They are integral parts of the entire drug discovery process from target validation, high throughput screening (HTS), lead optimization, preclinical and clinical development to NDA or BLA approval. They are used to measure the activity of a compound on an intended therapeutic target, or toxicity effect on an undesired off-target protein.
Often these assays are developed in vitro for accessibility and lower cost than animal based assays. Two key criteria for an assay are 1) to reflect the biology of the target in vivo and 2) in high throughput. In cell-based assays, the target activity can be measured in physiologically relevant host cells and signaling pathways. Therefore, live cell-based assays are strongly preferred over protein-based biochemical assays. However, it is challenging to develop cell-based assays that are amenable for high throughput screening.
Cell-based assay development service is among Multispan’s highly-valued offerings. A series of decisions need to be made before each assay development project. What cell line to use? What readout to use? What biological event to measure? What mode of action to measure?
Multispan combines cell engineering, assay development and HTS experience to succeed. 2 main categories of cell-based assays are routinely developed by the Multispan team, based on the cell types used: either primary cells expressing endogenous protein target or heterologous recombinant stable cell lines.
We have developed various HTS assays in hepatocytes, hiPSC derived microglial cells, human ventricle interstitial cardiomyocytes (hVIC), primary fibroblasts, PBMC, mouse islet cells, etc. In case of the 2nd category, we employ Multispan’s highly characterized MULTISCREENTM stable cell lines, designed, generated and QC’ed for high throughput screening assays. These assays are primarily cell signaling focused.
Here are the types of assays we have developed based on readout: ELISA, cell-based ELISA, reporter, binding, 2nd messenger, flow cytometry (FACS) in fluorescence, chemiluminescence, TR-FRET, absorbance, and radioisotope.
We have successfully applied these technologies in developing assays for serine/threonine kinases, receptor tyrosine kinases, nuclear hormone receptors, transporters, ion channels, etc. For G-protein coupled receptor (GPCR) targets, the specific assays include: Calcium, cAMP, β-arrestin, pGRK2, IP-1, pERK, pNFκB, radioligand binding, GTPγS, chemotaxis, cytokine secretion, insulin secretion, cell proliferation, luciferase reporter and internalization.
Notably, the latest MULTISCREENTM β-Arrestin Sensor makes it possible for the first time to assay orphan GPCRs (oGPCRs) in their native form in HTS. It also makes it possible to assay signaling biased ligands using untaggaed GPCRs in heterologous or primary cells in HTS for the first time.
In addition to human targets, assays with comparable pharmacology for mouse, rat, ferret, rabbit, dog, pig, and non-human primate ortholog targets have also been developed. To complement cell-based assays, radioligand binding assay and GTPγS assays are often performed using primary tissue homogenates such as human or rat, mouse and rabbit brain tissues from wildtype or transgenic animals.
Depending on the biology of each target and mechanism of therapeutic intervention, different modalities would be employed for HTS. Accordingly, assays would be developed in specific modes to meet the screening requirement such as agonist, partial agonist, antagonist, partial antagonist, inverse agonist, positive allosteric modulator (PAM) and negative allosteric modulator (NAM) modes. Tool compounds selected for each assay development will hence reflect such choice of mode of action.
Finally, to develop and optimize an assay, extensive optimization steps are often taken to arrive at high signal-to-background ratio, and pharmacologically relevant efficacy and potency, measured by tool compounds. Multispan’s assay experts not only rely on in-depth experience and knowhow accumulated over nearly 2 decades of practice, but also leverage the high quality MULTISCREENTM stable cell lines and assay reagents where possible. The majority of our cell-based assays are developed and validated in 384-well microtiter plate format. The minimum requirement of Z is 0.5 to ensure an assay HTS is compatible. For HTS performed at Multispan, our standard Z’ goal is 0.7.