Structural/functional studies of Trio provide insights into its configuration and show that conserved linker elements enhance its activity for Rac1.

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio.

Generation of Highly Selective, Potent, and Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors.

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate the desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating diseases such as heart failure and cancer. Previously, our work showed that Cys474, a GRK5 subfamily-specific residue located on a flexible loop adjacent to the active site, can be used as a covalent handle to achieve selective inhibition of GRK5 over GRK2 subfamily members. However, the potency of the most selective inhibitors remained modest.

Cryo-electron microscopy structure and analysis of the P-Rex1-Gβγ signaling scaffold.

PIP-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.

A Chemogenomic Screening Platform Used to Identify Chemotypes Perturbing HSP90 Pathways.

Compounds that modulate the heat shock protein (HSP) network have potential in a broad range of research applications and diseases. A yeast-based liquid culture assay that measured time-dependent turbidity enabled the high-throughput screening of different Saccharomyces cerevisae strains to identify HSP modulators with unique molecular mechanisms. A focused set of four strains, with differing sensitivities to Hsp90 inhibitors, was used to screen a compound library of 3680 compounds.

Discovery of antagonists of tick dopamine receptors via chemical library screening and comparative pharmacological analyses.

Ticks transmit a wide variety of disease causing pathogens to humans and animals. Considering the global health impact of tick-borne diseases, there is a pressing need to develop new methods for vector control. We are exploring arthropod dopamine receptors as novel targets for insecticide/acaricide development because of their integral roles in neurobiology.

Differential mitochondrial toxicity screening and multi-parametric data analysis.

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation.

High-throughput secondary screening at the single-cell level.

We have developed an automated system for drug screening using a single-cell-multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness.

A "genome-to-lead" approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1)-like dopamine receptors.

Background: Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.

Methodology/principal Findings: We describe a "genome-to-lead" approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome.

Revisiting absorbance at 230nm as a protein unfolding probe.

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230nm (A(230)) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed.

Robotic hierarchical mixing for the production of combinatorial libraries of proteins and small molecules.

We present a method to automatically plan a robotic process to mix individual combinations of reactants in individual reaction vessels (vials or wells in a multiwell plate), mixing any number of reactants in any desired stoichiometry, and ordering the mixing steps according to an arbitrarily complex treelike assembly protocol. This process enables the combinatorial generation of complete or partial product libraries in individual reaction vessels from intermediates formed in the presence of different sets of reactants. It can produce either libraries of chimeric genes constructed by ligation of fragments from different parent genes or libraries of chemical compounds constructed by convergent synthesis.

Inhibitors of anthrax lethal factor.

An inhibitor of anthrax lethal toxin mediated cell death (1) was identified by a medium throughput cell-based screen. This compound was determined to specifically inhibit anthrax lethal factor (LF), and subsequent SAR studies produced an even more potent inhibitor (4). Mechanistic studies identified these agents as uncompetitive inhibitors of LF with Ki values of 3.

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins.

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader.