Screening Of Small-molecule Inhibitors Research Articles

A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication

Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode.

Identification of Inhibitors of Yellow Fever Virus Replication Using a Replicon-Based High-Throughput Assay

Flaviviruses cause severe disease in humans and are a public health priority worldwide. However, no effective therapies or drugs are commercially available yet. Several flavivirus replicon-based assays amenable to high-throughput screening of inhibitors have been reported recently. We developed and performed a replicon-based high-throughput assay for screening small-molecule inhibitors of yellow fever virus (YFV) replication. This assay utilized packaged pseudoinfectious particles containing a YFV replicon that expresses Renilla luciferase in a replication-dependent manner. Several small-molecule compounds with inhibitory activity at micromolar concentrations were identified in the high-throughput screen. These compounds were subsequently tested for their inhibitory activities against YFV replication and propagation in low-throughput assays. Furthermore, YFV mutants that escaped inhibition by two of the compounds were isolated, and in both cases, the mutations were mapped to the NS4B coding region, suggesting a novel inhibitory target for these compounds. This study opens up new avenues for pursuing the nonenzymatic nonstructural proteins as targets for antivirals against YFV and other flaviviruses.

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide–substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.

Casein kinase 1 isoform 2 is essential for bloodstream form Trypanosoma brucei

Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death. A null mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro. A truncated form of TbCK1.2 expressed in Escherichia coli as a GST fusion produces catalytically active recombinant protein, facilitating screening for small molecule inhibitors. These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.

High-Throughput Screening for Small-Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

High-Throughput Screening for Small-Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

High-Throughput Screening for Small-Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

Functional Pseudotyping of Human Immunodeficiency Virus Type 1 Vectors by Western Equine Encephalitis Virus Envelope Glycoprotein

We investigated the ability of western equine encephalitis virus envelope glycoproteins (WEEV GP) to pseudotype lentiviral vectors. The titers of WEEV GP-pseudotyped human immunodeficiency virus type 1 (HIV) ranged as high as 8.0 x 10(4) IU/ml on permissive cells. Sera from WEEV-infected mice specifically neutralized these pseudotypes; cell transduction was also sensitive to changes in pH. The host range of the pseudotyped particles in vitro was somewhat limited, which is atypical for most alphaviruses. HIV vectors pseudotyped by WEEV GP may be a useful tool for characterizing WEEV cell binding and entry and screening for small-molecule inhibitors.

A Fluorescence-Based Assay for Monoacylglycerol Lipase Compatible with Inhibitor Screening

A novel fluorescence-based assay of monoacylglycerol lipase (MAGL) activity that is simple, sensitive, and amenable to the screening of small molecule inhibitors is described. Purified recombinant human MAGL protein and 7-hydroxycoumarinyl-arachidonate (7-HCA), a fluorogenic substrate for MAGL, were employed in the assay. MAGL protein catalyzes the hydrolysis of 7-HCA to generate arachidonic acid and the highly fluorescent 7-hydroxyl coumarin (7-HC). Release of 7-HC was measured using a fluorometer. MAGL protein catalyzed the hydrolysis of 7-HCA with an apparent K(m) of 9.8 microM and V(max) of 1.7 mmol/min/mg of protein. The assay is specific for MAGL as assay buffer alone or heat-denatured MAGL protein had no significant activity against 7-HCA. Furthermore, MAGL activity was inhibited in a dose-dependent manner by the specific inhibitor URB602 as well as N-arachidonyl maleimide with 50% inhibitory concentration values of 3.1 microM and 155 nM, respectively. The assay was further optimized under different conditions, including pH range and bovine serum albumin protein and dimethyl sulfoxide concentrations. The assay was found to be reproducible, having Z' values ranging from 0.7 to 0.9, and is therefore suitable for high-throughput screening.

Synthesis and screening of small molecule inhibitors of anthrax edema factor

The synthesis and development of a novel class of molecules that inhibit anthrax edema factor, an adenylyl cyclase, is reported. These molecules are derived from the initial discovery that histidine and imidazole adducts of the prostaglandin PGE 2 reduce the net secretory response of cholera toxin-challenged mice and act directly on the action of anthrax edema factor, a calmodulin-dependent adenylyl cyclase. The simple enones examined in this letter were prepared by palladium-catalyzed Suzuki reaction.

Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

Defining the levels of secreted non-structural protein NS1 after West Nile virus infection in cell culture and mice.

Infection with West Nile virus (WNV) causes a febrile illness that can progress to meningitis or encephalitis, primarily in humans that are immunocompromised or elderly. For successful treatment of WNV infection, accurate and timely diagnosis is essential. Previous studies have suggested that the flavivirus non-structural protein NS1, a highly conserved and secreted glycoprotein, is a candidate protein for rapid diagnosis. Herein, we developed a capture enzyme-linked immunosorbent assay (ELISA) to detect WNV NS1 using two anti-NS1 monoclonal antibodies (mAbs) that map to distinct sites on the protein. The capture ELISA efficiently detected as little as 0.5 ng/ml of soluble NS1 and exhibited no cross-reactivity for yellow fever, Dengue, and St. Louis encephalitis virus NS1. The capture ELISA reliably detected NS1 in plasma at day 3 after WNV infection, prior to the development of clinical signs of disease. As the time course of infection continued, the levels of detectable NS1 diminished, presumably because of interference by newly generated anti-NS1 antibodies. Indeed, treatment of plasma with a solution that dissociated NS1 immune complexes extended the window of detection. Overall, the NS1-based capture ELISA is a sensitive readout of infection and could be an important tool for diagnosis or screening small molecule inhibitors of WNV infection.

High-Throughput Screen for Small Molecule Inhibitors of Mint1-PDZ Domains

Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.

Screening for Selective Small Molecule Inhibitors of the Proteasome Using Activity‐Based Probes

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Label-Free Electrical Sensing of Small-Molecule Inhibition on Tyrosine Phosphorylation

Protein tyrosine kinases (PTKs) play a central role in human carcinogenesis and have emerged as the promising new targets. Small-molecule inhibitors of PTKs have shown impressive anticancer effects and are rapidly entering the clinic. PTK assays allow for high-throughput identification of small-molecule inhibitors. However, current methods of detecting kinase activity require the use of radioisotopes or expensive reagents; such as fluorescently labeled antibodies. We have developed a novel label-free approach for the quantitative detection of peptide tyrosine (Tyr) phosphorylation using the electrochemical oxidation current signal of Tyr. When the phosphorylation is achieved, the phosphorylated Tyr (Tyr-P) cannot be oxidized at approximately 0.65 V. However, when the phosphorylation is successfully inhibited using a small molecule, Tyr can be oxidized and result in a high current response on a multiwalled carbon nanotube-modified screen-printed carbon electrode. We determined the activity of cellular-sarcoma (c-Src) nonreceptor PTK, p60(c-Src), in combination with its highly specific substrate peptide, Raytide. Tyr kinase reactions were also performed in the presence of a well-defined small-molecule inhibitor, 4-amino-5-(4-chlorophenyl)-7- (tert-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Based on the dependency of Tyr oxidation signal on inhibitor concentration, IC50 value, half-maximal inhibition of the inhibitor, was estimated as 5 nM for PP2. Our label-free electrochemical method is a promising candidate for pharmaceutical research and development in screening small-molecule inhibitors of PTKs.

Genomic-based high throughput screening and identification of small molecule inhibitors targeting IFN α signaling pathways (131.17)

Abstract Multiple lines of evidence suggest that inhibition of Type I Interferons (IFNs), including IFN(, may provide a therapeutic benefit in treating a number of autoimmune diseases such as Systemic Lupus Erythematosus (SLE). Herein, we describe the generation and use of AvalonRx-HITS®, a proprietary genomic based high-throughput screen (HTS), followed by immunological assays in primary human cells to identify inhibitors of the IFN( pathway. In the HTS step, THP1 cells were stimulated with IFN( and 268 defined small molecule inhibitors targeting 41 different intracellular signaling pathways were screened for their ability to block a specific IFN(-evoked gene signature. Four classes of compounds emerged from the HTS: NF-(B, JAK/STAT, Ubiquitin/Proteasome and HDAC inhibitors. In secondary assays, these compounds blocked both IFN(-induced human monocyte activation and differentiation into dendritic cells, and IFNs-associated gene signature induced by SLE serum in human monocytes. In addition to their dose-dependent anti-inflammatory effects, inhibitors targeting NF- (B or JAK/STAT signaling did not modulate IFN( anti-viral effects in an in vitro HSV-1 replication assay. In summary, we have developed a novel and robust platform using genomic, immunological and viral assays that may lead to the identification of small molecule inhibitors for the treatment of IFN(-associated autoimmune disorders.

High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation

A library of ∼51,000 compounds was interrogated by high throughput screening (HTS) using a heparin-induced tau fibrillization assay. HTS was conducted with bacterially expressed recombinant tau fragment K18 and the reaction was monitored by thioflavine T fluorescence. Hits meeting criteria set for selection in HTS were further evaluated in a panel of assays designed (a) to confirm the initial results and (b) to identify possible false positives arising from non-specific mechanisms or assay-dependent artifacts. Two 2,3-di(furan-2-yl)-quinoxalines were confirmed as inhibitors of tau fibrillization with IC 50s in the low micromolar range (l–3 μM). Among false positive hits, members of the pyrimidotriazines, benzofurans, porphyrins, and anthraquinone, inhibited tau fibrillization by generating peroxides via catalytic redox cycles due to the reducing agent dithiothreitol (DTT) in the assay. This study delineates focused strategies for HTS of tau fibrillization inhibitors that are relevant to drug discovery for Alzheimer’s disease and related tauopathies.

Synthesis and Biological Activity of a Focused Library of Mitogen‐activated Protein Kinase Phosphatase Inhibitors

Mitogen-activated protein kinase phosphatase 1 is a tyrosine phosphatase superfamily member that dephosphorylates and inactivates mitogen-activated protein kinase substrates, such as p38, c-Jun-N-terminal kinase, and extracellular signal-related kinase. These mitogen-activated protein kinase substrates regulate many cellular processes associated with human diseases. In spite of this potential as a molecular target for chemotherapy, however, pharmacologically tractable inhibitors of mitogen-activated protein kinase phosphatase-1 have yet to be developed. Based on the results from a high-throughput screen for small molecule inhibitors of mitogen-activated protein kinase phosphatase-1, we designed, synthesized, and evaluated a focused library in an effort to further understand the structural requirements for mitogen-activated protein kinase phosphatase-1 inhibitory activity.

Elucidation of the CMP-pseudaminic acid pathway in Helicobacter pylori: synthesis from UDP-N-acetylglucosamine by a single enzymatic reaction

Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic acid (pseudaminic acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of pseudaminic acid, and its nucleotide-activated form CMP-pseudaminic acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-pseudaminic acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.