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Combining Biologically-Relevant Assays with Cell Engineering For GPCR Targets and Beyond

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Cell-based assays are essential throughout the drug discovery process. They are important for target validation, high throughput screening (HTS), rational drug design, and SAR lead optimization. Cell-based assays are also critical in the determination of safety and selectivity of new lead compounds. The most frequently used cells in cell-based assays are recombinant stable cell lines that heterologously express protein targets such as G-Protein Coupled Receptors (GPCRs). Stable cell lines are cornerstones in the discovery of new chemical entities.

Stable cell line providers often resort to heavily engineered targets, modified signaling components and overamplified protein expression, all of which destroy the targets’ natural pharmacology.

Multispan’s MultiScreen™ stable cell line/assay development platform is designed to reflect true target pharmacology for robust high throughput screening. The MultiScreen™ platform is built on a foundation of rigorous science and proprietary knowhow. Multispan’s team of experts ensures a reliable process that each stable cell line is both biologically and pharmacologically relevant, and ready for HTS. We take extensive measures to achieve these mission-critical properties in every stable cell line we develop. We will share a few of these important properties in this article.

Firstly, we identify the specific disease-relevant variant and mutants of research interest for the protein target (Table 1). Secondly, Multispan’s propriety universal MultiScreen™ expression vector is optimized for robust cell surface expression of any transmembrane proteins like GPCRs, receptor tyrosine kinase receptors and transporters.

GPCR Family GPCR Target Assay GPCR Family GPCR Target Assay
Ca++ cAMP Ca++ cAMP
Bile Acid GPBA Opioid mu
GPBA Mutant W83G mu Mutant Y150F
GPBA Mutant W83A mu Mutant Y150A
GPBA Mutant V88L mu Mutant M153A
GPBA Mutant Y89A mu Mutant H299N
GPBA Mutant Y89H mu Mutant H299A
GPBA Mutant F96A Kynurenic acid GPR35
GPBA Mutant W237A GPR35 (short form)
GPBA Mutant W240A GPR35 (long form) T108M
Cannabinoid CB1 Prokineticin PK2
CB1a PK2 mutant
CB1b Purinergic GPR17 (long form)
Free Fatty-Acid GPR120 GPR17 (short form)
Neuropeptide S NPS

Table 1. Matched Wild-type vs Mutant and Canonical vs Variant GPCR Stable Cell Lines and Cell-based Assays

We also believe in keeping the recombinant cell system as natural as possible. When using readouts measuring endogenous cell signaling second messengers and functional fluorescence readouts It is always important to maintain the native pharmacology.

Get what you screen for. Lastly, throughout the cell cloning and selection process, our team’s strong assay expertise in cell-based assays plays an important role. We leverage this expertise to enable clone selection directly in disease-relevant cell signaling pathways using tool compounds. In addition, 384-well high-throughput screening format is employed from the start.

As a result, each MultiScreen stable cell line is customized to closely resemble endogenous pharmacology in primary cells and uniquely positioned to deliver robust and physiologically relevant screening data.

In the past decade and a half, the Multispan team has delivered hundreds of stable clones to pharmaceutical researchers where detection of exquisitely small differences between compounds in HTS and SAR is needed. This unique capability at Multispan has been battle-tested and our team hasn’t failed to deliver, no matter how rare the specific phenotypic event is in the cell pool. Not only Multispan’s pharmaceutical partners, but also the Multispan team inhouse rely on these high-performance stable cell lines to support HTS and weekly SAR compound screening for our partners.

Here is an example that illustrates how Multispan’s approach and rigorous clone process can make disease-specific “Designer Pharmacology” possible (Figure 1).

Efficacy-gap Reduction between Partial Agonist and Full Agonists
in Dose-Response Curve cAMP Assay in D1-HEK293 Stable Clones
Correlation with Increased D1-Receptor Surface Expression
in the D1-HEK293 Stable Clones Measured by FACS
Figure 1. Example of How Surface Expression Impacts Dopamine Receptor D1 Pharmacology in Multispan’s Stable Cell Line Clones

Our stable cell lines expressing GPCRs offer the following unique benefits:

  • Preservation of all endogenous cell signaling pathways: Calcium, cAMP, beta-arrestin, pGRK2, IP-1, pERK, pNFκB, radioligand binding, GTPγS, chemotaxis, cytokine secretion, cell proliferation, Luciferase reporter and internalization
  • Gold standard for studying all GPCR cell signaling-biases in ONE cell line
  • Multi-screen capability in the same cell line across multiple assays for counter screens
  • Available as division-arrested cells and membranes
  • Rigorously validated in MultiScreen No Wash Calcium Assay and cAMP Assay Kits
  • Expression and pharmacology-matched human and ortholog protein target pairs, including rhesus monkey, cynomolgus monkey, dog, ferret, rabbit, rat, and mouse (Figure 2 and Table 2)

Collectively, Multispan’s stable cell line generation platform is highly differentiated to mimic native responses in multiple physiologically disease-relevant pathways by minimizing modifications in our MultiScreen expression vector. Your success is our priority. Multispan employs an assay-centric screening approach customized to meet each project’s need.

We enjoy learning the biology of each target and eveloping high-performance cell lines for disease-relevant assays. It serves our mission to help “make better medicine, one assay at a time”. ­

Figure 3. Pharmacology-Matched Human, Dog and Mouse Serotonin Receptor 5-HT2A MultiScreen™ Stable Cell Lines for Calcium Assays

Please email info@multispaninc.com for more. Let’s discuss how MultiScreen™ stable cell lines can uniquely empower your program.

Receptor Family Receptor Species Assay Receptor Family Receptor Species Assay
Ca++ cAMP Ca++ cAMP
Adenosine A2A human Leukotriene B4r BLT2 human
A2A mouse BLT2 Rhesus monkey  
A2B human BLT2 mouse  
A2B rat Lyso-phospholipid LPA1 human
Bile Acid GPBA human   LPA1 mouse
GPBA rat   LPA1 rat
GPBA mouse   LPA5 human  
Bombesin BB1 human   LPA5 mouse  
BB1 rat   S1P2 human  
Bradykinin B1 human   S1P2 mouse  
B1 mouse   S1P2 rat
B2 human S1P3 human  
B2 mouse   S1P3 mouse  
B2 rat   S1P3 rat  
B2 dog   S1P4 human  
B2 monkey   S1P4 mouse  
B2 guinea pig   S1P4 rat  
B2 rabbit   Melanin-Concentrating
MCH1 human  
Calcium-Sensing CaSR human   MCH1 rat  
CaSR rat   Melatonine MT2 human  
Cannabinoid CB1 human MT2 mouse
CB1 mouse   MT2 rat
CB1 mouse   Metabotropic Glutamate mGlu2 human  
Chemokine CCR6 human mGlu2 rat  
CCR6 mouse mGlu3 human
CCR8 human   mGlu4 dog  
CCR9 dog   mGlu4 human
CCR9 human   mGlu4 mouse  
CCR9 mouse   mGlu4 rat  
CCR9 rat   Muscarinic M3 human  
Endothelin ETA human M3 mouse  
ETA rat   M3 rat  
Free-Fatty Acid  GPR119 human   Neuropeptide S NPS human  
GPR119 mouse   NPS mouse  
GPR120 human   Neuropeptide W/B NPBW1 human  
GPR120 mouse   NPBW1 rat  
GPR120 rat   Neuropeptide Y2 human  
GPR40 human   Y2 rat  
GPR40 cynomolgus monkey   Opioid delta human  
GPR40 dog   delta rat
GPR40 mouse     kappa human
GPR40 rat   kappa rat
GPR40 rhesus monkey   mu human  
GPR41 human   mu rat
GPR41 mouse   NOP human
GPR41 rat NOP rat
GPR43 human Kynurenic acid GPR35 human  
GPR43 mouse GPR35 dog  
GPR43 rat GPR35 mouse  
GPR43 dog Oxysterol EBI2 human  
GPR43 cynomolgus monkey EBI2 mouse  
GPR43 rhesus monkey EBI2 mouse  
Ghrelin Ghrelin human   Prostanoid EP2 human
Ghrelin mouse   EP2 mouse  
Glucagon GIP human   EP2 rat  
GIP mouse   Purinergic GPR17 human
GIP rat   GPR17 mouse
GIP dog   GPR17 rat
GIP rabbit   P2Y2 human    
GIP rhesus monkey   P2Y2 rat  
GIP ferret   P2Y12 human  
GLP-1 human   P2Y12 rat  
GLP-1 cynomolgus monkey P2Y14 human  
GLP-1 rat   P2Y14 rat  
GLP-1 dog   P2Y14 Cynomolgus monkey  
GLP-1 mouse   Serotonin 5-HT2A human  
GLP-2 human   5-HT2A mouse  
GLP-2 rat   5-HT2A dog  
Glucagon human   5-HT2B mouse  
Glucagon cynomolgus monkey   5-HT4A human  
Glycoprotein Hormone TSH human   5-HT4A mouse  
TSH rat   Trace Amine TA1 human  
Histamine H1 human   TA1 rat
H1 mouse   Urotensin UT human  
H3 human   UT rat  
H3 mouse Vasopressin/ Oxytocin OT human  
H4 human   OT rabbit  
H4 mouse V1A human  
KiSS1-Derived Peptide KiSS1 human   V1A rabbit  
KiSS1 rat   V1B human  

Table 2. Examples of Multispan’s Ortholog GPCR Stable Cell Lines for Cell-Based Assays

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