Novel small molecule antagonists of NPBWR1 (GPR7) are herein reported. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 5-chloro-4-(4-methoxyphenoxy)-2-(p-tolyl)pyridazin-3(2H)-one as a NPBWR1 hit antagonist with micromolar activity. Design, synthesis and structure–activity relationships study of the HTS-derived hit led to the identification of 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule with submicromolar antagonist activity at the target receptor and high selectivity against a panel of therapeutically relevant off-target proteins. This lead molecule may provide a pharmacological tool to clarify the molecular basis of the in vivo physiological function and therapeutic utility of NPBWR1 in diverse disease areas including inflammatory pain and eating disorders.
NPBWR1 (GPR7); Selective small molecule antagonists; Feeding behavior and energy homeostasis; Inflammatory pain
NR2E3 is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. NR2E3-specific modulators may prolong photoreceptor survival in patients with dry age-related macular degeneration and other forms of retinal degeneration. To definitively establish NR2E3 as a photoreceptor protection target, identification of small-molecule NR2E3 modulators and their testing in animal models of retinal degeneration are required. Development of the high-throughput screen (HTS)-compatible screen for small-molecule NR2E3 modulators is the first step toward this goal.
Purification protocol for isolation of the functionally competent soluble NR2E3 protein after its expression in the insect Sf9 cells was developed. The time-resolved fluorescence energy-transfer (TR-FRET) assay assessing agonist-sensitive interaction between apo-NR2E3 and transcriptional corepressor RetCOR was used for characterization of the previously reported putative NR2E3 agonist, Compound 11a, and to conduct the HTS for novel small-molecule NR2E3 modulators (direct and inverse agonists). A counterscreen TR-FRET assay that measures the affect of test compounds on PPARγ interaction with corepressor NCOR was used for assessing the specificity of compounds identified in the HTS.
We developed the cell-free TR-FRET assay for small-molecule NR2E3 modulators, which is based on agonist-induced disruption of the interaction between GST-tagged apo-NR2E3 and MBP-tagged fragment of transcriptional corepressor RetCOR. Compound 11a, a putative NR2E3 agonist, did not affect the NR2E3–RetCOR interaction, as was established by its titration in the developed assay. The assay was miniaturized for an ultralow-volume 1,536-well format and automated into 3 simple pipetting steps. Consistent with excellent assay performance, the test runs established a Z′-score within the 0.6–0.8 range. Analysis of the mid-size National Institutes of Health collection of 315,001 structurally diverse drug-like compounds confirmed excellent assay performance, but did not reveal NR2E3-specific agonists or inverse agonists.
A robust and reliable TR-FRET assay for small-molecule NR2E3-specific modulators suitable for the analysis of million compound-strong HTS libraries was developed. A previously described putative NR2E3 agonist, Compound 11a, is unlikely to represent a direct NR2E3 agonist. Application of the developed assay for screening of a more abundant and diverse compound collection be required for identification of synthetic NR2E3 ligands.
High affinity and selective small molecule agonists of the S1P4 receptor (S1P4-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P4-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P4-R agonists exquisitely selective over the remaining S1P1–3,5–Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P4–R.
S1P4 receptor; selective small molecule S1P4–R agonists; autoimmune diseases; viral infections; thrombocytopenia
High affinity and selective S1P4 receptor (S1P4–R) small molecule agonists may be important proof-of-principle tools used to clarify the receptor biological function and effects to assess the therapeutic potential of the S1P4–R in diverse disease areas including treatment of viral infections and thrombocytopenia. A high-throughput screening campaign of the Molecular Libraries-Small Molecule Repository was carried out by our laboratories and identified (2Z,5Z)-5-((1-(2-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-3-methyl-2-(methylimino) thiazolidin-4-one as a promising S1P4–R agonist hit distinct from literature S1P4–R modulators. Rational chemical modifications of the hit allowed the identification of a promising lead molecule with low nanomolar S1P4–R agonist activity and exquisite selectivity over the other S1P1-3,5–Rs family members. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the effects of the S1P4–R signaling cascade and elucidate the molecular basis of the receptor function.
S1P4 receptor; selective small molecule S1P4–R agonists; thrombocytopenia; viral infections
The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the NIH’s public domain, the Molecular Libraries Probe Production Centers Network (MLPCN) library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was performed in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50<10 µM for KLF5 inhibition and EC50>10 µM for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized based on computational, Western blot, and cell viability analyses. Both of these compounds and two newly-synthesized structural analogs of CID 5951923 significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results demonstrate the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.
Colorectal cancer; KLF5; Ultrahigh-throughput screen; Luciferase; Cell viability; Small-molecule compounds
Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase (PEP) and the bacterial peptidase oligopeptidase B (OPDB). The biochemistry and biology of PREPL is of fundamental interest due to this enzyme’s homology to the biomedically important prolyl peptidases and its localization in the central nervous system (CNS). Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it crosses the blood-brain barrier, and may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL, and in eventually understanding the link between PREPL and HCS.
Prolyl peptidases; activity-based proteomics; fluopol; high-throughput screening; chemical inhibitors; Prolyl endopeptidase-like
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n ~ 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site binding inhibitors may be identified via HTS protocols utilizing such assays.
The Steroidogenic factor 1 (SF-1, also known as NR5A1) is a transcription factor belonging to the nuclear receptor superfamily. Whereas most of the members of this family have been extensively characterized, the therapeutic potential and pharmacology of SF-1 still remains elusive. Described here is the identification and characterization of selective inhibitory chemical probes of SF-1 by a rational ultra-high-throughput screening (uHTS) strategy. A set of 64,908 compounds from the National Institute of Health’s Molecular Libraries Small Molecule Repository (MLSMR) was screened in a transactivation cell-based assay employing a chimeric SF-1 construct. Two analogous isoquinolinones, SID7969543 and SID7970631, were identified as potent submicromolar inhibitors, yielding IC50 values of 760 nM and 260 nM. The compounds retained their potency in a more physiologic functional assay employing the full-length SF-1 protein and its native response element, yielding IC50 values of 30 and 16 nM, respectively. The selectivity of these isoquinolinones was confirmed via transactivation-based functional assays for RORA, VP-16 and LRH-1. Their cytotoxicity, solubility, permeability and metabolic stability were also measured. These isoquinolinones represent valuable chemical probes to investigate the therapeutic potential of SF-1.
The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-Luciferase fusion protein. To insure uHTS compatibility, the assay was scaled to 1,536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogenous assay demonstrated robust performance, with a calculated Z′ factor of 0.65±0.05. The assay was screened against a publicly available library of ~218,000 compounds in order to identify Wee1 stabilizers. Nonselective, cytotoxic and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of a N-cyclin B-Luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of four unrelated cell-permeable small molecules that showed selective Wee1-Luciferase stabilization with micromolar potency. One of these compounds, SID4243143, was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Our results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation, and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.
Wee1; degradation; stabilizer; reporter assay; transient transfection; cryopreserved cells; ubiquitin; proteasome
We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying the enzyme’s active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.
We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na+]. This synergism revealed existence of distinct and cooperative activation mechanisms, and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are non-responsive to the activators identified in this 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.
We have studied the Sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 vs. S1P3 in this closely related receptor family.
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, while the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen, hence aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase 2 (ADAMTS-5) is desirable. We have previously described collagen-model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multi-well formats. In the present study, a collagen-model FRET substrate has been examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n = 960) with known pharmacologic activity. Five compounds were identified that inhibited ADAMTS-4 >60% at a concentration of 1 μM. A secondary screen using RP-HPLC was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC50 value of 1 μM. Because the collagen-model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), non-active site binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow for a more definitive evaluation of this protease in osteoarthritis, as well as representing a potential next generation in metalloproteinase therapeutics.
To determine how subjects responded to alarms for hypo- and hyperglycemia while they were sleeping.
Research Design and Methods
Twenty subjects with type 1 diabetes (ages 4–17 years) were admitted to a clinical research center for approximately 24 hours. Each subject wore two GlucoWatch® G2TM Biographers (GW2B) and was videotaped using an infrared camera from 9 PM to 7 AM. The videotapes were reviewed to determine if the GW2B alarms were audible on the tape and to document the subject’s response to the alarms. Single or multiple alarms separated by at least ½ hour were considered as discreet alarm “events”.
Downloaded data from the biographers identified 240 individual alarms, 75% of which occurred while the subject was sleeping. Of the 240 alarms 68% were audible on the videotape. Subjects awoke to 29% of individual alarms and to 66% of alarm events. Subjects 4 to 6 years old responded to 17% of alarms; 7 to 11 year olds responded to 20% of alarms, adolescents responded to 53% of alarms, and parents to 37% of alarms. Subjects awoke to 40% of the first alarm during the night, but to only 28% of subsequent alarms. There were 11 events when the glucose was confirmed ≤70 mg/dL, and in each case the subject was awoken. Fifty-five percent of alarm events occurred when there was no hypo- or hyperglycemia confirmed by a reference glucose value.
Subjects awoke to 29% of individual alarms and to 66% of alarm events. Subjects awoke during all alarm events when hypoglycemia was confirmed, but there was a high incidence of false alarms.