MS Binding Assays for D₁and D₅Dopamine Receptors

Abstract

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol) was established and validated, using its 8-bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well-suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.

Development and validation of an LC‐ESI‐MS/MS method for the quantification of D‐84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET

“…In MS Binding Assays as well as in radioligand binding assays, separation of the target–marker complexes from nonbound marker is typically achieved by filtration (Chen et al, ; Grimm et al, ; Hess et al, ; Neiens et al, ; Zepperitz et al, ; R. Zhang & Xie, ). In contrast to radioligand binding assays, where the whole filter with the remaining target material and the bound marker is subjected to measurement of radioactivity, in MS Binding Assays the target‐bound marker remaining on the filter has to be eluted by means of an organic solvent to enable its quantification by LC–MS (see also Figure ).…”

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors

“…For the development of simultaneous MS Binding Assays using SCH23390 and raclopride as markers for the D 1 and the D 2 receptor, respectively, an LC–MS/MS method capable to quantify both markers at the low picomolar concentration level is required. Unfortunately, we were not able to quantify both raclopride and SCH23390 together at the required concentration level with the LC–ESI‐MS/MS method using a C 8 stationary phase recently developed for quantification of SCH23390 as a marker for D 1 and D 5 receptors in corresponding MS Binding Assays (data not shown) . Because previously published LC–MS methods for raclopride quantification as well were by far not sensitive enough for the intended purpose, we started to develop a new LC–ESI‐MS/MS method based on pneumatically assisted electrospray ionization (ESI) in the positive mode recording the known mass transitions of m / z 288/91 for SCH23390 and m / z 347/112 for raclopride with a standard performance triple quadrupole mass spectrometer (API3200, ABSciex).…”

“…In detail, SCH23390 shows a D 1 vs. D 2 subtype selectivity of about 5000, whereas for raclopride the corresponding D 2 vs. D 1 subtype selectivity is even at about 10000 . Additionally, for both compounds sensitive quantification by LC–MS/MS has already been demonstrated . Finally, it is worth to note that SCH23390 has already been successfully used as a non‐labeled marker in MS Binding Assays addressing the D 1 and the D 5 subtype and that raclopride has already been used to label D 2 receptors in vivo …”

“…The sensitivity of this method had to be high enough to monitor the binding of the markers toward their corresponding targets under conditions as they are typically applied in radioligand binding assays. We recently estimated the lowest concentration of bound marker to be quantified in saturation experiments to be <0.0091 of a marker's equilibrium dissociation constant K d . As the K d values published for [ 3 H]SCH23390 and [ 3 H]raclopride determined in radioligand binding assays addressing D 1 and D 2 receptors, respectively, are about 1 n m , the lowest concentration of bound marker to be quantified in saturation experiments can be assumed to be in the low picomolar range.…”

Determination of the enantiomeric purity of the selective dopamine transporter inhibitor (+)‐R,R‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol

“…As a powerful alternative to these radioligand binding assays, MS Binding Assays have been developed by our group. [14][15][16][17][18] In DAT radioligand binding assays, typically 23,24 and is therefore used in combination with agents blocking the binding to NET and SERT. 22 Also in MS Binding Assays, the reporter ligand represents an essential tool.…”