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Martinsried, Germany

Koster H.,University of Marburg | Craan T.,University of Marburg | Brass S.,University of Marburg | Herhaus C.,Merck KGaA | And 4 more authors.
Journal of Medicinal Chemistry

Druglike molecules are defined by Lipinski's rule of 5, to characterize fragment thresholds, they have been reduced from 5 to 3 (Astex's rule of 3). They are applied to assemble fragment libraries, and providers use them to select fragments for commercial offer. We question whether these rules are too stringent to compose fragment libraries with candidates exhibiting sufficient room for chemical subsequent growing and merging modifications as appropriate functional groups for chemical transformations are required. Usually these groups exhibit properties as hydrogen bond donors/acceptors and provide entry points for optimization chemistry. We therefore designed a fragment library (364 entries) without strictly applying the rule of 3. For initial screening for endothiapepsin binding, we performed a biochemical cleavage assay of a fluorogenic substrate at 1 mM. "Hits were defined to inhibit the enzyme by at least 40%. Fifty-five hits were suggested and subsequently soaked into endothiapepsin crystals. Eleven crystal structures could be determined covering fragments with diverse binding modes: (i) direct binding to the catalytic dyad aspartates, (ii) water-mediated binding to the aspartates, (iii) no direct interaction with the dyad. They occupy different specificity pockets. Only 4 of the 11 fragments are consistent with the rule of 3. Restriction to this rule would have limited the fragment hits to a strongly reduced variety of chemotypes. © 2011 American Chemical Society. Source

Rajagopala S.V.,J. Craig Venter Institute | Uetz P.,Proteros biostructures
Methods in Molecular Biology

The yeast two-hybrid (Y2H) system is a powerful tool to identify binary protein-protein interactions. Here, we describe array-based two-hybrid methods that use defined libraries of open reading frames (ORFs) and pooled prey library screenings that use random genomic or cDNA libraries. The array-based Y2H system is well-suited for interactome studies of existing ORFeomes or subsets thereof, preferentially in a recombination-based cloning system. Array-based Y2H screens efficiently reduce false positives by using built-in controls, retesting, and evaluation of background activation. Hands-on time and the amount of used resources grow exponentially with the number of tested proteins; this is a disadvantage for large genome sizes. For large genomes, random library screen may be more efficient in terms of time and resources, but not as comprehensive as array screens, and it requires significant sequencing capacity. Furthermore, multiple variants of the Y2H vector systems detect markedly different subsets of interactions in the same interactome. Hence, only multiple variations of the Y2H systems ensure comprehensive coverage of an interactome. © 2011 Springer Science+Business Media, LLC. Source

Schoop A.,Proteros biostructures | Dey F.,Roche Holding AG
Drug Discovery Today: Technologies

In the lead discovery process residence time has become an important parameter for the identification and characterization of the most efficacious compounds in vivo. To enable the success of compound optimization by medicinal chemistry toward a desired residence time the understanding of structure-kinetic relationship (SKR) is essential. This article reviews various approaches to monitor SKR and suggests using the on-rate as the key monitoring parameter. The literature is reviewed and examples of compound series with low variability as well as with significant changes in on-rates are highlighted. Furthermore, findings of kinetic on-rate changes are presented and potential underlying rationales are discussed. © 2015 Elsevier Ltd. All rights reserved. Source

Rajagopala S.V.,J. Craig Venter Institute | Casjens S.,University of Utah | Uetz P.,Virginia Commonwealth University | Uetz P.,Proteros biostructures
BMC Microbiology

Background: Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results: In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions: Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. © 2011Rajagopala et al; licensee BioMed Central Ltd. Source

Seemann K.M.,Julich Research Center | Kiefersauer R.,Proteros biostructures | Kuhn B.,Max Planck Institute of Biochemistry | Kuhn B.,Okinawa Institute of Science and Technology
Journal of Physical Chemistry B

The pH is one of the key parameters governing protein conformation and activity. In protein crystals, however, the pH is so far not accessible by experiment. Here, we report on the optical detection of the pH in a lysozyme crystal employing the pH-sensitive fluorescent dyes SNARF-1 and SNARF-4F. The molecular probes were loaded into the crystal by diffusion. Two-dimensional fluorescence spectra of the labeled protein crystal were recorded, and the average pH of the crystal at different bath pH's was determined by calibrating fluorescence peak ratios. In addition, we used two-photon microscopy to spatially resolve the pH inside a lysozyme crystal three-dimensionally and to follow pH changes in response to a pH change of the bath over time. At equilibrium at bath pH between 5.5 and 8.0, we found a pH in the water-filled crystal channels that was ΔpH = -0.3 to -1.0 lower than that of the bath. This corresponds to a 2- to 10-fold higher proton concentration in the crystal channels than in the bath. The lower pH at equilibrium in the crystal channels can be explained by slower proton diff usion in the channels than in the bath and a resulting proton accumulation in the crystal for conservation of mass and so an equilibrium of proton flux. © 2012 American Chemical Society. Source

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