Richard B. Rothman

Novel allosteric modulators of the dopamine transporter (DAT) have been identified. We have shown previously that SRI-9804 [N-(diphenylmethyl)-2-phenyl-4-quinazolinamine], SRI-20040 [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine], and SRI-20041 [N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine] partially inhibit [¹²⁵I]RTI-55 ([¹²⁵I]3β-(4′-iodophenyl)tropan-2β-carboxylic acid methyl ester) binding and [³H]dopamine ([³H]DA) uptake, slow the dissociation rate of [¹²⁵I]RTI-55 from the DAT, and allosterically modulate d-amphetamine–induced, DAT-mediated DA release. We synthesized and evaluated the activity of >500 analogs of these ligands and report here on 36 selected compounds. Using synaptosomes prepared from rat caudate, we conducted [³H]DA uptake inhibition assays, DAT binding assays with [³H]WIN35428 ([³H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane), and DAT-mediated release assays with either [³H]MPP⁺ ([³H]1-methyl-4-phenylpyridinium) or [³H]DA. We observed three groups of [³H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) full-efficacy agents with a two-site fit, and 3) partial-efficacy agents with a one-site fit—the focus of further studies. These agents partially inhibited DA, serotonin, and norepinephrine uptake, yet were much less potent at inhibiting [³H]WIN35428 binding to the DAT. For example, SRI-29574 [N-(2,2-diphenylethyl)-2-(imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine] partially inhibited DAT uptake, with an IC₅₀ = 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [³H]MPP⁺ in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC₅₀ or E_max value for DAT-mediated release of [³H]MPP⁺. These studies demonstrate the existence of potent DAT ligands that partially block [³H]DA uptake, without affecting DAT binding or d-amphetamine–induced [³H]MPP⁺ release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.

Several compounds have been identified that display low-efficacy, "partial substrate" activity. Here, we tested the hypothesis that the mechanism of this effect is a slower rate of induced neurotransmitter efflux than that produced by full substrates. Biogenic amine transporter release assays were carried out in rat brain synaptosomes and followed published procedures. [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) was used to assess release from dopamine (DA) and norepinephrine nerve terminals, whereas [(3)H]5-hydroxytryptamine (5-HT) was used to assess release from 5-HT nerve terminals. A detailed time-course evaluation of DA transporter (DAT)-mediated efflux was conducted by measuring the efflux of [(3)H]MPP(+) after the addition of various test compounds. In vivo microdialysis experiments compared the effects of the full substrates [(±)-1-(2-naphthyl)propan-2-amine (PAL-287) and (S)-N-methyl-1-(2-naphthyl)propan-2-amine (PAL-1046)], to that of a partial DAT/5-HT transporter substrate [(S)-N-ethyl-1-(2-naphthyl)propan-2-amine (PAL-1045)] on extracellular DA and 5-HT in the nucleus accumbens of the rat. The in vitro release assays demonstrated that partial substrate activity occurs at all three transporters. In the DAT efflux experiments, D-amphetamine (full substrate) promoted a fast efflux (K1 = 0.24 min(-1)) and a slow efflux (K2 = 0.008 min(-1)). For the partial DAT substrates, K1 = ∼0.04 min(-1), and K2 approximated zero. The in vivo microdialysis experiments showed that the partial substrate (PAL-1045) was much less effective in elevating extracellular DA and 5-HT than the comparator full substrates. We conclude that low-efficacy partial DAT substrates promote efflux at a slower rate than full substrates, and "partiality" reflects the ultra-slow K2 constant, which functionally limits the ability of these compounds to increase extracellular DA. We speculate that partial biogenic amine transporter substrates bind to the transporter but are less effective in inducing conformational changes required for reverse transport activity.

Evidence suggests that elevations in extracellular serotonin (5-HT) in the brain can diminish stimulant effects of dopamine (DA). To assess this proposal, we evaluated the pharmacology of amphetamine analogs (m-fluoroamphetamine, p-fluoroamphetamine, m-methylamphetamine, p-methylamphetamine), which display similar in vitro potency as DA releasers (EC₅₀ = 24–52 nM) but differ in potency as 5-HT releasers (EC₅₀ = 53–1937 nM). In vivo microdialysis was used to assess the effects of drugs on extracellular DA and 5-HT in rat nucleus accumbens, while simultaneously measuring ambulation (i.e., forward locomotion) and stereotypy (i.e., repetitive movements). Rats received two intravenous injections of drug, 1 mg/kg at time 0 followed by 3 mg/kg 60 min later. All analogs produced dose-related increases in dialysate DA and 5-HT, but the effects on DA did not agree with in vitro predictions. Maximal elevation of dialysate DA ranged from 5- to 14-fold above baseline and varied inversely with 5-HT response, which ranged from 6- to 24-fold above baseline. All analogs increased ambulation and stereotypy, but drugs causing greater 5-HT release (e.g., p-methylamphetamine) were associated with significantly less forward locomotion. The magnitude of ambulation was positively correlated with extracellular DA (p < 0.001) and less so with the ratio of DA release to 5-HT release (i.e., percentage DA increase divided by percentage 5-HT increase) (p < 0.029). Collectively, our findings are consistent with the hypothesis that 5-HT release dampens stimulant effects of amphetamine-type drugs, but further studies are required to address the precise mechanisms underlying this phenomenon.

The use of fenfluramines can increase the risk of developing pulmonary arterial hypertension (PAH) in humans, but the mechanisms responsible are unresolved. A recent study reported that female mice lacking the gene for tryptophan hydroxylase-1 (Tph1(−/−) mice) were protected from PAH caused by chronic dexfenfluramine, suggesting a pivotal role for peripheral serotonin (5-HT) in the disease process. Here we tested two alternative hypotheses which might explain the lack of dexfenfluramine-induced PAH in Tph1(−/−) mice. We postulated that: 1) Tph1(−/−) mice express lower levels of pulmonary 5-HT transporter (SERT) when compared to wild-type controls, and 2) Tph1(−/−) mice display adaptive changes in the expression of non-serotonergic pulmonary genes which are implicated in PAH. SERT was measured using radioligand binding methods, whereas gene expression was measured using microarrays followed by quantitative real time PCR (qRT-PCR). Contrary to our first hypothesis, the number of pulmonary SERT sites was modestly up-regulated in female Tph1(−/−) mice. The expression of 51 distinct genes was significantly altered in the lungs of female Tph1(−/−) mice. Consistent with our second hypothesis, qRT-PCR confirmed that at least three genes implicated in the pathogenesis of PAH were markedly up-regulated: Has2, Hapln3 and Retlna. The finding that female Tph1(−/−) mice are protected from dexfenfluramine-induced PAH could be related to compensatory changes in pulmonary gene expression, in addition to reductions in peripheral 5-HT. These observations emphasize the intrinsic limitation of interpreting data from studies conducted in transgenic mice that are not fully characterized.

The basal (constitutive) activity of G protein‐coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The μ‐opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist‐treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR‐CHO cells) were incubated for 20 h with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)‐9‐(benzoyloxy)‐2‐(3‐furanyl)dodecahydro‐6a,10b‐dimethyl‐4,10‐dioxo‐2H‐naphtho‐[2,1‐c]pyran‐7‐carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [³⁵S]‐GTP‐γ‐S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK‐treated hMOR‐CHO cells. All antagonists, including CTAP and 6β‐naltrexol, were inverse agonists. However, LTC‐274 ((−)‐3‐cyclopropylmethyl‐2,3,4,4α,5,6,7,7α‐octahydro‐1H‐benzofuro[3,2‐e]isoquinolin‐9‐ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [³⁵S]‐GTP‐γ‐S binding. Other efforts in this study identified KC‐2‐009 ((+)‐3‐((1R,5S)‐2‐((Z)‐3‐phenylallyl)‐2‐azabicyclo[3.3.1]nonan‐5‐yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK‐treated cells, KC‐2‐009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC‐2‐009) and a novel MOR antagonist (LTC‐274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC‐274 is a promising lead compound for developing a true MOR neutral antagonist. Synapse 64:280–288, 2010.

The prevalence of obesity in the United States is a major health problem associated with significant morbidity, mortality, and economic burden. Although obesity and drug addiction are typically considered distinct clinical entities, both diseases involve dysregulation of biogenic amine neuron systems in the brain. Thus, research efforts to develop medications for treating drug addiction can contribute insights into the pharmacotherapy for obesity. Here, we review the neurochemical mechanisms of selected stimulant medications used in the treatment of obesity and issues related to fenfluramine-associated cardiac valvulopathy. In particular, we discuss the evidence that cardiac valve disease involves activation of mitogenic serotonin 2B (5-HT_2B) receptors by norfenfluramine, the major metabolite of fenfluramine. Advances in medication discovery suggest that novel molecular entities that target 2 different neurochemical mechanisms, that is, “combination pharmacotherapy,” will yield efficacious antiobesity medications with reduced adverse side effects.

Background: The serotonin (5-HT) releasers (±)-fenfluramine and (+)-fenfluramine were withdrawn from clinical use owing to increased risk of valvular heart disease. One prevailing hypothesis (i.e.,...