of the eazyplex® BloodScreen GN as a simple and rapid molecular test for identification of Gram-negative bacteria from positive blood cultures

The LAMP-based eazyplex® BloodScreen GN was evaluated for the detection of frequent Gram-negatives directly from positive blood culture (BC) bottles. A total of 449 BCs were analyzed. Sensitivities and specificities were 100% and 100% for Escherichia coli, 95.7% and 100% for Klebsiella pneumoniae, 100% and 100% for bl

Shorter planning depth and higher response noise during sequential decision-making in old age

Abstract Forward planning is crucial to maximize outcome in complex sequential decision-making scenarios. In this cross-sectional study, we were particularly interested in age-related differences of forward planning. We presumed that especially older individuals would show a shorter planning depth to keep the costs of model-based decision-making within limits. To test this hypothesis, we developed a sequential decision-making task to assess forward planning in younger (age  60 years; n = 27) adults. By using reinforcement learning modelling, we inferred planning depths from participants' choices. Our results showed significantly shorter planning depths and higher response noise for older adults. Age differences in planning depth were only partially explained by well-known cognitive covariates such as working memory and processing speed. Consistent with previous findings, this indicates age-related shifts away from model-based behaviour in older adults. In addition to a shorter planning depth, our findings suggest that older adults also apply a variety of heuristical low-cost strategies

High-Throughput Crystallography : Reliable and Efficient Identification of Fragment Hits

Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods