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Articles by C. David Weaver in JoVE

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High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

1Department of Pharmacology, Vanderbilt University School of Medicine, 2Department of Anesthesiology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine

JoVE 4209

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Other articles by C. David Weaver on PubMed

A Thallium-sensitive, Fluorescence-based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells

Potassium channels have been identified as targets for a large number of therapeutic indications. The ability to use a high-throughput functional assay for the detection and characterization of small-molecule modulators of potassium channels is very desirable. However, present techniques capable of screening very large chemical libraries are limited in terms of data quality, temporal resolution, ease of use, and requirements for specialized instrumentation. To address these issues, the authors have developed a fluorescence-based thallium flux assay. This assay is capable of detecting modulators of both voltage and ligand-gated potassium channels expressed in mammalian cells. The thallium flux assay can use instruments standard to most high-throughput screening laboratories, and using such equipment has been successfully employed to screen large chemical libraries consisting of hundreds of thousands of compounds.

A Novel High-throughput PepT1 Transporter Assay Differentiates Between Substrates and Antagonists

PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

A Novel Assay of Gi/o-linked G Protein-coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors

The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with G(i/o)-coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the Gbetagamma subunits of G(i) and G(o) heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRK-mediated thallium flux strategy will provide a novel tool to advance the study of G(i/o)-coupled GPCR biology and promote ligand discovery and characterization.

Discovery, Characterization, and Antiparkinsonian Effect of Novel Positive Allosteric Modulators of Metabotropic Glutamate Receptor 4

Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (+/-)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.

Initial SAR Studies on Apamin-displacing 2-aminothiazole Blockers of Calcium-activated Small Conductance Potassium Channels

An initial SAR study on a series of apamin-displacing 2-aminothiazole K(Ca)2 channel blockers is described. Potent inhibitors such as N-(4-methylpyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (13) are disclosed, and for select members of the series, the relationship between the observed activity in a thallium flux, a binding and a whole-cell electrophysiology assay is presented.

Preliminary SAR Studies on Non-apamin-displacing 4-(aminomethylaryl)pyrrazolopyrimidine K(Ca) Channel Blockers

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine K(Ca) channel blockers is described and their selectivity against K(Ca) channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.

Discovery and Characterization of Novel Allosteric Potentiators of M1 Muscarinic Receptors Reveals Multiple Modes of Activity

Activators of M(1) muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M(1)-active compounds has resulted in nonselective activation of the highly related M(2) to M(5) mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M(1) with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M(1) that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M(1) activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M(1) receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M(1) agonist, 1-[1'-(2-tolyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M(1)-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M(1) is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M(1) potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.

Small-molecule Screen Identifies Inhibitors of the Neuronal K-Cl Cotransporter KCC2

KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl(-) concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-Cl cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in clinical medicine because they inhibit the loop of Henle Na-K-2Cl cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for high-throughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. Here, we report the characterization of a small number of inhibitors, some of which inhibit KCC2 activity in the submicomolar range without substantially affecting NKCC1 activity. Using medicinal chemistry, we synthesized a number of variants, tested their effect on KCC2 function, and provide an analysis of structure/activity relationships. We also used one of the compounds to demonstrate competitive inhibition in regard to external [K(+)] versus noncompetitive inhibition in respect to external [Cl(-)].

Discovery of the First Highly M5-preferring Muscarinic Acetylcholine Receptor Ligand, an M5 Positive Allosteric Modulator Derived from a Series of 5-trifluoromethoxy N-benzyl Isatins

This report describes the discovery and initial characterization of the first positive allosteric modulator of muscarinic acetylcholine receptor subtype 5 (mAChR5 or M5). Functional HTS, identified VU0119498, which displayed micromolar potencies for potentiation of acetylcholine at M1, M3, and M5 receptors in cell-based Ca(2+) mobilization assays. Subsequent optimization led to the discovery of VU0238429, which possessed an EC(50) of approximately 1.16 microM at M5 with >30-fold selectivity versus M1 and M3, with no M2 or M4 potentiator activity.

Discovery and SAR of Novel MGluR5 Non-competitive Antagonists Not Based on an MPEP Chemotype

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and the first example of a mode of pharmacology 'switch' to provide PAMs with a non-MPEP scaffold.

Synthesis and Evaluation of a Series of Heterobiarylamides That Are Centrally Penetrant Metabotropic Glutamate Receptor 4 (mGluR4) Positive Allosteric Modulators (PAMs)

We report the synthesis and evaluation of a series of heterobiaryl amides as positive allosteric modulators of mGluR4. Compounds 9b and 9c showed submicromolar potency at both human and rat mGluR4. In addition, both 9b and 9c were shown to be centrally penetrant in rats using nontoxic vehicles, a major advance for the mGluR4 field.

High-throughput Screening Reveals a Small-molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1

The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K(+) (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.

Discovery and Development of a Potent and Highly Selective Small Molecule Muscarinic Acetylcholine Receptor Subtype I (mAChR 1 or M1) Antagonist in Vitro and in Vivo Probe

This article describes the discovery and development of the first highly selective, small molecule antagonist of the muscarinic acetylcholine receptor subtype I (mAChR1 or M(1)). An M(1) functional, cell-based calcium-mobilization assay identified three distinct chemical series with initial selectivity for M(1) versus M(4). An iterative parallel synthesis approach was employed to optimize all three series in parallel, which led to the development of novel microwave-assisted chemistry and provided important take home lessons for probe development projects. Ultimately, this effort produced VU0255035, a potent (IC(50) = 130 nM) and selective (>75-fold vs. M(2)-M(5) and > 10 microM vs. a panel of 75 GPCRs, ion channels and transporters) small molecule M(1) antagonist. Further profiling demonstrated that VU0255035 was centrally penetrant (Brain(AUC)/Plasma(AUC) of 0.48) and active in vivo, rendering it acceptable as both an in vitro and in vivo MLSCN/ MLPCN probe molecule for studying and dissecting M(1) function.

Discovery and SAR of 6-substituted-4-anilinoquinazolines As Non-competitive Antagonists of MGlu5

A high-throughput cell-based screen identified a series of 6-substituted-4-anilinoquinazolines as non-competitive antagonists of metabotropic glutamate receptor 5 (mGlu(5)). This Letter describes the SAR of this series and the profile of selected compounds in selectivity and radioligand binding assays.

Synthesis and SAR of Novel, Non-MPEP Chemotype MGluR5 NAMs Identified by Functional HTS

This Letter describes the discovery and SAR of three novel series of mGluR5 non-competitive antagonists/negative allosteric modulators (NAMs) not based on manipulation of an MPEP/MTEP chemotype identified by a functional HTS approach. This work demonstrates fundamentally new mGluR5 NAM chemotypes with submicromolar potencies, and further examples of a mode of pharmacology 'switch' to provide PAMs with a non-MPEP scaffold.

MAOS Ls for the General Synthesis and Lead Optimization of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines

General, high-yielding MAOS protocols for the expedient synthesis of functionalized 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines are described amenable to an iterative analog library synthesis strategy for the lead optimization of an M1 antagonist screening hit. Optimized compounds proved to be highly selective M1 antagonists.

Context-dependent Pharmacology Exhibited by Negative Allosteric Modulators of Metabotropic Glutamate Receptor 7

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323:147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[ 4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative cooperativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.

Identification of Metabotropic Glutamate Receptor Subtype 5 Potentiators Using Virtual High-Throughput Screening

Selective potentiators of glutamate response at metabotropic glutamate receptor subtype 5 (mGluR5) have exciting potential for the development of novel treatment strategies for schizophrenia. A total of 1,382 compounds with positive allosteric modulation (PAM) of the mGluR5 glutamate response were identified through high-throughput screening (HTS) of a diverse library of 144,475 substances utilizing a functional assay measuring receptor-induced intracellular release of calcium. Primary hits were tested for concentration-dependent activity, and potency data (EC(50) values) were used for training artificial neural network (ANN) quantitative structure-activity relationship (QSAR) models that predict biological potency from the chemical structure. While all models were trained to predict EC(50), the quality of the models was assessed by using both continuous measures and binary classification. Numerical descriptors of chemical structure were used as input for the machine learning procedure and optimized in an iterative protocol. The ANN models achieved theoretical enrichment ratios of up to 38 for an independent data set not used in training the model. A database of approximately 450,000 commercially available drug-like compounds was targeted in a virtual screen. A set of 824 compounds was obtained for testing based on the highest predicted potency values. Biological testing found 28.2% (232/824) of these compounds with various activities at mGluR5 including 177 pure potentiators and 55 partial agonists. These results represent an enrichment factor of 23 for pure potentiation of the mGluR5 glutamate response and 30 for overall mGluR5 modulation activity when compared with those of the original mGluR5 experimental screening data (0.94% hit rate). The active compounds identified contained 72% close derivatives of previously identified PAMs as well as 28% nontrivial derivatives of known active compounds.

Synthesis and SAR of Novel, 4-(phenylsulfamoyl)phenylacetamide MGlu4 Positive Allosteric Modulators (PAMs) Identified by Functional High-throughput Screening (HTS)

Herein we disclose the synthesis and SAR of a series of 4-(phenylsulfamoyl)phenylacetamide compounds as mGlu(4) positive allosteric modulators (PAMs) that were identified via a functional HTS. An iterative parallel approach to these compounds culminated in the discovery of VU0364439 (11) which represents the most potent (19.8 nM) mGlu(4) PAM reported to date.

Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Reveals Chemical and Functional Diversity and in Vivo Activity in Rat Behavioral Models of Anxiolytic and Antipsychotic Activity

Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have robust activity in animal models that predict efficacy in the treatment of CNS disorders.

Discovery and Characterization of Novel Subtype-selective Allosteric Agonists for the Investigation of M(1) Receptor Function in the Central Nervous System

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M(1)-M(5). Of the mAChR subtypes, M(1) is among the most heavily expressed in regions that are critical for learning and memory, and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M(1) and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M(1). Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M(1) allosteric agonists. These compounds activate M(1) with EC(50) values in the 150 nM to 500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M(1) receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M(1) allosteric agonists provides critical research tools to allow dissection of M(1)-mediated effects in the CNS and potential leads for novel treatments for Alzheimer's disease and schizophrenia.

Discovery of N-Aryl Piperazines As Selective MGlu(5) Potentiators with Efficacy in a Rodent Model Predictive of Anti-Psychotic Activity

This Letter describes the discovery, SAR and in vitro and in vivo pharmacological profile of a novel non-MPEP derived mGlu(5) positive allosteric modulator (PAM) based upon an N-aryl piperazine chemotype. This mGlu(5) chemotype exhibits the ability to act as either a non-competitive antagonist/negative allosteric modulator (NAM) or potentiator of the glutamate response depending on the identity of the amide substituent, i.e., a 'molecular switch'. A rapidly optimized PAM, 10e (VU0364289), was shown to be potent and specific for the rat mGlu(5) receptor and subsequently demonstrated to be efficacious in a clinically relevant rodent model predictive of anti-psychotic activity, thus providing the first example of a centrally active mGluR(5) PAM optimized from an HTS-derived mGluR5 competitive antagonist.

Development of a Selective Small-molecule Inhibitor of Kir1.1, the Renal Outer Medullary Potassium Channel

The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2'-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target.

Discovery and Optimization of a Novel, Selective and Brain Penetrant M1 Positive Allosteric Modulator (PAM): the Development of ML169, an MLPCN Probe

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M(1) PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M(1) PAM with an in vitro profile comparable to the prototypical M(1) PAM, BQCA, but with an improved brain to plasma ratio.

Identification and Optimization of Small Molecules That Restore E-cadherin Expression and Reduce Invasion in Colorectal Carcinoma Cells

E-cadherin is a transmembrane protein that maintains intercellular contacts and cell polarity in epithelial tissue. The down-regulation of E-cadherin contributes to the induction of the epithelial-to-mesenchymal transition (EMT), resulting in an increased potential for cellular invasion of surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors as a result of somatic mutations, chromosomal deletions, silencing of the CDH1 gene promoter, and proteolytic cleavage. To date, no compounds directly targeting E-cadherin restoration have been developed. Here, we report the development and use of a novel high-throughput immunofluorescent screen to discover lead compounds that restore E-cadherin expression in the SW620 colon adenocarcinoma cell line. We confirmed restoration of E-cadherin using immunofluorescent microscopy and were able to determine the EC(50) for selected compounds using an optimized In-Cell Western assay. The profiled compounds were also shown to have a minimal effect on cell proliferation but did decrease cellular invasion. We have also conducted preliminary investigations to elucidate a discrete molecular target to account for the phenotypic behavior of these small molecules and have noted a modest increase in E-cadherin mRNA transcripts, and RNA-Seq analysis demonstrated that potent analogues elicited a 10-fold increase in CDH1 (E-cadherin) gene expression.

Discovery, Synthesis, and Structure-activity Relationship Development of a Series of N-(4-acetamido)phenylpicolinamides As Positive Allosteric Modulators of Metabotropic Glutamate Receptor 4 (mGlu(4)) with CNS Exposure in Rats

Herein we report the discovery, synthesis, and evaluation of a series of N-(4-acetamido)-phenylpicolinamides as positive allosteric modulators of mGlu(4). Compounds from the series show submicromolar potency at both human and rat mGlu(4). In addition, pharmacokinetic studies utilizing subcutaneous dosing demonstrated good brain exposure in rats.

Development of a High Throughput Screen for Allosteric Modulators of Melanocortin-4 Receptor Signaling Using a Real Time CAMP Assay

The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.

Synthesis and SAR of Centrally Active MGlu5 Positive Allosteric Modulators Based on an Aryl Acetylenic Bicyclic Lactam Scaffold

This Letter describes the hit-to-lead progression and SAR of a series of biphenyl acetylene compounds derived from an HTS screening campaign targeting the mGlu(5) receptor. 'Molecular switches' were identified that modulated modes of pharmacology, and several compounds within this series were shown to be efficacious in reversal of amphetamine induced hyperlocomotion in rats after ip dosing, a preclinical model that shows similar positive effects with known antipsychotic agents.

Solution-phase Parallel Synthesis and SAR of Homopiperazinyl Analogs As Positive Allosteric Modulators of MGluâ‚„

Using a functional high-throughput screening (HTS) and subsequent solution-phase parallel synthesis approach, we have discovered a novel series of positive allosteric modulators for mGluâ‚„, a G-protein coupled receptor. This series is comprised of a homopiperazine central core. The solution-phase parallel synthesis and SAR of analogs derived from this series will be presented. This series of positive allosteric modulators of mGluâ‚„ provide critical research tools to further probe the mGluâ‚„-mediated effects in Parkinson's disease.

High-throughput Quantification of Bioactive Lipids by MALDI Mass Spectrometry: Application to Prostaglandins

Analysis and quantification of analytes in biological systems is a critical component of metabolomic investigations of cell function. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We introduce a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted metabolomes. Proof-of-concept is demonstrated by analysis of prostaglandins and glyceryl prostaglandins. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increases the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15-2000 fmol on plate) and improved linearity (r(2) = 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated.

Identification of ML204, a Novel Potent Antagonist That Selectively Modulates Native TRPC4/C5 Ion Channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Discovery, Synthesis, and Structure-activity Relationship Development of a Series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides (VU0400195, ML182): Characterization of a Novel Positive Allosteric Modulator of the Metabotropic Glutamate Receptor 4 (mGlu(4)) with Oral Efficacy in an Antiparkinsonian Animal Model

There is an increasing amount of literature data showing the positive effects on preclinical antiparkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu(4)). However, most of the data generated utilize compounds that have not been optimized for druglike properties, and as a consequence, they exhibit poor pharmacokinetic properties and thus do not cross the blood-brain barrier. Herein, we report on a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides with improved PK properties with excellent potency and selectivity as well as improved brain exposure in rodents. Finally, ML182 was shown to be orally active in the haloperidol induced catalepsy model, a well-established antiparkinsonian model.

Discovery, Characterization, and Structure-activity Relationships of an Inhibitor of Inward Rectifier Potassium (Kir) Channels with Preference for Kir2.3, Kir3.x, and Kir7.1

The inward rectifier family of potassium (Kir) channels is comprised of at least 16 family members exhibiting broad and often overlapping cellular, tissue, or organ distributions. The discovery of disease-causing mutations in humans and experiments on knockout mice has underscored the importance of Kir channels in physiology and in some cases raised questions about their potential as drug targets. However, the paucity of potent and selective small-molecule modulators targeting specific family members has with few exceptions mired efforts to understand their physiology and assess their therapeutic potential. A growing body of evidence suggests that G protein-coupled inward rectifier K (GIRK) channels of the Kir3.X subfamily may represent novel targets for the treatment of atrial fibrillation. In an effort to expand the molecular pharmacology of GIRK, we performed a thallium (Tl(+)) flux-based high-throughput screen of a Kir1.1 inhibitor library for modulators of GIRK. One compound, termed VU573, exhibited 10-fold selectivity for GIRK over Kir1.1 (IC(50) = 1.9 and 19 μM, respectively) and was therefore selected for further study. In electrophysiological experiments performed on Xenopus laevis oocytes and mammalian cells, VU573 inhibited Kir3.1/3.2 (neuronal GIRK) and Kir3.1/3.4 (cardiac GIRK) channels with equal potency and preferentially inhibited GIRK, Kir2.3, and Kir7.1 over Kir1.1 and Kir2.1.Tl(+) flux assays were established for Kir2.3 and the M125R pore mutant of Kir7.1 to support medicinal chemistry efforts to develop more potent and selective analogs for these channels. The structure-activity relationships of VU573 revealed few analogs with improved potency, however two compounds retained most of their activity toward GIRK and Kir2.3 and lost activity toward Kir7.1. We anticipate that the VU573 series will be useful for exploring the physiology and structure-function relationships of these Kir channels.

The Discovery and Characterization of ML218: A Novel, Centrally Active T-Type Calcium Channel Inhibitor with Robust Effects in STN Neurons and in a Rodent Model of Parkinson's Disease

T-type Ca(2+) channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca(2+) channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca(2+) channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620) a selective T-Type Ca(2+) (Ca(v)3.1, Ca(v)3.2, Ca(v)3.3) inhibitor (Ca(v)3.2, IC(50) = 150 nM in Ca(2+) flux; Ca(v)3.2 IC(50) = 310 nM and Ca(v)3.3 IC(50) = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-Type calcium current, inhibition of low threshold spike and rebound burst activity. Based on the basal ganglia circuitry in Parkinson's disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca(2+) channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A(2A) antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca(2+) function in vitro and in vivo, and freely available.

Functional Impact of Allosteric Agonist Activity of Selective Positive Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 in Regulating Central Nervous System Function

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

Iterative Experimental and Virtual High-throughput Screening Identifies Metabotropic Glutamate Receptor Subtype 4 Positive Allosteric Modulators

Activation of metabotropic glutamate receptor subtype 4 has been shown to be efficacious in rodent models of Parkinson's disease. Artificial neural networks were trained based on a recently reported high throughput screen which identified 434 positive allosteric modulators of metabotropic glutamate receptor subtype 4 out of a set of approximately 155,000 compounds. A jury system containing three artificial neural networks achieved a theoretical enrichment of 15.4 when selecting the top 2 % compounds of an independent test dataset. The model was used to screen an external commercial database of approximately 450,000 drug-like compounds. 1,100 predicted active small molecules were tested experimentally using two distinct assays of mGlu(4) activity. This experiment yielded 67 positive allosteric modulators of metabotropic glutamate receptor subtype 4 that confirmed in both experimental systems. Compared to the 0.3 % active compounds in the primary screen, this constituted an enrichment of 22 fold.

A Biased Ligand for OXE-R Uncouples Gα and Gβγ Signaling Within a Heterotrimer

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.

Further Optimization of the K-Cl Cotransporter KCC2 Antagonist ML077: Development of a Highly Selective and More Potent in Vitro Probe

Further chemical optimization of the MLSCN/MLPCN probe ML077 (KCC2 IC(50)=537 nM) proved to be challenging as the effort was characterized by steep SAR. However, a multi-dimensional iterative parallel synthesis approach proved productive. Herein we report the discovery and SAR of an improved novel antagonist (VU0463271) of the neuronal-specific potassium-chloride cotransporter 2 (KCC2), with an IC(50) of 61 nM and >100-fold selectivity versus the closely related Na-K-2Cl cotransporter 1 (NKCC1) and no activity in a larger panel of GPCRs, ion channels and transporters.

Optimization of an Ether Series of MGlu5 Positive Allosteric Modulators: Molecular Determinants of MPEP-site Interaction Crossover

We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu(5)) positive allosteric modulators (PAMs) based on a simple acyclic ether series. Modifications led to a gain of MPEP site interaction through incorporation of a chiral amide in conjunction with a nicotinamide core. A highly potent PAM, 8v (VU0404251), was shown to be efficacious in a rodent model of psychosis. These studies suggest that potent PAMs within topologically similar chemotypes can be developed to preferentially interact or not interact with the MPEP allosteric binding site.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

Benchmarking Ligand-Based Virtual High-Throughput Screening with the PubChem Database

With the rapidly increasing availability of High-Throughput Screening (HTS) data in the public domain, such as the PubChem database, methods for ligand-based computer-aided drug discovery (LB-CADD) have the potential to accelerate and reduce the cost of probe development and drug discovery efforts in academia. We assemble nine data sets from realistic HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods. Each data set is public domain through PubChem and carefully collated through confirmation screens validating active compounds. These data sets provide the foundation for benchmarking a new cheminformatics framework BCL::ChemInfo, which is freely available for non-commercial use. Quantitative structure activity relationship (QSAR) models are built using Artificial Neural Networks (ANNs), Support Vector Machines (SVMs), Decision Trees (DTs), and Kohonen networks (KNs). Problem-specific descriptor optimization protocols are assessed including Sequential Feature Forward Selection (SFFS) and various information content measures. Measures of predictive power and confidence are evaluated through cross-validation, and a consensus prediction scheme is tested that combines orthogonal machine learning algorithms into a single predictor. Enrichments ranging from 15 to 101 for a TPR cutoff of 25% are observed.

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