Towards Functional Selectivity for α6®3γ2 GABAA Receptors: A Series of Novel Pyrazoloquinolinones (original) (raw)
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International Journal of Molecular Sciences
As a continuation of our study in the GABAA receptor modulators field, we report the design and synthesis of new 8-chloropyrazolo[1,5-a]quinazoline derivatives. Molecular docking studies and the evaluation of the ‘Proximity Frequencies’ (exploiting our reported model) were performed on all the final compounds (3, 4, 6a–c, 7a,b, 8, 9, 12a–c, 13a,b, 14–19) to predict their profile on the α1β2γ2-GABAAR subtype. Furthermore, to verify whether the information coming from this virtual model was valid and, at the same time, to complete the study on this series, we evaluated the effects of compounds (1–100 µM) on the modulation of GABAA receptor function through electrophysiological techniques on recombinant α1β2γ2L-GABAA receptors expressed in Xenopus laevis oocytes. The matching between the virtual prediction and the electrophysiological tests makes our model a useful tool for the study of GABAA receptor modulators.
GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one) elicits a strong and subtype-dependent enhancement of GABA-induced currents via a novel drug-binding site at extracellular ax+by- (x=1–6, y=1–3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA-induced currents of various GABAA receptor subtypes. Most of the compounds investigated were able to modulate GABA-induced currents of ab and abg receptors to a comparable extent, suggesting that the effect of these drugs is not dependent on the benzodiazepine site of GABAA receptors. Steric hindrance experiments demonstrated that these compounds exert their action predominantly via the ax+by- (x=1–6, y=1–3) interfaces. Whereas some compounds are unselectively modulating a broad range of receptor subtypes, other compounds feature remarkable functional selectivity for the a6b3g2 receptor, or behave as null modulators at some receptor subtypes investigated. Pyrazoloquinolinones and pyrazolopyridinones represent the first prototypes of drugs exerting benzodiazepine-like modulatory effects via the a+b- interface of GABAA receptors. The discovery of modulators with functional subtype selectivity at this class of binding sites provides a highly useful tool for the investigation of a6b2/3g2 receptor function, and may lead to novel therapeutic principles.
Synthesis of New GABAA Receptor Modulator with Pyrazolo[1,5-a]quinazoline (PQ) Scaffold
International Journal of Molecular Sciences, 2019
We previously published a series of 8-methoxypirazolo[1,5-a]quinazolines (PQs) and their 4,5-dihydro derivatives (4,5(H)PQ) bearing the (hetero)arylalkylester group at position 3 as ligands at the γ-aminobutyric type A (GABA A) subtype receptor. Continuing the study in this field, we report here the design and synthesis of 3-(hetero)arylpyrazolo[1,5-a]quinazoline and 3-(hetero)aroylpyrazolo[1,5-a]quinazoline 8-methoxy substituted as interesting analogs of the above (hetero)arylalkylester, in which the shortening or the removal of the linker between the 3-(hetero)aryl ring and the PQ was performed. Only compounds that are able to inhibit radioligand binding by more than 80% at 10 µM have been selected for electrophysiological studies on recombinant α1β2γ2L GABA A receptors. Some compounds show a promising profile. For example, compounds 6a and 6b are able to modulate the GABA A R in an opposite manner, since 6b enhances and 6a reduces the variation of the chlorine current, suggesting that they act as a partial agonist and an inverse partial agonist, respectively. The most potent derivative was 3-(4-methoxyphenylcarbonyl)-8-methoxy-4,5-dihydropyrazolo[1,5-a] quinazoline 11d, which reaches a maximal activity at 1 µM (+54%), and it enhances the chlorine current at ≥0.01 µM. Finally, compound 6g, acting as a null modulator at α1β2γ2L, shows the ability to antagonize the full agonist diazepam and the potentiation of CGS 9895 on the new α+/β− 'non-traditional' benzodiazepine site.
Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes
Scientific Reports
γ-Aminobutyric acid type A (GABA A) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular α+/β− interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced β1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent β1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of β1-containing receptor subtypes and the investigation of their abundance and distribution. GABA A receptors are pentameric ligand-gated ion channels that can be opened by GABA and alternative agonists, as well as modulated by multiple endogenous or exogenous allosteric ligands, some of which have high clinical importance 1. In the nervous system GABA A receptors are, among others, targets of certain sleeping aids, general anesthetics and antiepileptic medications. High affinity ligands of the benzodiazepine binding site of these receptors are also used as versatile CNS imaging tools 2. Specific receptor subtypes also occur in diverse peripheral tissues where their function is largely unknown 3, 4. A total of 19 genes encode, in mammalian species, GABA A receptor subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3) 5. Specific subunits assemble into homo-or hetero-pentameric arrangements, whereby a given pentamer with defined subunit composition and arrangement is referred to as receptor subtype. The receptor subtype composed of α1, β3 and γ2 subunits was shown to be arranged as β3-α1-γ2-β3-α1 6 , where each subunit interface by definition has a principal (plus) and a complementary (minus) side 7. The total number of pentameric arrangements that exist in mammalian species is still unknown 5 , but given the repertoire of 19 subunits, it could be large. The conserved cys-loop receptor structure harbors a large number of binding sites, including those for the generic agonist GABA, for channel blockers such as picrotoxin, and for a wide range of allosteric modulators 8. Each binding sites' ligand preferences are determined by the subunits that contribute to it. The ion channel pore is formed by the five transmembrane domain two segments (TM2) 9, 10. Agonist sites are at extracellular interfaces between specific subunits such as the bicuculline insensitive ρ+/ρ− and the bicuculline sensitive β+/α− sites 11. Allosteric sites have been described at interfaces and in other locations in the extracellular and transmembrane domains 8. Together, the staggering variety of receptor subtypes and the large number of binding sites on each subtype results in a highly complex pharmacology 1. Specific high affinity ligands of GABA A receptor subtypes are invaluable tools to study their abundance and distribution in tissues and to detect them in living organisms. Unselective
Tricyclic pyridones as functionally selective human GABAAα2/3 receptor-ion channel ligands
Bioorganic & Medicinal Chemistry Letters, 2004
A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the a 3 over the a 1 containing subtype of the human GABA A receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam. # 2004 Elsevier Ltd. All rights reserved.
Bioorganic Chemistry, 2012
Based on a pharmacophore model of the benzodiazepine binding site of the GABA A receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl-and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction between the hydrogen bond donor site H1 with the 3-carbonyl oxygen in 3-carbonylquinolin-4-ones can be replaced by an interaction between H1 and N-2 in the isothiazoloquinolin-4-ones, was confirmed. As with the 3-carbonylquinolin-4-ones, the length of the chain in position 3 is critical for an efficient interaction with the lipophilic pockets of the pharmacophore model. The most potent 3-alkyl derivative, 3-pentyl-6-methylisothiazoloquinolin-4-one, has an affinity (K i value) for the benzodiazepine binding site of the GABA A receptors of 13 nM. However, by replacing the 3-pentyl with a 3-butyramido group an even more potent compound was obtained, with a K i value of 2.8 nM, indicating that the amide function facilitates additional interactions with the binding site.
The Journal of pharmacology and experimental therapeutics, 2016
GABA-A receptors meet all the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6 dimethylcyclohexanol diastereomers were investigated on human γ-aminobutyric acid type A (GABA-A, α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis; trans,trans; and cis,trans isomers were isolated from commercially available 2,6 dimethylcyclohexanol and were tested for positive modulation of sub-maximal GABA responses. For example, the addition of 30 μM cis,cis isomer resulted in ~2-3 fold enhancement of the EC20 GABA current. Co-applications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > cis,trans isomer. In molecular modeling studies, the three cycl...
The GABAA Receptor a+/b- Interface: A Novel Target for Subtype Selective Drugs
GABAA receptors mediate the action of many clinically important drugs interacting with different binding sites. For some potential binding sites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular a1+b3- interface, which is homologous to the benzodiazepine binding site at the a1+g2- interface. On screening of >100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3–c]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents a a1b3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at a1b3g2 and a1b3 receptors. By mutating pocket-forming amino acid residues at the a1+ and the b3- side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect ofCGS9895. The inhibition was not caused by a general inactivation of GABAA receptors, because the GABA-enhancing effect of ROD 188 or the steroid a-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the a and b subunit types forming the interface. CGS9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the a+b- interface of GABAAreceptors. Since such binding sites are present at ab, abg, and abd receptors, such drugs will have a much broader action than benzodiazepines and might become clinical important for the treatment of epilepsy.