Spirocyclic replacements for the isatin in the highly selective,muscarinic M1 PAM ML137: The continued optimization of an MLPCN probe molecule

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.     

Discovery of ML326: The first sub-micromolar,selective M5 PAM

This Letter describes the further chemical optimization of the M₅ PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M₅ PAMs (M₅ EC₅₀ >10 μM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M₅ PAM, ML326 (VU0467903), (human and rat M₅ EC₅₀s of 409 nM and 500 nM, respectively) with excellent mAChR selectivity (M₁–M₄ EC₅₀s >30 μM) and a robust 20-fold leftward shift of the ACh CRC.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5 NAM with high CNS penetration and a desired short half-life in rat for addiction studies

This letter describes the continued optimization of M₅ NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t_1/2 = 80 h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M₅ NAM of comparable potency to ML375, but with a rat t_1/2 of less than 4 h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M₅ NAM, with high CNS penetration, excellent selectivity versus M_1–4 and the desired short half-life (t_1/2 = 2.3 h) in rat.     

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 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.     

Role of CREB in the regulatory action of sarsasapogenin on muscarinic M1 receptor density during cell aging

This work studied the role of cyclic AMP responsive element binding protein (CREB) in the up-regulation of M₁ muscarinic acetylcholine receptor (M₁ receptor) density by sarsasapogenin (ZMS) in CHO cells transfected with M₁ receptor gene (CHOm1 cells). During cell aging, sarsasapogenin elevated M₁ receptor density as well as CREB and phosphor-CREB (pCREB) levels. CREB peaked earliest, followed by pCREB and M₁ receptor density peaked last. When CREB synthesis was blocked by antisense oligonucleotides, the elevation effect of sarsasapogenin on M₁ receptor density was abolished. These results suggest that sarsasapogenin up-regulates M₁ receptor density in aged cells by promoting CREB production and phosphorylation. Furthermore, the results support the hypothesis that pCREB regulates M₁ receptor gene expression through heterodimer formation.     

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M₁-, M₃-, and M₅-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70 ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M₁- and M₅-acetylcholine receptors and the amplitude of these signals was larger at the M₁-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M₁-, M₃- and M₅-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.     

M1 Muscarinic Receptors Contribute to, whereas M4 Receptors Inhibit, Dopamine D1 Receptor-Induced [3H]-Cyclic AMP Accumulation in Rat Striatal Slices

In rat striatal slices labelled with [³H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [³H]-AMP ([³H]-cAMP) accumulation induced by the dopamine D₁ receptor agonist SKF-81297 (1 μM; 177±13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72±3%, IC₅₀ 0.30±0.06 μM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M₁ receptors, reduced the effect of SKF-81297 by 40±7% (IC₅₀ 251±57 pM) and enhanced the inhibitory action of a submaximal (1 μM) concentration of carbachol (69±4% vs. 40±7% inhibition, IC₅₀ 386±105 pM). The toxin MT-1, agonist at M₁ receptors, stimulated [³H]-cAMP accumulation in a modest but significant manner (137±11% of basal at 400 nM), an action additive to that of D₁ receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D₁ receptor-induced [³H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M₄ receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M₁ receptors and PKC stimulation.     

AGAP1/AP‐3‐dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M₅ is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M₅ and the AP‐3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP‐3 to M₅. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M₅ in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP‐3 or abrogation of AGAP1–M₅ interaction in vivo decreased the magnitude of presynaptic M₅‐mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor‐recycling pathway that may underlie mechanisms of G‐protein‐coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.     

Selective inactivation of muscarinic receptor subtypes

Muscarinic receptors exist in multiple subtypes, denoted as M₁, M₂ M₃ and M₄, encoded by four distinct but related genes. A fifth gene product, m5, has also been predicted although this sequence awaits a pharmacological equivalent. Many tissues express more than one muscarinic receptor subtype, which may couple to different intracellular effectors and thus have different physiological roles. One way to characterize the role of each receptor is to selectively inactivate one receptor population, thus pharmacologically ‘isolating’ the muscarinic receptor subtype of interest. Selective receptor inactivation can be achieved using either a selective, irreversible antagonist, or protection using a selective, reversible antagonist against a non-selective irreversible antagonist. Therefore, combination of these two approaches may provide optimal selective inactivation. Several muscarinic alkylating agents have been identified, including phenoxybenzamine, EEDQ (N-Ethoxycarbonyl-1-ethoxy-1,2-dihydroquinoline) and propylbenzilylcholine mustard. These irreversible antagonists do not, in general, discriminate between muscarinic receptor subtypes and are frequently used to estimate the affinity and relative efficacy of muscarinic agonists. Consequently, use of these irreversible antagonists provides estimations of the ‘receptor reserve’ associated with a response mediated by muscarinic receptor activation. In contrast, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)piperidine) selectively inactivates M₃ receptors, but will not discriminate between M₁ M ₂ or M₄ receptors. In the absence of highly selective alkylating agents, receptor protection by reversible antagonists may be used. Thus, reversible antagonists, such as pirenzepine, methoctramine or para-fluorohexahydrosiladifenidol, at appropriate fractional receptor occupancies, may protect M₁ M₂ or M₃ receptors against alkylation by phenoxybenzamine. Selective alkylation of M₃ receptors by 4-DAMP mustard is enhanced with concurrent M₂ protection. This approach has been applied to defining the role of these muscarinic receptor subtypes in the control of ileal smooth muscle tone. These data suggest that, in ileum, M₂ receptors may act to inhibit β-adrenoceptor activation, thereby offsetting relaxation, while M₃ receptors directly mediate contraction.     

Monovalent-ion carrier effects on transport of Rb86 and Cs137 into bush bean plants

Summary Bush bean plants were exposed to either Rb⁸⁶ or Cs¹³⁷ for 24 hours with different monovalent cations as carriers in single-salt solutions except for the presence of 10⁻⁴ M CaCl₂. Ratio of uptake of the radionuclides at 10⁻³ to 10⁻² M was used as an index of the carrier ability of various cations. Different monovalent cations decreased uptake of Cs¹³⁷ and its transport to shoots unequally when 10⁻² M salts were compared with 10⁻³ M salts. Rubidium and cesium salts decreased Cs¹³⁷ uptake equally but potassium salts were less effective in decreasing uptake when the ratios of the two concentrations were considered. All monovalent cations decreased uptake of Cs¹³⁷ at the 10⁻² M carrier concentration but some did not at 10⁻³ M. Nitrate nitrogen was a big factor in these results. Cesium and rubidium salts were most effective. Potassium appeared to increase Cs¹³⁷ transport to shoots particularly at 10⁻³ M KNO₃. Only cesium, rubidium, and potassium salts decreased uptake of Rb⁸⁶ when 10⁻² M salts were compared with 10⁻³ M. Rubidium and cesium salts decreased uptake essentially equally and potassium salts again were less effective. All nitrate salts tended to increase Rb⁸⁶ transport to shoots more consistently than with Cs¹³⁷. It is concluded that absorption and transport to shoots were not equivalent for potassium, rubidium, and cesium.     

The allosteric site regulates the voltage sensitivity of muscarinic receptors

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein–coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M₁-Rs and M₃-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G_q protein cycle. In the presence of ACh, M₁-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M₃-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed “allosteric site” M₃/M₁-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M₃-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.     

2′ Biaryl amides as novel and subtype selective M1 agonists. Part I: Identification,synthesis, and initial SAR

Biaryl amides were discovered as novel and subtype selective M₁ muscarinic acetylcholine receptor agonists. The identification, synthesis, and initial structure–activity relationships that led to compounds 3j and 4c, possessing good M₁ agonist potency and intrinsic activity, and subtype selectivity for M₁ over M_2–5, are described.     

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M<Subscript>4</Subscript> Receptor

The five muscarinic acetylcholine receptors (M₁–M₅) are differentially expressed in the brain. M₂ and M₄ are coupled to inhibition of stimulated adenylyl cyclase, while M₁, M₃ and M₅ are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2—affinity order M₁ ≥ M₄ >> others—and MT3—highly selective M₄ antagonist—did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M₂, M₄ would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M₄ receptor would be responsible for this regulation. Diana Jerusalinsky and Edgar Kornisiuk contributed equally to this paper.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

Melatonin Receptor-Mediated Stimulation of Phosphoinositide Breakdown in Chick Brain Slices

Abstract: The direct effect of melatonin and related agonists on Li⁺-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-³H]-inositol. The melatonin receptor agonist 6-chloromelatonin (10–100 µM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 µM) was rapid as transient increases in IP₃/IP₄ (maximal increase, 29% at 20 s) and IP₂ levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP₁ level (30% at 5 min), when the amount of IP₃/IP₄ and IP₂ had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 µM) in isolated myo-[2-³H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5′-O-(3-thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration-dependent manner the IP₁ accumulation stimulated by 6-chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5-hydroxytryptamine (10 µM)-, but not 6-chloromelatonin (10 µM)-, induced IP₁ accumulation. Melatonin receptor agonists increased IP₁ accumulation in a concentration-dependent manner reaching different maximal responses. N-Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP₁ accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5-hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.     

Roles of M3 receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Background: This study is to investigate the roles of muscarinic receptor 3 (M₃ receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC).

Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M₃ receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR.

Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M₃ receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M₃ receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M₃ receptor shRNA groups.

Conclusion: These results indicate that M₃ receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M₃ receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M₃ receptor.     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.