An iterative parallel synthesis effort identified a PLD2 selective inhibitor, ML298 (PLD1 IC50 >20,000 nM, PLD2 IC50 = 355 nM) and a dual PLD1/2 inhibitor, ML299 (PLD1 IC50 = 6 nM, PLD2 IC50 = 20 nM). SAR studies revealed a small structural change (incorporation of a methyl group) increased PLD1 activity within this classically PLD2-preferring core, and that the effect was enantiospecific. Both probes decreased invasive migration in U87-MG glioblastoma cells.
Phospholipase D; PLD1; PLD2; ML299; ML298; MLPCN probe
In this Letter, we describe a novel approach for the general and enantioselective synthesis of a diverse array of small to large 1-azabicyclo[m.n.0]alkyl ring systems with an embedded olefin handle for further functionalization. The stereochemistry is established via a highly diastereoselective indium-mediated allylation of an Ellman sulfinimine in greater than 9:1 dr., which is readily separable by column chromatography to afford a single diastereomer. This methodology allows for the rapid preparation of 1-azabicyclo[m.n.0]alkane ring systems that are not readily accessible through any other chemistry in excellent overall yields and, for many systems, the only enantioselective preparation reported to date.
azabicycle; olefin metathasis; enantioselective; allylation; alkaloid
This Letter describes the further optimization of an MLPCN probe molecule (ML137) through the introduction of 5- and 6- membered spirocycles in place of the isatin ketone. Interestingly divergent structure-activity relationships, when compared to earlier M1 PAMs, are presented. These novel spirocycles possess improved efficacy relative to ML137, while also maintaining high selectivity for the human and rat muscarinic M1 receptor subtype.
Muscarinic acetylcholine receptor 1; M1; Spirocyclic; Positive allosteric modulator (PAM); ML137; VU0413162
The medial prefrontal cortex (mPFC) serves executive cognitive functions such as decision-making that are impaired in neuropsychiatric disorders and pain. We showed previously that amygdala-driven abnormal inhibition and decreased output of mPFC pyramidal cells contribute to pain-related impaired decision-making (Ji et al., 2010). Therefore, modulating pyramidal output is desirable therapeutic goal. Targeting metabotropic glutamate receptor subtype mGluR5 has emerged as a cognitive-enhancing strategy in neuropsychiatric disorders, but synaptic and cellular actions of mGluR5 in the mPFC remain to be determined. The present study determined synaptic and cellular actions of mGluR5 to test the hypothesis that increasing mGluR5 function can enhance pyramidal cell output.
Whole-cell voltage- and current-clamp recordings were made from visually identified pyramidal neurons in layer V of the mPFC in rat brain slices. Both the prototypical mGluR5 agonist CHPG and a positive allosteric modulator (PAM) for mGluR5 (VU0360172) increased synaptically evoked spiking (E–S coupling) in mPFC pyramidal cells. The facilitatory effects of CHPG and VU0360172 were inhibited by an mGluR5 antagonist (MTEP). CHPG, but not VU0360172, increased neuronal excitability (frequency– current [F–I] function). VU0360172, but not CHPG, increased evoked excitatory synaptic currents (EPSCs) and amplitude, but not frequency, of miniature EPSCs, indicating a postsynaptic action. VU0360172, but not CHPG, decreased evoked inhibitory synaptic currents (IPSCs) through an action that involved cannabinoid receptor CB1, because a CB1 receptor antagonist (AM281) blocked the inhibitory effect of VU0360172 on synaptic inhibition. VU0360172 also increased and prolonged CB1-mediated depolarization-induced suppression of synaptic inhibition (DSI). Activation of CB1 with ACEA decreased inhibitory transmission through a presynaptic mechanism.
The results show that increasing mGluR5 function enhances mPFC output. This effect can be accomplished by increasing excitability with an orthosteric agonist (CHPG) or by increasing excitatory synaptic drive and CB1-mediated presynaptic suppression of synaptic inhibition (“dis-inhibition”) with a PAM (VU0360172). Therefore, mGluR5 may be a useful target in conditions of impaired mPFC output.
mGluR5; Positive allosteric modulator; PAM; CB1; Prefrontal cortex; Synaptic transmission
Utilizing a combination of high-throughput and multi-step synthesis, SAR in a novel series of M1 acetylcholine receptor antagonists was rapidly established. The efforts led to the discovery the highly potent M1 antagonists 6 (VU0431263), and 8f (VU0433670). Functional Schild analysis and radioligand displacement experiments demonstrated the competitive, orthosteric binding of these compounds; human selectivity data are presented.
Muscarinic acetylcholine receptor 1; M1 antagonist; VU0433670; VU0431263; Fluorination
Recent preclinical studies demonstrate a role for the prostaglandin E2 (PGE2) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antagonist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE2 subtype 3 (EP3) receptor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, displayed a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially novel therapeutic in the treatment of hypertension and diabetes mellitus.
Prostaglandin E2; EP1; EP3; Antagonist
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.
We previously reported the discovery of VU0364572 and
VU0357017 as M1-selective agonists that appear to activate
M1 through actions at an allosteric site. Previous studies
have revealed that chemical scaffolds for many allosteric modulators
contain molecular switches that allow discovery of allosteric antagonists
and allosteric agonists or positive allosteric modulators (PAMs) based
on a single chemical scaffold. Based on this, we initiated a series
of studies to develop selective M1 allosteric antagonists
based on the VU0364572 scaffold. Interestingly, two lead antagonists
identified in this series, VU0409774 and VU0409775, inhibited ACh-induced
Ca2+ responses at rat M1–5 receptor subtypes,
suggesting they are nonselective muscarinic antagonists. VU0409774
and VU0409775 also completely displaced binding of the nonselective
radioligand [3H]-NMS at M1 and M3 mAChRs with affinities similar to their functional IC50 values. Finally, Schild analysis revealed that these compounds inhibit
M1 responses through a fully competitive interaction at
the orthosteric binding site. This surprising finding prompted further
studies to determine whether agonist activity of VU0364572 and VU0357017
may also engage in previously unappreciated actions at the orthosteric
site on M1. Surprisingly, both VU0364572 and VU0357017
completely displaced [3H]-NMS binding to the orthosteric
site of M1–M5 receptors at high concentrations.
Furthermore, evaluation of agonist activity in systems with varying
levels of receptor reserve and Furchgott analysis using a cell line
expressing M1 under control of an inducible promotor was
consistent with an action of these compounds as weak orthosteric partial
agonists of M1. However, consistent with previous studies
suggesting actions at a site that is distinct from the orthosteric
binding site, VU0364572 or VU0357017 slowed the rate of [3H]-NMS dissociation from CHO-rM1 membranes. Together,
these results suggest that VU0364572 and VU0357017 act as bitopic
ligands and that novel antagonists in this series act as competitive
orthosteric site antagonists.
Acetylcholine; GPCR; allosteric; orthosteric; agonist; antagonist
The M1 muscarinic acetylcholine receptor is
to play an important role in memory and cognition, making it a potential
target for the treatment of Alzheimer’s disease (AD) and schizophrenia.
Moreover, M1 interacts with BACE1 and regulates its proteosomal
degradation, suggesting selective M1 activation could afford
both palliative cognitive benefit as well as disease modification
in AD. A key challenge in targeting the muscarinic acetylcholine receptors
is achieving mAChR subtype selectivity. Our lab has previously reported
the M1 selective positive allosteric modulator ML169. Herein
we describe our efforts to further optimize this lead compound by
preparing analogue libraries and probing novel scaffolds. We were
able to identify several analogues that possessed submicromolar potency,
with our best example displaying an EC50 of 310 nM. The
new compounds maintained complete selectivity for the M1 receptor over the other subtypes (M2–M5), displayed improved DMPK profiles, and potentiated the carbachol
(CCh)-induced excitation in striatal MSNs. Selected analogues were
able to potentiate CCh-mediated nonamyloidogenic APPsα release,
further strengthening the concept that M1 PAMs may afford
a disease-modifying role in the treatment of AD.
Muscarinic; acetylcholine; positive allosteric
modulator (PAM); ML169; Alzheimer’s disease
(AD); medium spiny neurons (MSNs); MLPCN
The first total synthesis of Aplidiopsamine A, a rare 3H-pyrrolo[2,3-c]quinolone alkaloid from the Aplidiopsis confluata, has been achieved following the proposed biosynthesis. This biomimetic synthesis requires only 5 steps and proceeds in 20.8% overall yield. Biological evaluation across large panels of discrete molecular targets identified that Aplidiopsamine A is a highly selective PDE4 inhibitor, a target for numerous CNS disorders.
An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.
Many orthosteric agonists differentially activate downstream effectors of GPCRs. Such defined induction of signaling has strongly supported the hypothesis termed ‘ligand-directed trafficking of receptor signaling’ (LDTRS). More recently, subtype-selective GPCR activators, such as allosteric agonists and positive allosteric modulators, have also exhibited the capacity to activate specific signaling pathways. Based on this finding, it may be possible to achieve ligand-specific receptor active states that optimize the biological responses specific to GPCRs. This review discusses recent studies in which both orthosteric and allosteric compounds have been demonstrated to induce LDTRS.
Allosteric agonist; GPCR; LDTRS; ligand-directed trafficking of receptor signaling; orthosteric agonist; positive allosteric modulator; receptor trafficking
This Letter describes the synthesis and structure–activity relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of a 1,3,8-triazaspiro[4,5]decan-4-one privileged structure, PLD inhibitors with nanomolar potency and an unprecedented 40-fold selectivity for PLD2 over PLD1 were developed. Interestingly, SAR for this diverged from our earlier efforts, and dual PLD1/2 inhibitors were also discovered within this series.
PLD; Phospholipase; Cancer; Privileged structure
Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents.
G-Protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle “molecular switches” that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites.
Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved.
Allosterism; AMPA; glycine; glutamate; NAMS; NMDA; PAMS; schizophrenia
This Letter describes the synthesis and structure–activity-relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of alternative halogenated piperidinyl benzimidazolone privileged structures, in combination with a key (S)-methyl group, novel PLD inhibitors with low nM potency and unprecedented levels of PLD1 isoform selectivity (~1700-fold) over PLD2 were developed.
Phospholipase D; Cancer; Isoform; PLD1; PLD2
This Letter describes the synthesis and SAR of two mGluR4 positive allosteric modulator leads, 6 and 7. VU001171 (6) represents the most potent (EC50 = 650 nM), efficacious (141% Glu Max) and largest fold shift (36-fold) of any mGluR4 PAM reported to date. However, this work highlights the challenges in hit-to-lead for mGluR4 PAMs, with multiple confirmed HTS hits displaying little or no tractable SAR.
mGluR4; PAM; Positive allosteric modulator; Parkinson’s Disease
The synthesis of phidianidines A and B, the first 1,2,4-oxadiazole-containing
alkaloid, from the marine opisthobranch mollusk Phidiana militaris is reported. The synthesis proceeds in six steps from known indole
acetic acids in 39.9% (phidianidine A) and 21% (phidianidine B) overall
yields from commercially available materials. Biological characterization
found that phidianidines A and B are selective inhibitors of DAT (versus
SERT and NET) and a selective, potent ligand and partial agonist of
the μ opioid receptor (versus δ- and κ-opioid receptors).
Moreover, neither phidianidines A and B are cytotoxic, and thus represent
an attractive starting point for chemical optimization; therefore,
we piloted a number of chemistries and prepared a diverse series of
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