High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

The described method is suitable for rapidly screening small and medium-sized sleep drugs. Currently, most mainstream high-throughput drug screening methods are based on biochemical and cellular levels. For example, the structure and properties of the receptor are examined to search for specific ligands that can bind to it²². Another approach involves analyzing the binding mode and strength of molecular fragments of selected drugs using Nuclear Magnetic Resonance (NMR) with mass spectrometry³⁵. However, these methods often have a relatively high screening error rate, and the drugs selected through them often show no effect in animal or clinical experiments. The efficacy of drugs in the body is influenced by various factors, such as drug absorption, distribution, metabolism, and excretion, leading to a high rate of false screening. In contrast, although our proposed method has a smaller screening scale compared to high-throughput methods, it offers a more straightforward and cost-effective approach by directly observing drug effects on phenotypes. This demonstrates the potential of using the Drosophila model for effective drug screening and identification of drug targets.

Drosophila possesses a conserved sleep regulation mechanism and exhibits sleep disturbances associated with aging. We observed that the sleep duration of 30-day-old female flies was significantly shorter than that of 7-day-old flies, while the sleep duration of 30-day-old male flies did not differ significantly from that of 7-day-old flies (Figure 6). Consequently, 30-day-old female flies were selected for the current experiments. The screening process in multiple rounds was conducted to minimize accidental factor interference. The drug concentration in the first round was set at 20 µM to avoid toxic side effects that could lead to fly mortality. In the second screening round, the drug concentration was increased to 50 µM to assess the effects of the drug at different concentrations. Drugs selected from the second round were administered to male flies at both 20 µM and 50 µM to evaluate sex differences in drug effects. This allowed one to screen for drugs that consistently demonstrated sleep-related effects. For instance, Reserpine has previously been shown to increase sleep in adult flies aged 4-6 days³¹. We successfully replicated this result in our model using older flies, where older females showed a significant increase in sleep after being administered Reserpine (Figure 5).

DMSO was used to dissolve the drugs, but its potential toxicity should be considered. Previous studies have shown that concentrations of 0.1% to 0.25% DMSO in the culture medium do not harm rat hair cells within 24 h, while concentrations of 0.5% to 6% significantly increase cell death³⁶. Similarly, it has been found that DMSO concentrations of 0.1% or less do not affect the expression of key drug metabolism-related enzymes or transporters in human hepatocytes. Still, higher concentrations can induce changes in expression³⁷. However, it should be noted that 0.1% DMSO has been found to significantly affect the lifespan of female flies but not males³⁸. Additionally, intraperitoneal administration of 15% and 20% DMSO has been shown to interfere with sleep in rats³⁹. To mitigate the potential toxicity of DMSO, we kept its concentration below 0.2%.

Currently, there are two main methods used to characterize the behavior of Drosophila. One method is based on video analysis, which provides a wealth of behavioral parameters, including fly position, speed, and subtle movements of body parts. The other method is based on infrared beam fracture, such as the DAM system.⁴⁰. However, it is important to note that certain video analysis tools like PySolo are designed for studying multiple single-resident flies, limiting the number of flies that can be placed under a camera⁴¹. Other tools like C-trax⁴² and JAABA⁴³ can perform population tracking but are computationally expensive and time-consuming. For high-throughput screening, capturing the overall sleep duration of flies is usually sufficient, and precise movement parameters are not necessary. Therefore, the widely used and highly scalable method based on infrared beam fracture is preferred. However, this method also has its limitations. For instance, if flies only move at one end of the tube without interrupting the infrared beam, the system may mistakenly record it as sleep, leading to an overestimation of sleep⁴⁴. Additionally, it is important to carefully test the motility of the fly strain before using it in screening to avoid unintended influences.

Here are some helpful tips for a successful setup: (1) To prevent food from sticking to the glass tube when removing it from the small beaker after solidification, one can try inserting the glass tube vertically into the bottom of the small beaker before the food solidifies. Gently pulling the glass tube back and forth, tapping the bottom of the beaker to allow air to enter, slowly rotating the beaker to remove all the food and the glass tube, and then carefully wiping off any remaining food on the outer wall of the glass tube can be effective. (2) When sealing the food end of the glass tube with paraffin film, it is recommended to use a water bath to slowly heat the film until the paraffin melts. This approach helps avoid the problem of the medicinal food splashing violently at high temperatures and contaminating the paraffin film. Alternatively, one can use small plastic caps for sealing, but ensure that air may enter during sealing, causing the food to push up overall. (3) It is worth considering that some potent sleep-promoting drugs may initially lead to the incorrect judgment of tested flies as dead. To overcome this issue, it is recommended to set a concentration gradient, allowing exploration of the optimal drug concentration and repeating the experiment. (4) Take into account that the odor of the drug may influence the amount of food consumed by the flies and their intake of the drug, potentially affecting the accuracy of the experimental results. Therefore, it can be beneficial to extend the duration of the experiment appropriately, ensuring that flies have ample time to consume as much drug as possible and enhancing the accumulation effect of the drug. (5) For data processing, while many universities and institutes have access to Matlab for public use, there are lower-cost alternatives available for individuals or research institutions that have not yet purchased the program. One recommended option is ShinyR-DAM v3.1 «Refresh»⁴⁵.

In conclusion, we have developed a step-by-step procedure for screening drugs to treat sleep disorders. Using an older fly model exhibiting a phenotype of shorter sleep duration, the efficacy of Reserpine in increasing sleep duration in older female flies is validated. This method offers a new approach to drug screening with significant application potential and serves as a foundation for further drug research. While drug effects are assessed based on phenotypes, the underlying mechanism of drug action remains unknown. Further studies will be conducted to investigate the pathology of sleep disorders and the molecular regulation of sleep, thereby shedding light on the pharmacological mechanisms involved. Although the circadian machinery in Drosophila bears similarities to human oscillators, differences in sleep control mechanisms between humans and flies should not be overlooked. This protocol provides a basic framework for drug screening for sleep disorders. However, future research will determine whether any of the screened drugs can be utilized for clinical treatment, as well as elucidate their mechanisms of action.