Assay Development

RNA editing by uridine insertion/deletion is essential for mitochondrial
gene expression in all trypanosomatids pathogens. Editing is catalyzed by multiprotein complexes, the editosomes. A key catalytic component of editosomes is RNA editing ligase 1 (REL1). REL1 has been validated as a drug target in T. brucei in vitro and in vivo. (Schnaufer et al., 2001). See also abstract #107. The 1.2 Ǻ crystal structure of REL1 revealed a deep ATP binding pocket with numerous opportunities for specific binding of small molecule inhibitors (Deng, Schnaufer et al., 2004). There are no close REL1 homologs in the mammalian host and thus this
enzyme represents a target of potentially high specificity. Computational screening efforts based on molecular dynamics simulations resulted in the identification of several REL1 inhibitors with
IC50 values in the single-digit μM range (Amaro, Schnaufer et al., 2008;
Durrant, Hall et al., 2010). These inhibitors are not drug-like, although
they share similarities with suramin.

Here we describe development of a high throughput screening (HTS)
compatible, fluorescence-based REL1 assay, identification with this
assay of novel small compound REL1 inhibitors in large chemical
libraries, and expansion of selected hits to increase potency.

High-throughput screening collaborations:

- Scripps Institute San Diego (LOPAC Collection), 1,280 cpds
- Dundee Drug Discovery Unit (DDU), ~23,000 cpds
- GlaxoSmithKline Tres Cantos Madrid (GSK), ~160,000 cpds
Hit confirmation on going
- The Scripps Research Institute Florida (NIH/TSRI), ~90,000 cpds
Medium-throughput

Curcumin is a polyphenolic diketone derived from turmeric and has been exploited for many applications. Earlier studies have demonstrated that curcumin can be used as a less toxic alternative to Coomassie Brilliant Blue to stain protein and to quantify protein by fluorometric assays, but the potential of curcumin to quantify proteins by absorbance assays has not been adequately explored. In this study, we examined curcumin under several conditions to determine whether it may be used to quantify proteins by either fluorometric or absorbance microassays. Fluorometric assays worked best in an acidic DMSO solution while absorbance assays worked best in an EtOH solution buffered by Tris.

We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR). We also create a single-site cysteine mutant of DHFR engineered within the Met20 catalytic loop region and study the protein's structural motion at this site. Using published x-ray crystal structures, we show that the changes in the SHG signals upon ligand binding are the result of structural motions that occur at the labeled sites between the apo and ligand-bound forms of the proteins, which are easily distinguished from each other. In addition, we demonstrate that different magnitudes of the SHG signal changes are due to different and specific ligand-induced conformational changes. Taken together, these data illustrate the potential of the SHG approach for detecting and measuring protein conformational changes for a wide range of biological applications.

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated–low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.