In recent years, there has been a resurgence of interest in the therapeutic potential of psychedelics, unlocking new possibilities in the treatment of mental health conditions. Here’s an overview of psychedelics, their importance in drug development, and how o2h discovery can contribute to this evolving field.

Understanding Psychedelics and their Connection to Mental Health

Psychedelics, such as psilocybin and LSD, are substances known for their mind-altering effects. Research suggests their potential in treating mental health conditions like depression, anxiety, and PTSD. These compounds act on serotonin receptors in the brain, influencing mood and perception. Today, the globe is experiencing an escalating mental health crisis. The NIMH (National Institute of Mental Health) in the United States estimates that the entire expenditures associated with serious mental illness, which affects around 6% of the adult population, exceed $300 billion each year.

The Role of Psychedelics in Drug Development

Hallucinogens such psilocybin and LSD, induce profound alterations of human emotion, and cognition. The discovery of the hallucinogenic effects of LSD and the observations that LSD and the endogenous ligand serotonin share pharmacological profiles led to the suggestion that serotonin was involved in the psychosis of mental disorders. Although they bind other G protein-coupled receptor (GPCR) subtypes, studies indicate that several effects of hallucinogens involve agonist activity at the serotonin 5-HT2A receptor.

Prototypical psychedelics activate heterotrimeric Gq stimulating intracellular release of Ca2+ and the receptor also interacts with β-arrestin proteins controlling the efficacy and duration of its signalling. The coupling mechanisms can vary from one brain region to another, differentially activating particular brain regions that may be associated with specific neurochemical, physiological, and behavioural responses. Understanding the biased mechanisms and outcome in the context of drug discovery for the 5HT receptor subtypes, provides a strategy to achieve superior therapeutic outcomes in treating neurological disorders.

How o2h discovery Supports Psychedelics Research?

At o2h, we have developed and screened a series of 5-HT2A-small molecules with varying potencies towards Gq and β-arrestin-biased signaling thererby shedding light on the dynamics associated with the 5-HT2A receptor. These serve as invaluable tools in elucidating the specific intracellular responses providing comprehensive understanding for drug discovery and pharmacological research.

o2h assay setup and cell lines:

• Our scientists can support in identifying compounds with differential signaling activities, downstream of the 5-HT receptor, stimulated via Gαq and beta-arrestin pathways
• Stable cell lines overexpressing the receptor of interest e.g. 5HT2A/2B/2C and for different species – human, rat and mouse can be developed in appropriate cellular background for screening. These cells can measure IP1 and Ca2+ as a stable downstream signal utilising FLIPR to address activation of the Gαq pathway
• PathHunter cell lines from Eurofins/Discoverx can be utilised for the screening of 5HT2A agonists, antagonists and partial agonists in bespoke assays with a transferable licence agreement with DiscoverX 
• These cells overexpress human 5-HT2A receptors and the β-arrestin2. These proteins are engineered to reconstitute β-galactosidase activity upon recruitment of β-arrestin2 to 5-HT2A receptor
• We have explored the PathHunter cell line for IP one measurements as well using HTRF-based IP1 kit from CisBio. A significant and reliable fold window is achieved for both IP1 and beta Arrestin with the PathHunter cell line, making it convenient to study biased signalling
• o2h can also support in exploratory studies using BRET-based protein-protein interactions and complementation assays (an area that is currently gaining interest). The study will involve cloning into appropriate vector systems (e.g. LgBiT-SmBiT), based on your biology of interest and customise them to your needs

Figure 1: (A) Chemiluminescent detection of β-Arrestin recruitment in U2OS PathHunter cells in response to 5-HT (serotonin). The increase in serotonin concentration results in elevated luminescence, demonstrating a dose-dependent β-arrestin recruitment to the 5-HT2A receptor. The analysis was performed using non-linear regression, agonist vs. response variable slope (four parameters).
(B) U2OS PathHunter cells stimulated with indicated concentration of compounds and assayed for β-Arrestin recruitment.
Compounds exhibit differential, dose-dependent increase in luminescence – indicating their ability to recruit β-Arrestin to 5-HT2A receptor

Figure 2: (A, B) Stably expressing 5HT2A cells stimulated with an Increasing concentration of serotonin, and measuring intracellular Ca2+ (by FLIPR) and IP1 levels (by HTRF kit) respectively
(C) Compounds classified as agonists exhibiting different potencies and efficacies

Figure 3: (A) Cells treated with increasing concentration of the antagonist ketanserin exhibiting reduced IP1 generation upon stimulation with EC80 concentration of 5-HT (serotonin)
(B, C) Screening of compounds for their antagonistic activity by measuring intracellular levels of IP1 and Ca2+ respectively

Figure 4: NanoBiT assay for the interaction of 5HT2a receptor with beta arrestin 2 (barr2). (A) Schematic representation of the NanoBiT assay. The 5HT2a receptor is C-terminally tagged with LgBiT whilst the barr2 is N-terminally tagged with SmBiT. The addition of native ligand 5HT (serotonin) causes an interaction of the two proteins and structural complementation of luciferase to generate a luminescent signal.
(B) Luminescence readings from 2 hr kinetic read on cells transfected with 5HT2aR-LgBiT and SmBiT-barr2. Solid blue lines represent samples with 1uM 5HT added at 0 mins, whilst dashed blue lines represent control samples with only buffer added. The total DNA amount transfected (5HT2aR and barr2 plasmids) was reduced as a 2-fold dilution and presented as a blue heatmap showing decreasing DNA amount.
(C) Relative area under the curve (AUC) of graphs in B. Note whilst the total DNA decreases, the fold change upon addition of 5HT remains consistent. Negative control is cells transfected with 5HT2aR-LgBiT and HaloTag-SmBiT which show no interaction upon 5HT addition.

Our commitment to advancing psychedelics research reflects our dedication to innovation in drug discovery services and leaving a positive impact on human health. For more information or collaboration opportunities, please reach out to us at discovery@o2h.com.