Radioligand binding assays in the drug discovery process: Potential pitfalls of high throughput screenings

Radioligand binding assays evaluating directly the ability of a drug to Int eract with a defined molecular target is part of the drug discovery process . The need for a high throughput rate in screening drugs Is actually leadin g to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of fre quent multiplicity of (interacting] binding sites. To clearly illustrate th is situation, three different assays used in the academic drug discovery pr ogram of the authors were selected because they are rich of intrinsic artif acts: II) (20 mmol/l caffeine almost duplicated [H-3]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mu mol/l free calcium but did not exert any effect when using a high (107 mu mol/l) free calcium, as mostly used in ryanodine binding assays; [II) Rn agonist for the ionotr opic glutamate receptor of the kainate type can distinctly affect [H-3]kain ate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/ l), had no effect (at 200 nmol/l) or even progressively decreased [at 0.3-2 mu mol/l) the binding of 5 nmol/l [H-3]kainate, emphasizing the risk of us ing a single concentration for screening a drug; (III) In a classical [3H]f lunitrazepam binding assay, the stimulatory effect of a GABA (gamma -aminob utyric acid) agonist was only observed when using extensively washed rat br ain synaptosomes (10 mu mol/l GABA increased flunitrazepam binding by 90%). On the other hand, the Inhibitory effect of a GABA antagonist was only obs erved when using crude synaptosomes (10 mu mol/l bicuculine reduced [3H]flu nitrazepam binding by 40%). It can be concluded that carefully designed rad ioligand assays which can be performed in an academic laboratory are approp riate for screening a small number of drugs, especially if these are potent ial hits because of their rational design. Therefore, the low throughput ra te could be partially balanced by a higher performance when compared to wha t is done in a robotic high throughput screening where simplification of as say conditions can lead to False negative results.