Artificial Neural Networks (ANNs) are trained using High Throughput Screening (HTS) data to recover active compounds from a large data set. Improved classification performance was obtained on combining predictions made by multiple ANNs. The HTS data, acquired from a Methionine Aminopeptidases Inhibition study, consisted of a library of 43,347 compounds, and the ratio of active to non-active compounds, RA/N, was 0.0321. Back-propagation ANNs were trained and validated using Principal Components derived from the physico-chemical features of the compounds. On selecting the training parameters carefully, an ANN recovers one-third of all active compounds from the validation set with a three-fold gain in RA/N value. Further gains in RA/N values were obtained upon combining the predictions made by a number of ANNs. The generalization property of the back-propagation ANNs was utilized to train those ANNs with the same training samples, after being initialized with different sets of random weights. As a result, only 10% of all available compounds were needed for training and validation, and the rest of the data set was screened with more than a ten-fold gain of the original RA/N value. Thus, ANNs trained with limited HTS data might become useful in recovering active compounds from large data sets.
pattern classification; neural networks; generalization property
The standard (STD) 5 × 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors. Application of the STD 5 × 5 HMF to the primary screen raw data reduced background signal deviation, thereby improving the assay dynamic range and hit confirmation rate. While this HMF can correct gradient vectors, it does not properly correct periodic patterns that may present in other screening campaigns. To address this issue, 1 × 7 median and a row/column 5 × 5 hybrid median filter kernels (1 × 7 MF and RC 5 × 5 HMF) were designed ad hoc, to better fit periodic error patterns. The correction data show periodic error in simulated MTP data arrays is reduced by these alternative filter designs and that multiple corrective filters can be combined in serial operations for progressive reduction of complex error patterns in a MTP data array.
Pesticides currently in widespread use often lack species specificity and also become less effective as resistance emerges. Consequently, there is a pressing need to develop novel agents that are narrowly targeted and safe to humans. A cell-based screening platform was designed to discover compounds that are lethal to mosquito (Anopheles and Aedes) cells but show little or no activity against other insect (Drosophila) or human cell lines. Mosquito-specific, aqueous-stable cytotoxins were recovered at rare frequencies. Three of these were profiled for structure-activity relationships and also assessed in whole-animal toxicity assays. In at least one test case, species-specific cytotoxicity seen in culture effectively translated to the whole-animal level, with potent toxicity against Anopheles yet none against Drosophila. Therefore, this initiative has the potential to advance novel mosquitocidal agents and, in a broader sense, could establish a versatile platform for developing customized pesticides that selectively target other disease vectors as well.
agricultural; animal health; cell-based assays; high-content screening; phenotypic drug discovery
Tumor marker endothelial 8 (TEM8) is a receptor for the Protective Antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared to normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo.
Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z-prime value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complimentary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for anti-anthrax and anti-angiogenic diseases.
High-throughput screening; FRET; anthrax; angiogenesis; Tumor endothelial marker 8
Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes mRNAs, leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung and colon cancer. Small molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z’ factor = 0.60) FAMA-based high throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic non-specific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high throughput screen was demonstrated by identification of the first small molecule IMP-1 inhibitor; a lead compound that selectively inhibits proliferation of IMP-1 positive cancer cells with very little or no effect on proliferation of IMP-1 negative cells.
Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.
Xenopus; zebrafish; in vivo screening; mass spectrometry; high-content screening
We developed a homogeneous phenotypic fluorescence endpoint assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 μM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 μM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the MAP kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.
Cytotoxic T lymphocytes; exocytosis; flow cytometry; high-throughput screen; phenotypic assay
Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two E. coli reporter fusions; one to identify potential RhaS inhibitors, and a second to eliminate non-specific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not impact bacterial cell growth at the concentrations used in this study. In vitro DNA binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.
High throughput screening; Cell-based assays; AraC family activators; Antibacterial agents; Inhibitor
The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.
phenotypic drug discovery; imaging technologies; label-free technologies; cell-based assays
Elevation of amyloid β-peptide (Aβ) is critically associated with Alzheimer’s disease (AD) pathogenesis. Aβ-induced synaptic abnormalities, including altered receptor trafficking and synapse loss, have been linked to cognitive deficits in AD. Recent work implicates a lipid critical for neuronal function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], in Aβ-induced synaptic and behavioral impairments. Synaptojanin 1 (Synj1), a lipid phosphatase mediating the breakdown of PI(4,5)P2, has been shown to play a role in synaptic vesicle recycling and receptor trafficking in neurons. Heterozygous deletion of Synj1 protected neurons from Aβ-induced synaptic loss and restored learning and memory in a mouse model of AD. Thus, inhibition of Synj1 may ameliorate Aβ-associated impairments, suggesting Synj1 as a potential therapeutic target. To this end, we developed a screening assay for Synj1 based on detection of inorganic phosphate liberation from a water-soluble, short chain PI(4,5)P2. The assay displayed saturable kinetics and detected Synj1’s substrate preference for PI(4,5)P2 over PI(3,4,5)P3. The assay will enable identification of novel Synj1 inhibitors which have potential utility as chemical probes to dissect the cellular role of Synj1 as well as potential to prevent or reverse AD-associated synaptic abnormalities.
Lipid phosphatase; phosphatidylinositol-4,5-bisphosphate; Synaptojanin 1; src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2); small molecule screening; Alzheimer’s disease; drug discovery
Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.
HIV-1; HIV-1 Nef; fluorescence complementation; BiFC; YFP; high-content screening
Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed 1) a high throughput assay for measuring aminoacyl-tRNA synthetase activity and 2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product, and yielded a Z′-factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 of 0.6μM. Enzymology studies were also performed with rIleRS and its Km and kcat determined as 3.97 × 10−5 mol/L and 142 S−1, respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.
aminoacyl-tRNA synthetase; high throughput assay; malachite green; tRNA; aminoacylation
Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, while depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. We used a radiometric proximity assay in a high-throughput screen to identify small molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabelled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microtitre plates. Seventy non-aggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualised using pericentrin antibodies confirmed that two of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.
Cell cycle; mitosis; centrosome separation; Nek2 kinase inhibitors; automated immunofluorescence
The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. In order to identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high throughput screening and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small molecule inhibitors of MEKK2 activity.
MEKK2; MAP3K2; ATPase; crizotinib; bosutinib
Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed ATP-binding pockets make ideal targets for small molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of a HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.
LIM kinase; assay; luminescence; high-throughput screen
TDP-43 is an RNA binding protein found to accumulate in the cytoplasm of brain and spinal cord from patients affected with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Nuclear TDP-43 protein regulates transcription through several mechanisms, and under stressed conditions it forms cytoplasmic aggregates that co-localize with stress granule (SG) proteins in cell culture. These granules are also found in the brain and spinal cord of patients affected with ALS and FTLD. The mechanism through which TDP-43 might contribute to neurodegenerative diseases is poorly understood. In order to investigate the pathophysiology of TDP-43 aggregation and to isolate potential therapeutic targets, we screened a chemical library of 75,000 compounds using high content analysis with PC12 cells that inducibly express human TDP-43 tagged with GFP. The screen identified 16 compounds that dose-dependently decreased the TDP-43 inclusions without significant cellular toxicity or changes in total TDP-43 expression levels. To validate the effect of the compounds, we tested compounds by Western Blot analysis and in a model that replicates some of the relevant disease phenotypes. The hits from this assay will be useful for elucidating regulation of TDP-43, stress granule response, and possible ALS therapeutics.
Amyotrophic lateral sclerosis; RNA granule; RNA binding protein; aggregation; high throughput screen; protein synthesis
Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-mRNA splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB) is overexpressed in ovarian tumors, compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high throughput screening HTS assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high throughput assay could be used to identify small molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
PTB; ovarian cancer; fluorescence methods; cell-based HTS; alternative splicing
Wnt/β-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/β-catenin signaling in disease relevant contexts are needed. The use of human patient-derived induced pluripotent stem cell (iPSC) models provides ideal disease relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/β-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose responsive stimulation by several known Wnt/β-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1,500 compounds from a library of FDA-approved drugs and known bioactives, and confirmed HTS hits, revealing multiple chemical and biological classes of novel small molecule probes of Wnt/β-catenin signaling. Generating this type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorder therapeutics.
induced pluripotent stem cell (iPSC); neural progenitor cell (NPC); Wnt/β-catenin signaling; neuropsychiatric disorders; human neurons
von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL.
The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed.
Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds.
786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
VHL upregulation; proteostasis; high-throughput screen; Prestwick
Eya proteins are essential co-activators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be anti-tumorigenic and anti-metastatic, as well as sensitize cancer cells to DNA damage inducing therapies. In this paper, we identified a previously unknown chemical series using high throughput screening that inhibits the Eya2 phosphatase activity with IC50s ranging from 1.8 to 79 μM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anti-cancer drugs in the future.
Phosphatase; Eyes Absent 2; Eya2; Eya2 inhibitor; Six1
Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate we identified a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused SAR-driven chemistry effort we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2 over-expressing tumor model. At least two analogs significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
Multi-drug resistance; ABC Transporter; ABCG2; ABCB1; Efflux inhibition
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
calcium pump; calcium transport; phospholamban; reconstituted membrane; fluorescence lifetime
Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by progressive loss of motor neuron function. It is caused by the homozygous loss of the SMN1 (survival of motor neuron 1) gene and a decrease in full-length SMN protein. SMN2 is a nearly identical homolog of SMN1 that, due to alternative splicing, expresses predominantly truncated SMN protein. SMN2 represents an enticing therapeutic target. Increasing expression of full-length SMN from the SMN2 gene might represent a treatment for SMA. We describe a newly designed cell-based reporter assay that faithfully and reproducibly measures full-length SMN expression from the SMN2 gene. This reporter can detect increases of SMN protein by an array of compounds previously shown to regulate SMN2 expression and by the overexpression of proteins that modulate SMN2 splicing. It also can be used to evaluate changes at both the transcriptional and splicing level. This assay can be a valuable tool for the identification of novel compounds that increase SMN2 protein levels and the optimization of compounds already known to modulate SMN2 expression. We present here preliminary data from a high-throughput screen using this assay to identify novel compounds that increase expression of SMN2.
spinal muscular atrophy; survival of motor neuron; SMN1; SMN2; cell-based assay; high-content screening; HTS
The generation of an action potential is a complex process in excitable cells which involves the temporal opening and closing of several voltage-dependent ion channels in the cell membrane. The shape of an action potential can carry information concerning the state of the involved ion channels and their relationship to cellular processes. Alteration of these ion channels by the administration of toxins, drugs, and biochemicals can change the action potential’s shape in a specific way which can be characteristic for a given compound. Thus, action potential shape analysis could be a valuable tool to classify toxins and measure drug effects based on their mechanism of action. In an effort to begin classifying the effect of toxins on the shape of intracellularly recorded action potentials, patch clamp experiments were performed on NG108-15 hybrid cells in the presence of Veratridine, TEA, and quinine. To analyze the effect a computer model of the action potential mechanism was generated to determine to what extent each ion channel was affected during administration of these compounds based on the changes in the model parameters. Our work is a first step towards establishing a new assay system to detect and identify toxins by action potential shape analysis.
high-throughput; toxicity; cell models; electrophysiology; mechanisms
Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to our understanding of phagocyte biology. Due to lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. To investigate the feasibility of functionally cloning eat-me signals by phage display, we characterize the biological behavior of T7 phages displaying two well-known eat-me signals: growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). Gas6-phage binds to all three known Gas6 receptors, Mer, Axl and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and non-phagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia and retinal pigment epithelium (RPE) cells, but not in non-phagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library.
Eat-me signals; phagocytosis; phage display; Gas6; MFG-E8