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Abstract: The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [3H]nisoxetine. However, traditional [3H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [3H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [3H]CFT binding to whole cells (at physiological [Na+] and 25°C) were similar to potencies observed in the [3H]NE uptake assay. Inhibition constants (K i) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R 2 =0.99, p <0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [3H]nisoxetine binding to membrane preparations (4°C for 3h, [Na+] at 295mM), the potency of nisoxetine and desipramine in inhibiting [3H]CFT binding became greater than that measured in a functional assay of [3H]NE uptake at physiological [Na+]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [3H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [3H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter. [Copyright &y& Elsevier]

Abstract: In receptor binding assays with ultra-high-affinity radioligands, it is difficult, in practice, to adhere the golden rule that the receptor concentration in the assay should be substantially (at least 10-fold) lower than the dissociation constant (K d) of the radioligand and inhibition constant (K i) of compound. Especially for low specific activity radioligands (usually tritiated ligands of a couple of TBq/mmol), routinely applied in concentrations at around or below the K d, the use of extremely small amounts of receptor protein per assay will result in low levels of bound radioactivity; the alternative use of larger assay volumes will make it difficult to apply 96-well filtration devices. For assessing the inhibition constant (K i) of competitive inhibitors under conditions violating the above golden rule, equations are available incorporating both [receptor] and [ligand] versus K d; however, their application requires precise knowledge of [receptor] or initial bound/free [radioligand] ratio. In this study, we present the theoretical basis for determining the K i for a competitive inhibitor in a new protocol at high [protein] and high [radioligand] with the simple Cheng–Prusoff correction without the need to correct for [receptor] or initial bound/free [radioligand] ratio. In addition, we present results on the binding of the ultra-high-affinity ligand [3H]spiperone to dopamine D2 and D3 receptors validating the K i values calculated with the new protocol for competitive inhibitors as compared with those calculated with the most comprehensive equation available to date, that of . Binding was measured at varying [radioligand] and [receptor], test compounds (including (−)5-OH-DPAT, (±)7-OH-DPAT, and ropinirole) were run with varying [receptor], and simulations were done at vastly varying [radioligand] for inhibitors with vastly different K is. The modified high [radioligand] protocol presented here removes a major hindrance in the proper execution of binding assays with ultra-high-affinity tritiated ligands with K d values in the sub-nanomolar range, allowing the use of 96-well plates with small volumes of 100–200μl per binding assay. [Copyright &y& Elsevier]

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25°C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4°C for 3h, [Na(+)] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter., (Published by Elsevier B.V.)