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. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.

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. Herein, we describe a successful campaign to adapt an indolinone scaffold with covalent warheads, resulting in a series of 2-haloacetyl-containing compounds that react quickly and exhibit three orders of magnitude selectivity for GRK5 over GRK2 and low nanomolar potency. They however retain a similar selectivity profile across the kinome as the core scaffold, which was based on Sunitinib.

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

PIP3-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 3.2 Å cryo-EM structure of the P-Rex1-Gßγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gßγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gßγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gßγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

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. Computed turbidity curve functions were used to classify strain responses and sensitivity to chemical effects across the compound library. Filtering based on single-strain selectivity identified nine compounds as potential heat shock modulators, including the known Hsp90 inhibitor macbecin. Haploid yeast deletion strains (360), mined from previous Hsp90 inhibitor yeast screens and heat shock protein interaction data, were screened for differential sensitivities to known N-terminal ATP site-directed Hsp90 inhibitors to reveal functional distinctions. Strains demonstrating differential sensitivity (13) to Hsp90 inhibitors were used to prioritize primary screen hit compounds, with NSC145366 emerging as the lead hit. Our follow-up biochemical and functional studies show that NSC145366 directly interacts and inhibits the C-terminus of Hsp90, validating the platform as a powerful approach for early-stage identification of bioactive modulators of heat shock-dependent pathways.

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. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC(50) curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar 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. Herein, we developed a screening assay for dopamine receptor antagonists to further characterize the pharmacological properties of the two D1-like dopamine receptors (Isdop1 and Isdop2) identified in the Lyme disease vector, Ixodes scapularis, and develop a screening assay for receptor antagonists. A cell-based, cyclic AMP luciferase reporter assay platform was implemented to screen the LOPAC(1280) small molecule library for Isdop2 receptor antagonists, representing the first reported chemical library screen for any tick G protein-coupled receptor. Screening resulted in the identification of 85 "hit" compounds with antagonist activity at the Isdop2 receptor. Eight of these chemistries were selected for confirmation assays using a direct measurement of cAMP, and the effects on both Isdop1 and Isdop2 were studied for comparison. Each of these eight compounds showed antagonistic activity at both Isdop1 and Isdop2, although differences were observed regarding their relative potencies. Furthermore, comparison of the pharmacological properties of the tick dopamine receptors with that of the AaDOP2 receptor from the yellow fever mosquito and the human dopamine D1 receptor (hD1) revealed species-specific pharmacological profiles of these receptors. Compounds influencing dopaminergic functioning, such as the dopamine receptor antagonists discovered here, may provide lead chemistries for discovery of novel acaricides useful for vector control

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. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.

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. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2) from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D(1)-like (Gα(s)-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC(50): AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM). Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC(50) = 5.8±1.5 nM) and norepinephrine (EC(50) = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as "hits," and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D(1) dopamine receptor (hD(1)) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2-selective compounds. In subsequent Ae. aegypti larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as "leads" for insecticide discovery. CONCLUSIONS/SIGNIFICANCE: This research provides a "proof-of-concept" for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with in vivo toxicity toward mosquitoes.

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. Here we evaluate A(230) as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A(230). Significant changes in A(230) were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A(230) in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A(230) under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A(230) allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A(230) is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.

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. Given concentrations of the input reactants and desired amounts or volumes of the products, our algorithm, RoboMix, computes the required reactant volumes and the resulting product concentrations, along with volumes and concentrations for all intermediate combinations. It outputs a sequence of robotic liquid transfer steps that ensures that each combination is correctly mixed even when individualized stoichiometries are employed and with any fractional yield for a product. It can also account for waste in robotic liquid handling and residual volume needed to ensure accurate aspiration. We demonstrate the effectiveness of the method in a test mixing dyes with different UV-vis absorption spectra, verifying the desired combinations spectroscopically.

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 3.0 and 1.7 microM, respectively, with good cell potency and low cytotoxicity.

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. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.