B-973, a Novel α7 nAChR Ago-PAM: Racemic and Asymmetric Synthesis, Electrophysiological Studies, and <i>in Vivo</i> Evaluation

Garai, Sumanta; Raja, Krishnamohan S.; Papke, Roger L.; Deschamps, Jeffrey R.; Damaj, M. Imad; Thakur, Ganesh A.

doi:10.1021/acsmedchemlett.8b00407

Cited by 14 publications

(13 citation statements)

References 26 publications

(44 reference statements)

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“…In this tight pocket stereochemical constraints appear to be very important. This was shown to be the case for three functionally diverse analogs in the TQS family, TQS itself (Stokes et al, 2019), the ago-PAM 4BP-TQS (Papke et al, 2014;Thakur et al, 2013), and the allosteric antagonist presented in this work, 2,3,5,6TMP-TQS, as well as for the structurally unrelated ago-PAM B-973B (Garai et al, 2018;Quadri et al, MOL#119958 2019). Additionally, a recently published study of a family of sulfonamide-containing PAMs showed that the stereochemical orientation of side groups around a critical central cyclopropyl ring determine whether enantiomers function as Type I or Type II a7 PAMs (Harvey et al, 2019;Wang et al, 2020).…”

Section: Discussionsupporting

confidence: 56%

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

et al. 2020

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Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all of the desired activity comes from a single enantiomer. We have previously shown this to be the case with the a7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) TQS, and the a7 ago-PAM 4BP-TQS. 2,3,5,6TMP-TQS, previously published as a "silent allosteric modulator" and an antagonist of a7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type a7 and the non-orthosterically activatible C190A a7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast (+)2,3,5,6TMP-TQS proved to be an a7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of a7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive a4b2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with a7T106, and allosteric activation of a7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity.

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Section: Discussionsupporting

confidence: 56%

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

et al. 2020

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“…Acetylcholine chloride, atropine, and other chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO), and (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinoline-8-sulfonamide (GAT107) (Kulkarni and Thakur, 2013;Thakur et al, 2013), 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl) piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (Garai et al, 2018), and PNU-120596 (Williams et al, 2011a) were synthesized as described previously. Fresh ACh stock solutions were made in Ringer's solution each day of experimentation.…”

Section: Commercial Reagentsmentioning

confidence: 99%

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

Stokes

Garai

Kulkarni

et al. 2019

J Pharmacol Exp Ther

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Homomeric a7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by a7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to a7 at the 159 position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L159M) in heteromeric nAChR receptors containing a4 and b2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the a7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c] quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3yl)-urea. The presence of the mutation in the b2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the a4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant b subunit, regardless of the position of the mutant b subunit within the pentameric complex. Similar results were obtained when b2L159M was coexpressed with a2 or a3 and when the L159M mutation was placed in b4 and coexpressed with a2, a3, or a4. Functional receptors were not observed when b1L159M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the a4b2L159M receptor compared with a7 and the availability of high-resolution a4b2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of a7 subunits but are insensitive to PAMs, which greatly increase the open probability of a7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in a7. Specifically, the mutation of the TM2 159 leucine to methionine in a subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal b subunits allows heteromeric receptors to respond to select a7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

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“…Acetylcholine chloride (ACh), atropine, and other chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). GAT107 ((3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9btetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) (Kulkarni and Thakur, 2013;Thakur et al, 2013), (Garai et al, 2018), and PNU-120596 (N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea) (Williams et al, 2011a) were synthesized as described previously. All compounds were confirmed by NMR to have a purity ≥ 95%.…”

Section: Reagentsmentioning

confidence: 99%

“…PAMs like PNU-120596 and TQS can increase a7 channel activity by two or more orders of magnitude. While PAMs like PNU-120596 and TQS require ACh, choline, or an exogenous agonist such as nicotine to produce channel activation, other compounds, for example GAT107 and B-973B, have been identified as allosteric agonist-positive allosteric modulators (ago-PAMs) and are capable of producing high levels of allosteric activation without additional orthosteric agonists (Garai et al, 2018;Horenstein et al, 2016). It has been a concern however, that such strong enhancement in the activity of a receptor with high calcium permeability may actually present a risk for the therapeutic development of PAMs and ago-PAMs due to calcium overload (Williams et al, 2011b).…”

Section: Introductionmentioning

confidence: 99%

Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium

et al. 2020

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The currents of a7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of a7 receptors differ from normal a7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of a7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the ago-PAMs GAT107 and B-973B showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively compared to currents activated by ACh alone, indicative of reduced calcium permeability. TQS-potentiated currents were also linear and showed no calciumdependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically-activated a7 nAChR may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx.

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B-973, a Novel α7 nAChR Ago-PAM: Racemic and Asymmetric Synthesis, Electrophysiological Studies, and in Vivo Evaluation

Cited by 14 publications

References 26 publications

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium

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