Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.     

High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity

The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.     

Microplate-based screening for small molecule inhibitors of neuropilin-2/vascular endothelial growth factor-C interactions

Vascular endothelial growth factor-C (VEGF-C) is a secreted growth factor essential for lymphangiogenesis. VEGF-C functions in both physiological and pathological lymphangiogenesis, particularly in tumor metastasis, making it an attractive therapeutic target. Members of two families of cell surface receptors transduce VEGF-C signals: neuropilin-2 (Nrp2) and VEGF-receptor (VEGFR)-2/3. Nrp2 is a promising target for inhibition because it is highly expressed in lymphatic vessels. Here we describe a microplate-based assay for discovery of VEGF-C/Nrp2 inhibitors. We optimize this assay for use in screening an inhibitor library and identify three novel Nrp2/VEGF-C binding inhibitors from the National Institutes of Health (NIH) Clinical Collection small molecule library.     

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.     

2-Aminobenzimidazoles as potent Aurora kinase inhibitors

This Letter describes the discovery and key structure–activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.     

High-throughput screening for human galactokinase inhibitors

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z' factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 microM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia.     

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.     

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.     

High-throughput screening assay for D-amino acid oxidase

A membrane-based high-throughput screening (HTS) assay for active d-amino acid oxidase (DAAO) in liquid samples as well as in intact Escherichia coli cells has been developed and optimized. The detection limit of the assay was less than 1 ng per sample. The method proposed can be used for quantitative DAAO determination in the range of 0.13 to 3.60 ng enzyme per probe. The protocol was successfully tested to screen a library of E. coli clones containing mutant DAAOs active toward target substrates.     

High-throughput cell-based screening using scintillation proximity assay for the discovery of inositol phosphatase inhibitors

Inositol monophosphatase is a potential drug target for developing lithium-mimetic agents for the treatment of bipolar disorder. Enzyme-based assays have been traditionally used in compound screening to identify inositol monophosphatase inhibitors. A cell-based screening assay in which the compound needs to cross the cell membrane before reaching the target enzyme offers a new approach for discovering novel structure leads of the inositol monophosphatase inhibitor. The authors have recently reported a high-throughput measurement of G-protein-coupled receptor activation by determining inositol phosphates in cell extracts using scintillation proximity assay. This cell-based assay has been modified to allow the determination of inositol monophosphatase activity instead of G-protein-coupled receptors. The enzyme is also assayed in its native form and physiological environment. The authors have applied this cell-based assay to the high-throughput screening of a large compound collection and identified several novel inositol monophosphatase inhibitors.     

High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5′-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3′-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.     

High-throughput screening for Hsp90 ATPase inhibitors

Recently, we reported a useful assay for the determination of yeast Hsp90 ATPase activity. Using this assay, high-throughput screening of approximately 10,000 compounds was performed to determine the feasibility of this assay on large scale. Results from high-throughput screening indicated that the assay was reproducible (av Z-factor = 0.80) and identified 0.57% of the compounds as Hsp90 inhibitors that exhibited IC50s less than 20 microM. The structures of several of these inhibitory scaffolds are reported along with their IC50 values.     

Perturbation of biological processes with small molecule kinase inhibitors

The reversible phosphorylation of substrates mediated by kinases and phosphatases affects their subcellular localization, catalytic activity, and/or interaction with other molecules. It is essential for signal transduction and the regulation of nearly all cellular processes, such as proliferation, apoptosis, metabolism, motility, and differentiation. Small molecule kinase inhibitors (SMKIs) have served as critical chemical probes to reveal the biological functions and mechanisms of kinases and their potential as therapeutic targets. In this review, we focused on a few novel SMKIs and their recent application in biological and preclinical studies to showcase how highly selective and potent SMKIs can be developed and utilized to propel the investigations on kinases and the biology behind.     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.     

High-throughput screening of small molecules for bioactivity and target identification in Caenorhabditis elegans

This protocol describes a procedure for screening small molecules for bioactivity and a genetic approach to target identification using the nematode Caenorhabditis elegans as a model system. Libraries of small molecules are screened in 24-well plates that contain a solid agar substrate. On top of the agar mixture, one small-molecule species is deposited into each well, along with worm food (E. coli), and two third-stage or fourth-stage larval worms using a COPAS (Complex Object Parametric Analyzer and Sorter) Biosort. Three to five days later the plates are screened for phenotype. Images of the wells are acquired and archived using a HiDI 2100 automated imaging system (Elegenics). Up to 2,400 chemicals can be screened per week. To identify the predicted protein target of a bioactive molecule, wild-type worms are mutagenized using ethylmethanesulfonate (EMS). Progeny are screened for individuals resistant to the molecules effects. The candidate mutant target that confers resistance is then identified. Target identification might take months.     

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.     

High-throughput screening system for inhibitors of human Heat Shock Factor 2

Development of novel anti-cancer drug leads that target regulators of protein homeostasis is a formidable task in modern pharmacology. Finding specific inhibitors of human Heat Shock Factor 1 (hHSF1) has proven to be a challenging task, while screening for inhibitors of human Heat Shock Factor 2 (hHSF2) has never been described. We report the development of a novel system based on an in vivo cell growth restoration assay designed to identify specific inhibitors of human HSF2 in a high-throughput format. This system utilizes a humanized yeast strain in which the master regulator of molecular chaperone genes, yeast HSF, has been replaced with hHSF2 with no detrimental effect on cell growth. This replacement preserves the general regulatory patterns of genes encoding major molecular chaperones including Hsp70 and Hsp90. The controlled overexpression of hHSF2 creates a slow-growth phenotype, which is the basis of the growth restoration assay used for high-throughput screening. The phenotype is most robust when cells are cultured at 25 °C, while incubation at temperatures greater than 30 °C leads to compensation of the phenotype. Overexpression of hHSF2 causes overexpression of molecular chaperones which is a likely cause of the slowed growth. Our assay is characterized by two unique advantages. First, screening takes place in physiologically relevant, in vivo conditions. Second, hits in our screen will be of medically relevant potency, as compounds that completely inhibit hHSF2 function will further inhibit cell growth and therefore will not be scored as hits. This caveat biases our screening system for compounds capable of restoring hHSF2 activity to a physiologically normal level without completely inhibiting this essential system.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0605-0) contains supplementary material, which is available to authorized users.