Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-β hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 absent PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.
Lipolysis involves interactions between ATGL, ABHD5, and PLIN1 or PLIN5. Sanders et al. identify synthetic ligands that quickly release ABHD5 from PLIN proteins to directly activate fat and muscle lipolysis, bypassing canonical PKA-dependent signaling. Long-chain acyl-CoAs act as endogenous allosteric ligand of ABHD5 to suppress lipolysis.
Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M4 (M4), we developed a homogenous, multiparametric, 1,536 well assay to measure M4 receptor agonism, positive allosteric modulation (PAM) and antagonism in a single well. This assay yielded a Z’ of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M1 and M5 subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M4 selective primary agonists, 1,450 PAMs and 2,389 antagonist hits. Concentration response analysis identified 25 selective agonists, 4 PAMs and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.
The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP suggesting that it is a valid target for new anti-malaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network (MLPCN) collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository (MLSMR)). A Cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from HTS. Two structurally related compounds, CID 6852389 and CID 23724194, yield micromolar potency and are inactive in CTSL1 titration experiments (IC50 >59.6 μM). As measured by Ki assay, both compounds demonstrate micromolar non-competitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrate potency in malaria growth assays (IC50 4 μM and 1.3 μM, respectively).
Malaria; Plasmodium falciparum; Aspartyl aminopeptidase; PfM18AAP; parasite; exopeptidase; 1536 well; QFRET
high throughput screen identified a novel chemotype
for the positive allosteric modulation (PAM) of the muscarinic acetylcholine
receptor (mAChR) subtype 5 (M5). Application of rapid analog,
iterative parallel synthesis efficiently optimized M5 potency
to arrive at the most potent M5 PAMs prepared to date and
provided tool compound 8n (ML380) demonstrating modest
CNS penetration (human M5 EC50 = 190 nM, rat
M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).
Of the five G-protein-coupled muscarinic acetylcholine receptors (mAChRs or M1-M5), M5 is the least explored and understood due to a lack of mAChR subtype selective ligands. We recently performed a high-throughput functional screen and identified a number of weak antagonist hits that were selective for M5. An iterative parallel synthesis and detailed molecular pharmacologic profiling effort, led to the discovery of the first highly selective, CNS penetrant M5 orthosteric antagonist tool compound, with submicromolar potency (hM5 IC50 = 450 nM, hM5 Ki = 340 nM, M1-M4 IC50s >30 μM), enantiospecific inhibition and an acceptable DMPK profile for in vitro and electrophysiology studies.
muscarinic; M5; antagonist; orthosteric; acetylcholine
We describe a phenotypic high throughput screening (HTS) calcium flux assay designed to identify pharmacoperones for the gonadotropin releasing hormone receptor (GnRHR). Pharmacoperones are target-specific, small molecules that diffuse into cells, rescue misfolded protein mutants, and restore them to function. Rescue is based on correcting the trafficking of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function correctly. This approach identifies drugs with a significant degree of novelty, relying on cellular mechanisms that are not currently exploited. Development of such assays is important, since the extensive use of agonist/antagonist screens alone means that useful chemical structures may be present in existing libraries but have not been previously identified using existing methods. Our assay utilizes cell lines stably expressing a GnRHR mutant under the control of a tetracycline (OFF) transactivator. This allows us to quantitate the level of functional and properly trafficked G protein coupled receptors present in each test well. Furthermore, since we are able to turn receptor expression on and off, we can rapidly eliminate the majority of false positives from our screening results. Our data show that this approach is likely to be successful in identifying hits from large chemical libraries.
Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.
Virtually all transcription factors partner with coactivators that
recruit chromatin remodeling factors and interact with the basal transcription
machinery. Coactivators have been implicated in cancer cell proliferation,
invasion and metastasis, including the p160 steroid receptor coactivator (SRC)
family comprised of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3
(AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent
candidate molecular targets for new chemotherapeutics. Here we report on the
results of a high throughput screening effort which identified the cardiac
glycoside bufalin as a potent small molecule inhibitor for SRC-3 and SRC-1.
Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer
cell growth at nanomolar concentrations. When incorporated into a nanoparticle
delivery system, bufalin was able to reduce tumor growth in a mouse xenograft
model of breast cancer. Our work identifies bufalin as a potentially
broad-spectrum small molecule inhibitor for cancer.
Natural products; screening strategies; nuclear receptors; structure and function; new targets
Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools we have developed high throughput screening (HTS) assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (LOPAC, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β. Kinetic studies revealed competitive (PPNDS) and mixed competitive/non-competitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin β active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin β reported to the date. These results demonstrate the ability of meprin α and β assays to identify selective compounds and discard artifacts of primary screening.
Meprin; zinc metalloproteinase; HTS; inhibitors; Actinonin; PPNDS; NF449; suramin
Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahaemolyticus, catalyzes the transfer of AMP on to the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization based high-throughput-screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g. calmidazolium, GW7647 and MK886) with Kis ranging from 6–50 µM and upwards of 30-fold selectivity versus HYPE, the only known human AMPylator.
The protein arginine deiminases (PADs)
catalyze the post-translational
hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process
termed deimination or citrullination. PADs likely play a role in the
progression of a range of disease states because dysregulated PAD
activity is observed in a host of inflammatory diseases and cancer.
For example, recent studies have shown that PAD2 activates ERα
target gene expression in breast cancer cells by citrullinating histone
H3 at ER target promoters. To date, all known PAD inhibitors bind
directly to the enzyme active site. PADs, however, also require calcium
ions to drive a conformational change between the inactive apo-state
and the fully active calcium bound holoenzyme, suggesting that it
would be possible to identify inhibitors that bind the apoenzyme and
prevent this conformational change. As such, we set out to develop
a screen that can identify PAD2 inhibitors that bind to either the
apo or calcium bound form of PAD2. Herein, we provide definitive proof
of concept for this approach and report the first PAD inhibitor, ruthenium
red (Ki of 17 μM), to preferentially
bind the apoenzyme.
Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R–S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.
S1P3 receptor; Allosteric agonist; Cardiovascular functions
A functional high throughput screen and subsequent multi-dimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with sub-micromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1–4 IC50 >30 μM), excellent multi-species PK, high CNS penetration, and enantiospecific inhibition.
Muscarinic acetylcholine receptor; M5; negative allosteric modulator (NAM); ML375; MLPCN probe
Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar non-covalent 3CLpro inhibitors retaining a single amide bond.
3CLpro; severe acute respiratory syndrome; SARS; MERS; coronavirus
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.
Muscarinic acetylcholine receptors; M5; Positive allosteric modulator (PAM); ML326
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.
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.
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.