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1.  An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening 
Bioorganic & medicinal chemistry  2013;21(11):3138-3146.
Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z’ factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z’ factor of ≥ 0.8, good signal / baseline ratios (> 3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-hloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.
doi:10.1016/j.bmc.2013.03.031
PMCID: PMC3651783  PMID: 23591260
2.  Discovery of ML326: the first sub-micromolar, selective M5 PAM 
This letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M5 PAMs (M5 EC50 >10 μM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409 nM and 480 nM, respectively) with excellent mAChR selectivity (M1-M4 EC50s <30 μM) and a robust 20-fold leftward shift of the ACh CRC.
doi:10.1016/j.bmcl.2013.03.032
PMCID: PMC3634896  PMID: 23562060
Muscarinic acetylcholine receptors; M5; Positive allosteric modulator (PAM); ML326
3.  Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents 
Molecular cancer therapeutics  2013;12(5):610-620.
Acetyltransferase p300 (KAT3B) plays key roles in signaling cascades that support cancer cell survival and sustained proliferation. Thus, p300 represents a potential anticancer therapeutic target. To discover novel anticancer agents that target p300, we conducted a high-throughput screening campaign. A library of 622,079 compounds was assayed for cytotoxicity to the triple-negative breast cancer (TNBC) cell line MDA-MB-231 but not to the human mammary epithelial cells. The resulting compounds were tested in a biochemical assay for inhibiting the enzymatic activity of p300. One compound (L002, NSC764414) displayed an IC50 of 1.98 μM against p300 in vitro, inhibited acetylation of histones and p53, and suppressed STAT3 activation in cell-based assays. L002 could be docked to the active site of the p300 catalytic domain. Biochemical tests of a series of related compounds revealed functional groups that may impact inhibitory potency of L002 against p300. Interestingly, these analogs showed inhibitory activities against CBP (the cellular paralog of p300), PCAF and GCN5, but not to other acetyltransferases (KAT5, KAT6B and KAT7), histone deacetylases (HDACs) and histone methyltransferases. Among the NCI-60 panel of cancer cell lines, leukemia and lymphoma cell lines were extremely sensitive to L002, whereas it is toxic to only a limited number of cell lines derived from solid tumors. Notably, breast cancer cell lines, especially those derived from TNBC, were highly susceptible to L002. In vivo, it potently suppressed tumor growth and histone acetylation of MDA-MB-468 xenografts. Thus, these new acetyltransferase inhibitors are potential anticancer therapeutics.
doi:10.1158/1535-7163.MCT-12-0930
PMCID: PMC3651759  PMID: 23625935
4.  Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor 
PLoS ONE  2014;9(4):e95243.
Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.
doi:10.1371/journal.pone.0095243
PMCID: PMC3990629  PMID: 24743578
5.  Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A2 
Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA2 has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA2 have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA2 inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA2 and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA2 in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA2 function in biological systems.
doi:10.1016/j.bmcl.2012.11.061
PMCID: PMC3549684  PMID: 23260346
Phospholipase; Inhibitor; Screening; Proteomics
6.  Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent non-covalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease 
Journal of medicinal chemistry  2013;56(2):534-546.
A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a non-covalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multi-component Ugi reaction was utilized to rapidly explore structure activity relationships within S1′, S1, and S2 enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a non-covalent mechanism of action.
doi:10.1021/jm301580n
PMCID: PMC3569073  PMID: 23231439
7.  Design, synthesis and SAR analysis of novel potent and selective small molecule antagonists of NPBWR1 (GPR7) 
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.
doi:10.1016/j.bmcl.2012.09.074
PMCID: PMC3601546  PMID: 23079522
NPBWR1 (GPR7); Selective small molecule antagonists; Feeding behavior and energy homeostasis; Inflammatory pain
8.  Confirming Target Engagement for Reversible Inhibitors In Vivo by Kinetically Tuned Activity-Based Probes 
Journal of the American Chemical Society  2012;134(25):10345-10348.
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
doi:10.1021/ja303400u
PMCID: PMC3392194  PMID: 22690931
9.  Small-molecule Inhibitors of Bacterial AddAB and RecBCD Helicase-nuclease DNA Repair Enzymes 
ACS Chemical Biology  2012;7(5):879-891.
The AddAB and RecBCD helicase-nucleases are related enzymes prevalent among bacteria but not eukaryotes and are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although these enzymes have been extensively studied both genetically and biochemically, inhibitors specific for this class of enzymes have not been reported. We developed a high-throughput screen based on the ability of phage T4 gene 2 mutants to grow in Escherichia coli only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized this screen for use in 1536-well plates and screened 326,100 small molecules in the NIH molecular libraries sample collection for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli recBCD deletion strain. Secondary screening used assays with cells expressing AddAB or RecBCD and a viability assay that measured the effect of compounds on cell growth without phage infection. From this screening campaign, 12 compounds exhibiting efficacy and selectivity were tested for inhibition of purified AddAB and RecBCD helicase and nuclease activities and in cell-based assays for recombination; seven were active in the 0.1 – 50 μM range in one or another assay. Compounds structurally related to two of these were similarly tested, and three were active in the 0.1 – 50 μM range. These compounds should be useful in further enzymatic, genetic, and physiological studies of these enzymes, both purified and in cells. They may also lead to useful antibacterial agents, since this class of enzymes is needed for successful bacterial infection of mammals.
doi:10.1021/cb300018x
PMCID: PMC3356449  PMID: 22443934
10.  In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3 
Abstract
Purpose
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1089/jop.2012.0135
PMCID: PMC3613967  PMID: 23098562
11.  Discovery, Design and Synthesis of Novel Potent and Selective Sphingosine-1-Phosphate 4 Receptor (S1P4-R) Agonists 
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.
doi:10.1016/j.bmcl.2011.10.096
PMCID: PMC3248976  PMID: 22119461
S1P4 receptor; selective small molecule S1P4–R agonists; autoimmune diseases; viral infections; thrombocytopenia
12.  Identification of Novel, Exosite-Binding Matrix Metalloproteinase-13 Inhibitor Scaffolds 
Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). Compounds that inhibit the metalloproteinase at the Zn binding site typically lack specificity among MMP family members. Analogs of the low-micromolar lead MMP-13 inhibitor 4, discovered through high-throughput screening, were synthesized to investigate structure activity relationships in this inhibitor series. Systematic modifications of 4 led to the discovery of MMP-13 inhibitors 20 and 24 which are more selective than 4 against other MMPs. Compound 20 is also approximately 5-fold more potent as an MMP-13 inhibitor than the original HTS-derived lead compound 4.
doi:10.1016/j.bmcl.2011.09.077
PMCID: PMC3210410  PMID: 22018790
13.  Discovery, synthesis and SAR analysis of novel selective small molecule S1P4–R agonists based on a (2Z,5Z)-5-((pyrrol-3-yl)methylene)-3-alkyl-2-(alkylimino)thiazolidin-4-one chemotype 
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.
doi:10.1016/j.bmcl.2011.09.049
PMCID: PMC3209756  PMID: 21982495
S1P4 receptor; selective small molecule S1P4–R agonists; thrombocytopenia; viral infections
14.  Identification of Small-Molecule Inhibitors of the Colorectal Cancer Oncogene Krüppel-Like Factor 5 Expression by Ultrahigh-Throughput Screening 
Molecular cancer therapeutics  2011;10(11):2043-2051.
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.
doi:10.1158/1535-7163.MCT-11-0550
PMCID: PMC3213326  PMID: 21885866
Colorectal cancer; KLF5; Ultrahigh-throughput screen; Luciferase; Cell viability; Small-molecule compounds
15.  Potent and Selective Inhibitors of Glutathione S-transferase Omega 1 that Impair Cancer Drug Resistance 
Journal of the American Chemical Society  2011;133(41):16605-16616.
Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents
doi:10.1021/ja2066972
PMCID: PMC3226709  PMID: 21899313
16.  Monitoring of HTS Compound Library Quality via a High-Resolution Image Acquisition and Processing Instrument 
Journal of laboratory automation  2011;16(3):197-203.
This report presents the high-resolution image acquisition and processing instrument for compound management applications (HIAPI-CM). The HIAPI-CM combines imaging spectroscopy and machine vision analysis to perform rapid assessment of HTS compound library quality. It has been customized to detect and classify typical artifacts found in HTS compound library microtiter plates (MTPs). These artifacts include: (a) insufficient volume of liquid compound sample, (b) compound precipitation, and (c) colored compounds that interfere with HTS assay detection format readout. The HIAPI-CM is also configured to automatically query & compare its analysis results to data stored in a LIMS or corporate database, aiding in the detection of compound registration errors. To demonstrate its capabilities, several compound plates (n =5760 wells total) containing different artifacts were measured via automated HIAPI-CM analysis, and results compared to those obtained by manual (visual) inspection. In all cases, the instrument demonstrated high fidelity (99.8% empty wells; 100.1% filled wells; 94.4% for partially filled wells; 94.0% for wells containing colored compounds), and in the case of precipitate detection, the HIAPI-CM results significantly exceeded the fidelity of visual observations (220.0%). As described, the HIAPI-CM allows for noninvasive, nondestructive MTP assessment with a diagnostic throughput of about one minute per plate, reducing analytical expenses and improving the quality and stewardship of HTS compound libraries.
doi:10.1016/j.jala.2011.02.004
PMCID: PMC3417353  PMID: 21609702
compound management; HTS library; machine vision; precipitate detection; colored compound; volume detection
17.  A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors 
Journal of the American Chemical Society  2011;133(30):11665-11674.
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.
doi:10.1021/ja2036095
PMCID: PMC3145007  PMID: 21692504
Prolyl peptidases; activity-based proteomics; fluopol; high-throughput screening; chemical inhibitors; Prolyl endopeptidase-like
18.  Small Molecule Proteostasis Regulators for Protein Conformational Diseases 
Nature Chemical Biology  2011;8(2):185-196.
Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging, and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting a promising therapeutic approach. We describe the results of a ∼900,000 small molecule screen that identified novel classes of small molecule proteostasis regulators (PRs) that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. The beneficial effects to proteome stability are mediated by HSF-1, DAF-16/FOXO, SKN-1/Nrf-2, and the chaperone machinery through mechanisms that are distinct from current known small molecule activators of the HSR. We suggest that modulation of the proteostasis network by PRs represents a promising therapeutic approach for the treatment of a variety of protein conformational diseases.
doi:10.1038/nchembio.763
PMCID: PMC3262058  PMID: 22198733
heat shock; high-throughput screening; small molecules; protein misfolding
19.  High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate 
Bioorganic & medicinal chemistry  2008;17(3):990-1005.
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.
doi:10.1016/j.bmc.2008.03.004
PMCID: PMC3298815  PMID: 18358729
20.  Potent, selective and cell penetrant inhibitors of SF-1 by functional uHTS 
Molecular pharmacology  2008;73(6):1776-1784.
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.
doi:10.1124/mol.108.045963
PMCID: PMC3228235  PMID: 18334597
21.  A High-Throughput Screening Compatible Assay for Activators and Inhibitors of Methionine Sulfoxide Reductase A 
Abstract
The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.
doi:10.1089/adt.2009.0263
PMCID: PMC2978062  PMID: 20515413
22.  An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors 
Journal of biomolecular screening  2010;15(8):907-917.
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.
doi:10.1177/1087057110375848
PMCID: PMC3082437  PMID: 20660794
Wee1; degradation; stabilizer; reporter assay; transient transfection; cryopreserved cells; ubiquitin; proteasome
23.  NH-1,2,3-Triazole-based Inhibitors of the VIM-2 Metallo-β-Lactamase: Synthesis and Structure-Activity Studies 
ACS medicinal chemistry letters  2010;1(4):150-154.
Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here are potent and selective small molecule inhibitors of VIM-2 containing the arylsulfonyl-NH-1,2,3-triazole chemotype that potentiate the efficacy of the ß-lactam, imipenem, in E. coli.
doi:10.1021/ml900022q
PMCID: PMC2901175  PMID: 20625539
24.  Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9) 
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.
doi:10.1016/j.bmcl.2010.02.011
PMCID: PMC2843764  PMID: 20207142
25.  Small molecule activators of TRPML3 
Chemistry & biology  2010;17(2):135-148.
Summary
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.
doi:10.1016/j.chembiol.2009.12.016
PMCID: PMC2834294  PMID: 20189104

Results 1-25 (31)