Interconnectivity of Disparate Nonclinical Data Silos for Drug Discovery and Development.

Pharmaceutical research and development generates enormous amounts of nonclinical and clinical data related to safety and efficacy, and the ability to manage and utilize these data is critical for discovering and developing new drugs. Information systems exist that store and analyze relationships among seemingly disparate data sets (ie, data silos); however, to fully utilize the potential of these informatics systems, it is necessary to define basic parameters about the data and to develop concepts regarding "interconnectivity," or relationships among disparate data sets. To explore these issues, the Nonclinical Data Interconnectivity Sub-Group was chartered: a component of the Non-Clinical Road-Map and Impacts on Implementation Working Group associated with the US FDA-PhUSE (Pharmaceutical Users Software Exchange) Computational Sciences initiative. As a starting point, the group defined the meaning of data interconnectivity. Nonclinical data types were then identified and challenges and opportunities for interconnectivity explored. Specific-use cases were identified to provide examples of the value for interconnecting data across disciplines or silos.

FDA Engages Collaborators to Address Nonclinical Data Challenges.

FDA and PhUSE cohosted a Computational Science Symposium (CSS) in 2012 that brought stakeholders from the pharmaceutical industry and government to work collaboratively to solve common needs and challenges. A nonclinical informatics workgroup was formed, dedicated to improving nonclinical assessments and regulatory science by identifying, collecting, and prioritizing key needs and challenges in the field and then establishing an innovative framework for addressing them in a collaborative manner. This paper discusses the process and outcomes of the nonclinical informatics workgroup during the CSS and describes an approach which crossed organizational barriers to optimize computational science for nonclinical assessment.

Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase.

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

Crystallization of integral membrane proteins.

Over the last 20 years, the use of X-ray crystallography has become a viable technique for the structure determination of integral membrane proteins. However, standard crystallizaton protocols must be modified to account for difficulties involved in handling membrane proteins, which arise primarily from having detergent present. This unit provides protocols that can be used to crystallize a purified membrane protein, including detergent exchange, sample concentration, initial screening using a crystallization robot, and finally, optimization of crystallization conditions to obtain diffraction-quality crystals. These protocols were established for outer membrane proteins, but can be used for inner membrane proteins as well. Advice on alternative protocols, detergent selection, and optimization of crystallization conditions is provided.