High Throughput Screening Cascade To Identify Human Aspartate N-Acetyltransferase (ANAT) Inhibitors for Canavan Disease.

Canavan disease (CD) is a progressive, fatal neurological disorder that begins in infancy resulting from a mutation in aspartoacyclase (ASPA), an enzyme that catalyzes the deacetylation of N-acetyl aspartate (NAA) into acetate and aspartate. Increased NAA levels in the brains of affected children are one of the hallmarks of CD. Interestingly, genetic deletion of N-acetyltransferase-8-like (NAT8L), which encodes aspartate N-aceyltransferase (ANAT), an enzyme responsible for the synthesis of NAA from l-aspartate and acetyl-CoA, leads to normalization of NAA levels and improvement of symptoms in several genetically engineered mouse models of CD. Therefore, pharmacological inhibition of ANAT presents a promising therapeutic strategy for treating CD. Currently, however, there are no clinically viable ANAT inhibitors. Herein we describe the development of fluorescence-based high throughput screening (HTS) and radioactive-based orthogonal assays using recombinant human ANAT expressed in E. coli. In the fluorescence-based assay, ANAT activity was linear with respect to time of incubation up to 30 min and protein concentration up to 97.5 ng/µL with Km values for l-aspartate and acetyl-CoA of 237 µM and 11 µM, respectively. Using this optimized assay, we conducted a pilot screening of a 10 000-compound library. Hits from the fluorescence-based assay were subjected to an orthogonal radioactive-based assay using L-[U-14C] aspartate as a substrate. Two compounds were confirmed to have dose-dependent inhibition in both assays. Inhibitory kinetics studies of the most potent compound revealed an uncompetitive inhibitory mechanism with respect to l-aspartate and a noncompetitive inhibitory mechanism against acetyl-CoA. The screening cascade developed herein will enable large-scale compound library screening to identify novel ANAT inhibitors as leads for further medicinal chemistry optimization.

High-throughput genetic screen for synaptogenic factors: identification of LRP6 as critical for excitatory synapse development.

Genetic screens in invertebrates have discovered many synaptogenic genes and pathways. However, similar genetic studies have not been possible in mammals. We have optimized an automated high-throughput platform that employs automated liquid handling and imaging of primary mammalian neurons. Using this platform, we have screened 3,200 shRNAs targeting 800 proteins. One of the hits identified was LRP6, a coreceptor for canonical Wnt ligands. LRP6 regulates excitatory synaptogenesis and is selectively localized to excitatory synapses. In vivo knockdown of LRP6 leads to a reduction in the number of functional synapses. Moreover, we show that the canonical Wnt ligand, Wnt8A, promotes synaptogenesis via LRP6. These results provide a proof of principle for using a high-content approach to screen for synaptogenic factors in the mammalian nervous system and identify and characterize a Wnt ligand receptor complex that is critical for the development of functional synapses in vivo.

Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator.

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.

hERGCentral: a large database to store, retrieve, and analyze compound-human Ether-à-go-go related gene channel interactions to facilitate cardiotoxicity assessment in drug development.

The unintended and often promiscous inhibition of the cardiac human Ether-à-go-go related gene (hERG) potassium channel is a common cause for either delay or removal of therapeutic compounds from development and withdrawal of marketed drugs. The clinical manifestion is prolongation of the duration between QRS complex and T-wave measured by surface electrocardiogram (ECG)-hence Long QT Syndrome. There are several useful online resources documenting hERG inhibition by known drugs and bioactives. However, their utilities remain somewhat limited because they are biased toward well-studied compounds and their number of data points tends to be much smaller than many commercial compound libraries. The hERGCentral ( www.hergcentral.org ) is mainly based on experimental data obtained from a primary screen by electrophysiology against more than 300,000 structurally diverse compounds. The system is aimed to display and combine three resources: primary electrophysiological data, literature, as well as online reports and chemical library collections. Currently, hERGCentral has annotated datasets of more than 300,000 compounds including structures and chemophysiological properties of compounds, raw traces, and biophysical properties. The system enables a variety of query formats, including searches for hERG effects according to either chemical structure or properties, and alternatively according to the specific biophysical properties of current changes caused by a compound. Therefore, the hERGCentral, as a unique and evolving resource, will facilitate investigation of chemically induced hERG inhibition and therefore drug development.

Discovery, Synthesis, and Structure Activity Relationship of a Series of N-Aryl- bicyclo[2.2.1]heptane-2-carboxamides: Characterization of ML213 as a Novel KCNQ2 and KCNQ4 Potassium Channel Opener.

Herein we report the discovery, synthesis and evaluation of a series of N-Aryl-bicyclo[2.2.1]heptane-2-carboxamides as selective KCNQ2 (K(v)7.2) and KCNQ4 (K(v)7.4) channel openers. The best compound, 1 (ML213) has an EC(50) of 230 nM (KCNQ2) and 510 nM (KCNQ4) and is selective for KCNQ2 and KCNQ4 channels versus a large battery of related potassium channels, as well as affording modest brain levels. This represents the first report of unique selectivity profile for KCNQ2 and KCNQ4 over the other channels (KCNQ1/3/5) and as such should prove to be a valuable tool compound for understanding these channels in regulating neuronal activity.

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4ß by µ-opioid receptors. ML204 inhibited TRPC4ß-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 µm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4ß currents activated through either µ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 µm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Selective inhibition of the K(ir)2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR, and pharmacological characterization of ML133.

The K(ir) inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (K(ir)1-K(ir)7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective K(ir)2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of K(ir)2.1 function. Here we report one potent K(ir)2.1 inhibitor, ML133, which inhibits K(ir)2.1 with an IC(50) of 1.8 µM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on K(ir)1.1 (IC(50) > 300 µM) and displays weak activity for K(ir)4.1 (76 µM) and K(ir)7.1 (33 µM), making ML133 the most selective small molecule inhibitor of the K(ir) family reported to date. Because of the high homology within the K(ir)2 family-the channels share a common design of a pore region flanked by two transmembrane domains-identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent K(ir) inhibitors. Using chimeric channels between K(ir)2.1 and K(ir)1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of K(ir)2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of K(ir)1.1 to those of K(ir)2.1 (N171D and C175I) transplants ML133 inhibition to K(ir)1.1. Together, the combination of a potent, K(ir)2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of K(ir)2 inward rectifier potassium channels.

Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We identified a large number of unanticipated PDIs for known TFs, as well as for previously uncharacterized TFs. We also found that over three hundred unconventional DNA-binding proteins (uDBPs)--which include RNA-binding proteins, mitochondrial proteins, and protein kinases--showed sequence-specific PDIs. One such uDBP, ERK2, acts as a transcriptional repressor for interferon gamma-induced genes, suggesting important biological roles for such proteins.

Interrogation of phosphor-specific interaction on a high-throughput label-free optical biosensor system-Epic system.

The Epic system, a high-throughput label-free optical biosensor system, is applied for the biochemical interrogation of phosphor-specific interactions of the 14-3-3 protein and its substrates. It has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. A perspective of biochemical applications for diagnostics and biomarker discovery, as well as cell-based applications for endogenous receptors and viral infection characterization, are also provided.

Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins.

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.