This article describes the discovery and development of the first highly selective, small molecule antagonist of the muscarinic acetylcholine receptor subtype I (mAChR1 or M1). An M1 functional, cell-based calcium-mobilization assay identified three distinct chemical series with initial selectivity for M1 versus M4. 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 (IC50 = 130 nM) and selective (>75-fold vs. M2-M5 and > 10 μM vs. a panel of 75 GPCRs, ion channels and transporters) small molecule M1 antagonist. Further profiling demonstrated that VU0255035 was centrally penetrant (BrainAUC/PlasmaAUC 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 M1 function.
muscarinic; acetylcholine; mAChR; M1; antagonist
functional multiaddition high throughput screen and
subsequent iterative parallel synthesis effort identified the first
highly selective and CNS penetrant glucagon-like peptide-1R (GLP-1R)
positive allosteric modulator (PAM). PAM (S)-9b potentiated low-dose exenatide to augment insulin secretion
in primary mouse pancreatic islets, and (S)-9b alone was effective in potentiating endogenous GLP-1R to
reverse haloperidol-induced catalepsy.
G-protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.
conditional knockout; GIRK; Kir3; morphine; rostromedial tegmental area; ventral tegmental area
Metabotropic glutamate receptor 7
(mGlu7) is a member
of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both
excitatory and inhibitory synapses, and activation of the receptor
regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological
and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia.
Here we report two new pan-group III mGlu positive allosteric modulators,
VU0155094 and VU0422288, which show differential activity at the various
group III mGlus. Additionally, both compounds show probe dependence
when assessed in the presence of distinct orthosteric agonists. By
pairing studies of these nonselective compounds with a synapse in
the hippocampus that expresses only mGlu7, we have validated
activity of these compounds in a native tissue setting. These studies
provide proof-of-concept evidence that mGlu7 activity can
be modulated by positive allosteric modulation, paving the way for
future therapeutics development.
Allosteric modulator; metabotropic glutamate
receptor; electrophysiology; hippocampus
Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.
High-throughput screening; glucagon-like peptide-1 receptor; calcium; positive allosteric modulator; agonist
allosteric modulators (PAMs) of metabotropic glutamate
receptor 5 (mGlu5) represent a promising therapeutic strategy
for the treatment of schizophrenia. Starting from an acetylene-based
lead from high throughput screening, an evolved bicyclic dihydronaphthyridinone
was identified. We describe further refinements leading to both dihydronaphthyridinone
and tetrahydronaphthyridine mGlu5 PAMs containing an alkoxy-based
linkage as an acetylene replacement. Exploration of several structural
features including western pyridine ring isomers, positional amides,
linker connectivity/position, and combinations thereof, reveal that
these bicyclic modulators generally exhibit steep SAR and within specific
subseries display a propensity for pharmacological mode switching
at mGlu5 as well as antagonist activity at mGlu3. Structure–activity relationships within a dihydronaphthyridinone
subseries uncovered 12c (VU0405372), a selective mGlu5 PAM with good in vitro potency, low glutamate fold-shift,
acceptable DMPK properties, and in vivo efficacy in an amphetamine-based
model of psychosis.
The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac©), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl+) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z′=0.75±0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC50 of ∼6 μM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble “loose patch” recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC50=10 μM), VU717 (IC50=6 μM), and structurally related calcium channel blocker prenylamine (IC50=6 μM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl+ flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels.
Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound's bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides.
Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical ‘toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.
Starting from a singleton chromanone high throughput screening (HTS) hit, we describe a focused medicinal chemistry optimization effort leading to the identification of a novel series of phenoxymethyl-dihydrothiazolopyridone derivatives as selective positive allosteric modulators (PAMs) of the metabotropic glutamate 5 (mGlu5) receptor. These dihydrothiazolopyridones potentiate receptor responses in recombinant systems. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) evaluation allowed us to select compound 16a for its assessment in a preclinical animal screen of possible antipsychotic activity. 16a was able to reverse amphetamine-induced hyperlocomotion in rats in a dose-dependent manner without showing any significant motor impairment or overt neurological side effects at comparable doses. Evolution of our medicinal chemistry program, structure activity, and properties relationships (SAR and SPR) analysis as well as a detailed profile for optimized mGlu5 receptor PAM 16a are described.
G-protein activated, inward-rectifying potassium (K+) channels,
“GIRKs”, are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research
interest for nearly two decades. GIRKs are comprised of various homo-
and heterotetrameric combinations of four different subunits. These
subunits are expressed in different combinations in a variety of regions
throughout the central nervous system and in the periphery. The body
of GIRK research implicates GIRK in processes as diverse as controlling
heart rhythm, to effects on reward/addiction, to modulation of response
to analgesics. Despite years of GIRK research, very few tools exist
to selectively modulate GIRK channels’ activity and until now
no tools existed that potently and selectively activated GIRKs. Here
we report the development and characterization of the first truly
potent, effective, and selective GIRK activator, ML297 (VU0456810).
We further demonstrate that ML297 is active in two in vivo models
of epilepsy, a disease where up to 40% of patients remain with symptoms
refractory to present treatments. The development of ML297 represents
a truly significant advancement in our ability to selectively probe
GIRK’s role in physiology as well as providing the first tool
for beginning to understand GIRK’s potential as a target for
a diversity of therapeutic indications.
GIRK; G-protein; inward rectifier; potassium channel; epilepsy; activator
This letter describes a multi-dimensional SAR campaign based on a potent, efficacious and selective GIRK1/2 activator (~10-fold versus GIRK1/4 and inactive on nonGIRK 1-containing GIRKs, GIRK 2 or GIRK2/3). Further chemical optimization through an iterative parallel synthesis effort identified multiple ‘molecular switches’ that modulated the mode of pharmacology from activator to inhibitor, as well as engendering varying selectivity profiles for GIRK1/2 and GIRK1/4. Importantly, these compounds were all inactive on nonGIRK1 containing GIRK channels. However, SAR was challenging as subtle structural modifications had large effects on both mode of pharmacology and GIRK1/2 and GIRK1/4 channel selectivity.
GIRK; Kir3.x; Activators; Inhibitors; Thallium flux
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.