This Letter describes a novel series of GIRK activators identified through an HTS campaign. The HTS lead was a potent and efficacious dual GIRK1/2 and GIRK1/4 activator. Further chemical optimization through both iterative parallel synthesis and fragment library efforts identified dual GIRK1/2 and GIRK1/4 activators as well as the first examples of selective GIRK1/4 activators. Importantly, these compounds were inactive on GIRK2 and other non-GIRK1 containing GIRK channels, and SAR proved shallow.
GIRK; Kir3.x; Activators; Thallium flux
Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.
Biochemistry; Issue 71; Molecular Biology; Chemistry; Cellular Biology; Chemical Biology; Pharmacology; Molecular Pharmacology; Potassium channels; drug discovery; drug screening; high throughput; small molecules; fluorescence; thallium flux; checkerboard analysis; DMSO; cell lines; screen; assay; assay development
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 Ca2+ mobilization assays. Subsequent optimization led to the discovery of VU0238429, which possessed an EC50 of approximately 1.16 µM at M5 with >30-fold selectivity versus M1 and M3, with no M2 or M4 potentiator activity.
Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu5 PAM chemotype.
metabotropic glutamate receptor 5; mGlu5; positive allosteric modulator (PAM); non-MPEP
Current therapies to enhance CNS cholinergic function
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 KM with no change
in Vmax. 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.
Choline; transporter; hemicholinium-3; uptake; allosterism; assay; electrogenic
We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) 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.
Metabotropic glutamate receptor 5; mGlu5; Positive allosteric modulator (PAM); Non-MPEP
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 mGlu4 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.
Metabotropic glutamate receptor subtype 4; Virtual high-throughput screening; Machine learning; Quantitative structure-activity relationship; Enrichment
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.
virtual screening; machine learning; quantitative structure-activity relations (QSAR); high-throughput screening (HTS); cheminformatics; PubChem; BCL
Further chemical optimization of the MLSCN/MLPCN probe ML077 (KCC2 IC50 = 537 nM) proved to be challenging as the effort was characterized by steep SAR. However, a multidimensional 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 IC50 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.
Potassium-chloride co-transporter 2; KCC2; NKCC1; antagonist
This Letter describes the discovery, SAR and in vitro and in vivo pharmacological profile of a novel non-MPEP derived mGlu5 positive allosteric modulator (PAM) based upon an N-aryl piperazine chemotype. This mGlu5 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 mGlu5 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 mGluR5 PAM optimized from an HTS-derived mGluR5 competitive antagonist.
mGluR; potentiator; positive allosteric modulator; schizophrenia; hyperlocomotion
T-type Ca2+ 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 Ca2+ channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca2+ 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 Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 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 Ca2+ channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A2A antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca2+ function in vitro and in vivo, and freely available.
T-Type calcium channel; inhibitor; electrophysiology; Parkinson’s disease
T-Type Ca2+ channel inhibitors hold tremendous
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 Ca2+ channel inhibitors
free from IP constraints, and freely available through the MLPCN,
for use by the biomedical community to study T-type Ca2+ 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 Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM
in Ca2+ flux; Cav3.2 IC50 = 310 nM;
and Cav3.3 IC50 = 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 Ca2+ channel inhibitors, and ML218 was found to be orally efficacious
in haloperidol-induced catalepsy, a preclinical PD model, with comparable
efficacy to an A2A antagonist, a clinically validated PD
target. ML218 proves to be a powerful new probe to study T-type Ca2+ function in vitro and in vivo, and freely available.
T-Type calcium channel; inhibitor; electrophysiology; Parkinson’s disease
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 mGlu5 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 i.p. dosing, a preclinical model that shows similar positive effects with known antipsychotic agents.
There is an increasing amount of literature data showing the positive effects on preclinical anti-Parkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu4).1 However, most of the data generated utilize compounds that have not been optimized for drug-like 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 anti-Parkinsonian model.
metabotropic glutamate receptors; mGlu4; positive allosteric modulators; Parkinson’s disease; haloperidol-induced catalepsy; structure-activity relationship (SAR); oral efficacy; brain penetration
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 fmol – 2000 fmol on plate) and improved linearity (r2= 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated.
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 EC50 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.
The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 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 MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate 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= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR 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 an HTS for allosteric modulators for a Gs protein coupled receptor.
This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M1 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 M1 PAM with an in vitro profile comparable to the prototypical M1 PAM, BQCA, but with an improved brain to plasma ratio.
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 mGlu4, 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 mGlu4 provide critical research tools to further probe the mGlu4-mediated effects in Parkinson’s disease.
Herein we report the discovery, synthesis and evaluation of a series of N-(4-acetamido)-phenylpicolinamides as positive allosteric modulators of mGlu4.a Compounds from the series show submicromolar potency at both human and rat mGlu4. In addition, pharmacokinetic studies utilizing subcutaneous dosing demonstrated good brain exposure in rats.
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 (IC50 = 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.
GIRK; pharmacology; screening; thallium flux; fluorescence; electrophysiology; high throughput
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.
Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1-M5. Of the mAChR subtypes, M1 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 M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 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 M1 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 M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia.
mAChR; muscarinic; allosteric; agonist; cognition
This Letter describes the discovery and SAR of three novel series of mGluR5 noncompetitive 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.
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.