Identification and X-ray Co-crystal Structure of a Small-Molecule Activator of LFA-1-ICAM-1 Binding

The integrin Leucocyte function associated antigen 1 (LFA-1) is a heterodimeric immune receptor ubiquitously expressed on all leucocytes. Its interaction with Intercellular adhesion molecule 1 (ICAM-1) provides a critical recognition event between T-cells and antigen presenting cells in the immune systems efforts to pull off an early stage cell mediated immune response.[1–3] The LFA-1/ICAM-1 axis has thus been explored as a target interaction for drug discovery.[4–7] Furthermore, the structural changes of LFA-1 upon activation and interaction with ICAM-1 also make the LFA-1/ICAM-1 interaction an interesting example of protein-protein interaction (PPI) inhibition by small molecule inhibitors.[8,9] While protein-protein interaction inhibition by small molecules is considered to be the ultimate art in drug design, even fewer examples of true agonists of PPIs have been reported.[10–12] As for LFA-1, such activators would have interesting applications in rare hereditary genetic disorders called Leucocyte adhesion deficiency (LAD) or as potential enhancers of tumour immunotherapy.[13,14] Although, one such activator has been described recently, closer biological investigation has shown that it ultimately worked as an inhibitor on a cellular level by locking the LFA-1/ICAM-1 interaction when reversibility was needed for detachment of immune cells from endothelial surfaces and tissue infiltration.[15] Herein we describe the identification and structural biology of IBE-667, an ICAM-1 binding enhancer for LFA-1 from on-bead screening of tagged one-bead one-compound combinatorial libraries by confocal nanoscanning and bead picking (CONA).[16] Cellular assays demonstrate the activity of IBE-667 in promoting the binding of LFA-1 on activated immune cells to ICAM-1. X-ray structure based analysis did not only allow us to explain the molecular features of IBE-667 binding to LFA-1 but also offers an explanation for its mode of action

Single Bead Labeling Method for Combining Confocal Fluorescence On-Bead Screening and Solution Validation of Tagged One-Bead One-Compound Libraries

SummaryScreening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution

Identification of a Small Molecule Inhibitor of Importin β Mediated Nuclear Import by Confocal On-Bead Screening of Tagged One-Bead One-Compound Libraries

In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably,leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin beta with high nanomolar affinity and specifically inhibits importin alpha/beta mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding-experiments suggest that karyostatin 1A acts by disrupting the interaction between importin p and the OPase Ran. As a selective inhibitor of the importin alpha/beta import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.</p

Single-bead, single-molecule, single-cell fluorescence: technologies for drug screening and target validation.

According to many current reports, the pharmaceutical business will hit a wall over the next few years. The generic competition is expected to wipe out a double-digit billion-dollar amount from top companies' annual sales between 2007 and 2012 (Wall Street Journal, online, December 6, 2007). The industry's science engine has stalled, new blockbusters are lacking, and patent expirations are a big problem. Also, the U.S. Food and Drug Administration is pulling back on approvals, requesting larger safety studies. Among the different approaches taken throughout the industry to improve productivity and to reduce the attrition rate of compounds in the drug discovery process, an extended application of quantitative biology and biophysical methods is ranked very high. Fluorescence spectroscopy and imaging represented the main detection technologies for assays and screening methods in recent years. Today, label-free detection methods, such as isothermal titration calorimetry, differential scanning calorimetry, tandem mass spectrometry (MS(n)), light scattering, or interferometry, start to provide viable alternative readouts for physicochemical characterization of leads and hit list triaging. However, the multidimensional nature of fluorescence along with its high sensitivity and single-molecule resolution remains an unparalleled source of molecular parameters to extract all different kinds of information on molecules and ligand-protein complexes in solution. Although fluorescence-based methods are currently applied throughout the different stages of the industrial drug discovery process, they are usually applied in an unconnected way. We have developed a fully integrated hit and lead discovery process combining bead-based synthesis and screening methods with confocal fluorescence microspectroscopy. The primary on-bead screening process provides fluorescent ligands that after a multistep characterization process ultimately leads to fully mechanistically characterized cellularly validated binders and inhibitors of target protein interactions. The unlabeled small-molecular inhibitors represent chemical starting points in drug discovery and target validation

Determining changeover cost and holding cost of finished goods for the EPQ inventory model in two brick production plants in the USA.

The following report deals with the total changeover and holding costs of General Shale in two of its production plants, which are in Rome in Georgia and in Spring City in Tennessee. The company has cost statements, but these should be taken under the hat to find out if these values and the 10% holding cost rule of the company correspond to reality. First of all, the report deals with the company per se in order to get an overview of the facts. Then the problem was explained in detail and a research question was formulated. The approach to the problem is then explained as well as theoretical terms and models are explained to provide an understanding and the necessary basic knowledge for further chapters. In order to be able to deep dive into the matter, many data records had to be collected first. Therefore, all the cost factors that have an impact in the changeover process and the inventory holding were first brought together, what allowed to gain an overview of all, even the smallest, cost components. As a result, the current situation could be determined, recorded in numbers and, consequently, analyzed in detail. In several steps, scenarios were developed comparing the costs assumed so far with the cost estimated by this project. It could be concluded that the costs General Shale assumed so far for Changeover Cost calculations are underestimated and for the Holding Cost are overestimated. In addition, a perfect production scenario was simulated, which allowed to identify that there is still room for improvement in the production scheduling. Finally, recommendations were made for future production planning, inventory management, as well as how to further optimize production quantity by allowing to save costs at the same time

The chemical hunt for the identification of drugable targets.

Chemical biology has emerged as a new scientific discipline to change the way scientists approach and study the interface between chemistry, biology, and physics. By integrating the knowledge base of the human genome with the power of diverse and flexible chemical technology platforms, the ultimate goal is to define the 'rules of engagement' for small molecules and their use in basic biology and in drug discovery. Herein, we highlight the current counterpoles of the chemical biology philosophy in the framework between conformational diversity and informational complexity. Expanding the growing molecular recognition information matrix into classification of diseases and immediate mechanistic in-vivo proof of concept models represent the next development phase in a field that, unlike any other due to its multidisciplinary nature, unifies basic scientists and drug discoverers

Terminal adenosyl transferase activity of posttranscriptional regulator HuR revealed by confocal on-bead screening

Posttranscriptional regulation and RNA metabolism have become central topics in the understanding of mammalian gene expression and cell signalling, with the 3' untranslated region emerging as the coordinating unit. The 3' untranslated region trans-acting factor Hu protein R (HuR) forms a central posttranscriptional pathway node bridging between AU-rich element-mediated processes and microRNA regulation. While (m)RNA control by HuR has been extensively characterized, the molecular mode of action still remains elusive. Here we describe the identification of the first RRM3 (RNA recognition motif 3) targeted low molecular weight HuR inhibitors from a one-bead-one-compound library screen using confocal nanoscanning. A further compound characterization revealed the presence of an ATP-binding pocket within HuR RRM3, associated with enzymatic activity. Centered around a metal-ion-coordinating DxD motif, the catalytic site mediates 3'-terminal adenosyl modification of non-polyadenylated RNA substrates by HuR. These findings suggest that HuR actively contributes to RNA modification and maturation and thereby shed an entirely new light on the role of HuR in RNA metabolism

In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe.

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load