Renee Sharon Martin

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Background and purpose:  Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain‐related diseases. The current work describes the in vitro pharmacological characteristics of AF‐353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist.

Effects of cannabinoid receptor agonists on neuronally-evoked contractions of urinary bladder tissues isolated from rat, mouse, pig, dog, monkey and human

This study investigated the cannabinoid receptor, known to inhibit neuronally‐evoked contractions of the mouse isolated urinary bladder, in bladder sections isolated from mouse, rat, dog, pig non‐human primate or human. The CB1‐like pharmacology of the cannabinoid receptor in mouse isolated bladder observed previously was confirmed in this study by the rank order of agonist potencies: CP 55940WIN 55212‐2>HU 210>JWH 015>anandamide, the high affinity of the CB1 selective antagonist, SR 141716A (apparent pKB 8.7), and the low affinity of the CB2 antagonist, SR 144528 (apparent pKB<6.5). In these studies, SR 141716A (10–100 nM) significantly potentiated electrically‐evoked contractions in this tissue by an undetermined mechanism. A similar rank order of agonist potencies was determined in rat isolated bladder sections (CP 55, 940WIN 55212‐2>JWH 015). In this tissue, the maximal inhibitory effect of all agonists was lower than in the mouse bladder. Indeed, the effects of both HU 210 and anandamide were too modest to quantify potency accurately. In the rat isolated bladder, SR 141716A (30 nM) or SR 144528 (100 nM), reversed the inhibitory effect of WIN 55212‐2 (apparent pKB=8.4 and 8.0, respectively) or JWH 015 (apparent pKB=8.2 and 7.4, respectively). These findings may demonstrate pharmacological differences between the rat and mouse orthologues of the CB1 receptor. Alternatively, they may be attributed to a mixed population of CB1 and CB2 receptors that jointly influence neurogenic contraction of the rat bladder, but cannot be differentiated without more selective ligands. WIN 55212‐2 had no effect on electrically‐evoked contractions of bladder sections isolated from dog, pig, cynomolgus monkey and human. These findings suggest that the effect of cannabinoid agonists to inhibit neurogenic contraction of the mouse and rat bladder is not conserved across all mammalian species.

Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism

Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099–3108, 1973). Two different SERT-selective radioligands, [3H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [3H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [3H]DASB and [3H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC50) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-β-carboxy-3-β-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.

Highly potent, non-basic 5-HT6 ligands. Site mutagenesis evidence for a second binding mode at 5-HT6 for antagonism.

A series of 5-HT(6) ligands derived from (R)-1-(amino)methyl-6-(phenyl)sulfonyltetralin was prepared that yielded several non-basic analogs having sub-nanomolar affinity. Ligand structure-activity relationships, receptor point mutation studies, and molecular modeling of these novel ligands all combined to reveal a new alternative binding mode to 5-HT(6) for antagonism.

Mechanism of subendocardial cell proliferation in the rat and relevance for understanding drug-induced valvular heart disease in humans

A number of drugs and drug candidates, including fenfluramine and ergot derivatives, are associated with valvulopathy in humans; however, these responses are poorly predicted from animal studies. In vitro and in vivo evidence suggests that these compounds exert their pathological effect through activation of serotonin 2B receptor (5HT2BR) signaling. However, the variable effect of fenfluramine and other 5HT2BR agonists in rodents has cast doubt on the relevance of animal findings to predicting human risk. Herein, a candidate compound, RO3013, induced subendocardial cell proliferation in the mitral and tricuspid valves in rats after only 3 days of daily dosing. Additionally, there was a treatment-related increase in immunostaining of the proliferation marker Ki67, and phosphorylated Smad3 in the heart indicative of TGFβ signaling co-localized with 5HT2BR expression. To substantiate the hypothesis that RO3013-induced valvular proliferation is secondary to 5HT2BR activation, the compound was evaluated in vitro and found to bind to the human 5HT2BR with a K(i) of 3.8μM; however, it was virtually devoid of agonist activity in a functional assay in human cells. By contrast, RO3013 bound to the rat 5HT2BR with a K(i) of 1.2μM and activated the receptor with an EC50 of 0.5μM. This agonist potency estimate is in good agreement with the free plasma concentrations of RO3013 at which valvular proliferation was observed. These results suggest that the rat may be susceptible to 5HT2BR-mediated valvular proliferation similar to humans; yet, the significant differences between binding and functional activities can be a possible explanation for the observed species-selective receptor responses.

Pharmacological Comparison of a Recombinant CB1 Cannabinoid Receptor with Its Ga16 Fusion Product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB1 agonist-mediated signal transduction via an engineered Gaα16 system is different than that of the Gi/o coupling normally preferred by the CB1 receptor, we transfected the human recombinant CB1 receptor (hCB1) or a fusion protein comprising the hCB1 receptor and Gα16 (hCB1-Gα16) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB1-Gaα16 fusion protein was found to be similar to that for hCB1: HU 210 > CP 55,940 ≥ SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [35S]GTPγS binding or inhibited forskolin-stimulated cAMP, presumably by coupling to Gi/o, in cells expressing hCB1 but not hCB1-Gα16. However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCBq-Gaα16 but not hCB1. These data demonstrate that ligand affinities for the hCB1, receptor are not affected by fusion to the Gα16 subunit. Furthermore, there is essentially no difference in the function of the hCB1, receptor when coupled to Gi/o, or Gα16 .

Characterization of human recombinant α2A-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes

Human α2A‐adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand‐induced extracellular acidification rate changes. In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 μM. In α2A‐adrenoceptor transfected cells the rank order of agonist potency was A‐54741 (mean pEC50=8.96)>dexmedetomidine (8.88)>UK‐14304 (8.42)>B‐HT 920 (7.05)>noradrenaline (6.92). A‐54741, UK‐14304 and noradrenaline had the same maximum response while dexmedetomidine and B‐HT 920 behaved as partial agonists. The selective α2‐adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pKB) against noradrenaline was 8.43. The selective α1‐adrenoceptor ligands prazosin and doxazosin (each 3 μM) had no effect on noradrenaline responses. Acidification rate changes induced by each agonist were abolished by pre‐treatment of cells with pertussis toxin. These data suggest that agonist‐induced acidification rate responses in CHL cells transfected with the human α2A‐adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive Gi/o proteins.