1.1 Direct proof of melatonin binding

In order to define a new target of melatonin, a sine qua non condition is to provide direct proof of their physical interaction. An illustrative example of the long process of characterization of a new melatonin target is the NQO2 enzyme (formerly described as MT₃ binding site; Table 2). Several techniques such as radioligand binding, co-crystallization, isothermal titration calorimetry, equilibrium microdialysis, enzymatic activity, and NMR were applied to conclude on the nature of a new melatonin binding site⁵ (see Table 2 for details). The first and mandatory step in this process is the determination of the dissociation constant or equivalent (K_d/K_m) for melatonin binding. Commercially available and validated radioligands are 2-I[¹²⁵I]iodomelatonin⁸ or triply tritiated melatonin.^{9, 10}

1.2 Pharmacological characterization

Once the apparent affinity of melatonin for a biological sample is determined, the pharmacological profile of this “site” should be determined. The most straightforward way is to determine the capacity of a few analogue compounds to displace the radioligand in competition binding experiments. The choice of compounds will allow to compare the profile to established ones. Typical examples of compounds are found in the following pharmacological studies^{11, 12} and the latest IUPHAR review on melatonin pharmacology.⁴

1.3 Isolation and cloning of melatonin targets

In the case a previously unknown melatonin binding site is identified, the isolation of the corresponding protein or gene will be necessary to determine the nature of the target and to recapitulate the functional properties of the “binding site” previously characterized in a specific cell or tissue. Illustrative examples are the cloning/purification of melatonin receptors^13-15 and NQO2¹⁶ (see Table 2 for details). If the suspected molecular target is already available, experiments described in chapters 1-1 and 1-2 should be recapitulated with the recombinant protein or in cells transfected with the cDNA of the candidate protein.

2.1 In vitro experiments

Once a melatonin effect has been detected in a cellular system, a concentration-response curve has to be established. The range of concentrations should include the range of reported physiological melatonin concentrations, in any case below 0.1 µmol/L. Treatment of cells with melatonin should not to exceed concentrations higher than 1000 × K_d of the suspected target, unless justified by specific circumstances. When the K_d is unknown, no conclusion on the involvement of a particular target in an effect can be reached. Appropriate controls to rule out solvent effects are essential. The specificity of melatonin toward the observed effect should be tested with compounds characteristic for known melatonin targets including described antagonists and with biologically relevant compounds with structural similarity to melatonin, that is, indoles such as tryptophan, kynurenines, serotonin, or indole acetic acid. In case an effect is observed, EC₅₀ values have to be determined. The nec plus ultra proof that a compound is active through a particular protein target is to use cells or tissues either knockout or naturally devoid of the expression of the suspected melatonin target.

In the case that the suspected melatonin target corresponds to one of the already known targets (MT₁, MT_2, or NQO2), further indirect, functional evidence should be provided as confirmation. Examples of functional assays are the inhibition of intracellular cAMP levels, activation of the extracellular signal-regulated kinases 1/2, and beta-arrestin recruitment for melatonin receptors^{17, 18} or reactive oxygen species (ROS) production for NQO2.¹⁹ Use of known antagonist of the suspected target (ie, luzindole, 4P-PDOT, S20928 for melatonin receptors) and knockout animals/cells of known targets will help to define the specificity of the effect observed in these assays. In case the melatonin target is unknown, pharmacological inhibitors and targeted siRNA approaches can provide further information on the signaling pathways and the primary melatonin target involved. Melatonin effects observed at <0.1 µmol/L concentrations can be of physiological relevance, whereas effects observed at concentrations >0.1 µmol/L should be considered pharmacologically relevant. Use of very high (>100 µmol/L) melatonin concentrations is not recommended to avoid solubility (~2.5 mmol/L solubility limit of melatonin in water) and specificity problems.⁷ The effect of melatonin at concentrations equal or higher than 10 µmol/L should be shown on the viability of cells under the specific experimental conditions.

2.2 In vivo

In as much as cellular and in vitro validations of melatonin targets are essential, ultimate evidence to link melatonin effects to specific targets will require in vivo investigations, ideally using animal models with genetic deletion of the target. The first challenge is to reproduce the melatonin effect in an in vivo context. The choice of the melatonin dose to administer in vivo is crucial to draw reliable conclusions on the molecular target involved. Huge differences in doses of melatonin administration to rodents, ranging from µg/kg to up to 100 mg/kg, are observed in the literature,²⁰ making it difficult to compare different studies and conclusions. We suggest using a minimum of two to three melatonin doses in initial exploratory experiments before in-depth studies. Of note, maximal physiological plasmatic concentrations of melatonin are in the range of 50-100 pg/mL (210-420 pmol/L) at its peak during the night. If a study design aims to assess a precise melatonin effect that is expected to occur at physiological levels, and probably through melatonin receptors, we suggest a maximal dose of 3 mg/kg, as this is the highest dose reported to trigger the well-characterized phase-shift effect of melatonin on circadian rhythms in a receptor-dependent manner.²¹ This dose corresponds to a daily human equivalent dose of melatonin of 35 mg for a 75 kg adult calculated by normalization of body surface area.²² The time of melatonin administration in respect to the light/dark cycle should always be indicated.

Another frequently used method to provide melatonin in animal studies is to add melatonin in the drinking water during the night period only, in order to mimic the endogenous melatonin rhythm. In this respect, it has been shown that a dosage of 0.3 µg/mL is enough to restore nocturnal plasma melatonin levels in pinealectomized rats,²³ while 10 times higher dosage results in more than 2500 pg/mL of melatonin in the plasma.²⁴ Melatonin doses higher than 3 mg/kg or 0.3 µg/mL administrated intraperitoneally or in the drinking water, respectively, should thus be justified, as in the case of reaching a (validated) low-affinity melatonin target. In all cases, it is highly recommended to determine the amount of melatonin in the suspected target tissue (mg/Kg) or fluid (mg/mL) by sensitive techniques, as previously reported.²⁵ Data obtained with pharmacological tools, that is, luzindole or 4P-PDOT, have to be interpreted with care as their functional properties (antagonist vs partial agonist vs inverse agonist) may be dependent on the dose, the signaling pathway and the cellular context studied.