Cell surface receptors respond to a plethora of extracellular stimuli under several physiological or pathological conditions. Key components of the network, connect and diversify into multiple signal transduction pathways, further amplified by enzymes, cofactors and transcription factors. o2h scientists can quantitatively access drug-target engagement by measuring a variety of intra-cellular signaling events.

Some examples include cAMP, Ca²⁺, inositol phosphates, phosphoproteins, transcriptional regulation and sub-cellular protein translocation. These events can be sensitively captured using kit-based or bespoke assay designs utilizing different detection platforms like FLIPR, high-content imaging, western blotting, real time qPCR and plate readers.

As a case example, we have highlighted below our experience in GPCR biology wherein there might be an opportunity to screen compounds that are agnosts, partial agonists or antagonists for neuropsychiatric disorders that target serotonin receptors.

A)                                                                                       

Fig A: Chemiluminescent detection of β-Arrestin recruitment in U2OS cells in response to 5-HT (serotonin). Increase in serotonin leads to an increase in luminescence, indicating a dose-dependent β-arrestin recruitment to 5-HT2A receptor. Analysis performed using non-linear regression agonist vs response variable slope (four parameters).

B)                                                                                          C)

Fig (B, C): Cells stimulated with an Increasing concentration of 5-HT, serotonin measuring intracellular Ca2+ with FLIPR and IP1 levels  detected by HTRF.

D)

Fig (D): Cells treated with increasing concentration of the antagonist ketanserin exhibiting reduced IP1 generation upon stimulation with EC80 concentration of 5-HT (serotonin).

Serotonin 2A receptor (5-HT2A) plays a key role in mood regulation, cognition, and perception and is activated by the neurotransmitter serotonin. A prototypical psychedelics active Gq/11-mediated activation of PLC, leading to the formation of inositol phosphates, diacylglycerol, followed by Ca2+ release, and β-arrestin2 making their respective roles still unclear.

At o2h discovery, 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 discovery assay setup and cell lines

• Our o2h 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 down stream signal utilizing FLIPR to address activation of Gαq pathway.
• PathHunter-U2OS-HTR2A cell lines from Eurofins/Discoverx can be utilized for the screening of 5HT2A agonists, antagonists and partial agonists in bespoke assays with a transferable license agreement with DiscoverX.
• These cells overexpress human 5-HT2A receptor and the β-arrestin2. These proteins are engineered to reconstitute β-galactosidase activity upon recruitment of β-arrestin2 to 5-HT2A receptor.
• We have in the past 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.

To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.