G protein-coupled receptors (GPCRs) remain one of the most pharmacologically successful drug targets, with 35% of all commercial drugs and 19% of newly approved drugs targeting this protein superfamily.1  Despite the continued importance of GPCRs and the large potential of CRISPR technology, the application of CRISPR within GPCR-based drug discovery has just scratched the surface. As of May 2020, over 11,400 publications are cited in PubMed associated with GPCRs, while only a mere 37 publications appear upon “GPCR and CRISPR” searches.

This article discusses the role CRIPSR technology has played in strengthening our understanding of GPCR biology, its transformative potential for all phases of GCPR-targeted drug development, and the drivers of successfully implementing CRISPR initiatives.

Advancing GPCR biology & drug discovery

Over the last several decades new tools and technologies have significantly changed the way drug discovery is performed. Even with these advances, the development of new therapeutics remains risky with a greater than 90% estimated failure rate, requiring over a decade and a billion dollars to commercialize a single new drug.

In 2013 a new disruptive technology enabling simple yet precise gene editing called CRISPR-Cas was reported that has had far-reaching impacts from revolutionizing cell and gene therapy to an infusion of new discovery and drug development tools.

CRISPR’s ability to directly insert, delete, or introduce precise mutations in genes within physiologically relevant cell types provides immense opportunities to improve how we identify, qualify, and translate candidate drugs to the clinic.

Specifically, CRISPR can address high attrition rates and therapeutic development timelines by enabling improved understanding of normal and disease-associated target biology, more precise delineation of target and candidate mechanisms of action, and the creation of more powerful in vitro and in vivo discovery tools and toxicity models.

Getting to better know GPCRs

GPCRs continue to be of intense interest as drug targets due to their considerable role in a diverse array of normal and disease-associated physiological processes as well as their established pharmacological tractability.  Although a large number of drugs target GPCRs across a spectrum of diseases, only a small number of individual GPCRs have been extensively studied and/or targeted by current drugs relative to the total number of potential GPCRs in the human genome.

Figure 1

Thus, there is large untapped potential for furthering GPCR drug development through broader, more in-depth understanding of GPCR pathophysiology and the identification and characterization of orphan GPCRs.

The effects of GPCRs on cellular behaviors are mediated through an extensive array of complex processes including ligand binding and receptor conformational changes, G protein and/or β-arrestin activation, receptor desensitization, and recycling, stimulation of secondary signaling pathways, and ultimately altered gene expression (Figure 1).

Irregularities in any of these processes can lead to disease exemplified by the wide array of diseases that GPCRs have been linked to, ranging from CNS disorders, cardiovascular disease, metabolic disorders, immunoinflammatory disease, and cancer.

The direct and precise nature of CRISPR genomic alterations can provide clear and compelling data with profound implications in our understanding of GPCR biology.  As early as 2014, CRISPR/Cas technology was applied to GPCR research to aid in the identification of the function of GPR107, an orphan GPCR.2

Although limited in nature, since then, researchers have harnessed CRISPR technology to knockout (KO) GPCRs and/or GPCR-associated signaling proteins, knock-in normal or mutated GPCRs, generate panels of isogenic cell lines, as well as create cellular and animal, disease models.

CRISPR-based GPCR initiatives to date have:

  • Identified the function of orphan receptors2–5
  • Assessed ligand/receptor interactions and receptor dimerization6
  • Elucidated receptor trafficking, desensitization/reactivation7, 8
  • Deconvoluted and/or identified signal pathway usage8–16
  • Delineated mechanisms of action16–18
  • Defined their role in normal or disease states19–22

Seeing the Subtleties

Further advances in CRISPR/Cas9 technology have resulted in ‘dead’ Cas9 (dCas9) variants that recognize and bind specific DNA sequences in combination with guide RNA but cannot perform double-stranded DNA breaks.  By fusing these dCas9 with promoters or inhibitors of transcription, researchers can now easily and precisely regulate the expression levels of individual genes or groups of genes using methods termed CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation).23  As drugs generally do not completely abrogate cellular functions, future CRISPR a/i studies may prove to be even more therapeutically relevant.

Two particularly telling examples of CRISPR’s influence on our understanding of GPCRs and the doors that this knowledge can open for new disease intervention include the identification and characterization of an orphan receptor in epithelial cancers and the elucidation of a natural model of biased agonism.

Targeting the unknown

A 2017 publication reported that just over 100 different GPCRs are targeted by currently approved drugs, which represents a small fraction of the potential 800 GPCRs in the human genome.1 Furthermore, each established GPCR drug target was found to be addressed by an average of 10 drugs signifying a relative saturation of the market for currently targeted GPCRs.

The large number of uncharacterized and/or orphan GPCRs represents a significant pool of new drug targets that researchers look to. In fact, 36% of drug candidates in clinical trials target new GPCRs, several of which are orphan receptors with no known natural ligand.

The broad utility of CRISPR to knockout or knock-in specific GPCRs provides the opportunities to directly identify the function of uncharacterized GPCRs and begin to unravel their mechanisms of action.

CRISPR in Action:

GPCR Class C, Group 5, Member A (GPRC5A), an orphan GPCR, has been implicated in several epithelial cancers, with expression levels in some of these cancers correlating with tumor invasiveness and/or poor survival rates. Early research indicated that inhibition of GPRC5A has a growth-suppressive effect on cancer cells; however, its direct mechanism of action remained unclear.

Studies by Bulanova et al. in 2017 used CRISPR/Cas9 gene KO to identify the role of GPRC5A in the regulation of cellular adhesion and migration.4  GPRC5A KO cells displayed impaired integrin-mediated adhesion to a number of extracellular matrix proteins that correlated with reduced levels of integrin β1.  Their studies went on to show the first evidence for a direct interaction between GPRC5A and EPH receptor A2 (EphA2), a focal adhesion kinase (FAK)-regulating receptor tyrosine kinase.  Overall, the data suggest GPRC5A is a positive regulator of epithelial cell adhesion, representing a novel target in human epithelial cancers.

More recent studies in 2020 by Sawada et al. used CRISPR-mediated GPRC5A KO to characterize the role it plays in prostate cancer.5  Their data analysis indicated that patients with high GPRC5A expression have significantly shorter overall survival suggesting it may play a role in cancer progression.  GPRC5A KO in PC3 cancer cells resulted in reduced cell proliferation in both in vitro and in vivo studies.  Moreover, the GPRC5A-negative cells failed to establish bone metastasis in a xenograft mouse model.  The authors further noted that GPRC5A expression in clinical samples from prostate cancer patients significantly correlated with the presence of bone metastasis as well as the patient’s Gleason score (GS) confirming the clinical relevance of their findings and suggesting that GPRC5A may be a new therapeutic target as well as a prognostic marker of progressive prostate cancer.

Pinpointing biased agonism

Intense research concerning GPCR receptor activation brought to light the concept of biased agonism in which different ligands preferentially activate specific signaling pathways over others. Biased agonism reinforces the idea that GPCR-targeted candidates can be engineered to elicit fine-tuned cellular responses, thereby improving both a drug’s efficacy and safety profile.  To fully take advantage of the therapeutic potential of biased agonism, researchers have looked to gain a full understanding of the complete repertoire of GPCR signaling.

CRISPR in Action:

Free-fatty acid receptor-4 (FFA4), a GPCR previously known as GPR120, is found as two alternative splice isoforms, FFA4-S (short) and FFA4-L (long), that differ in length by 16 amino acids.  FFA4 receptor regulates a variety of activities, including metabolic processes and anti-inflammatory responses, and has been implicated in cancer progression.

It is known that FFA4-S activation preferentially stimulates Gαq/11 proteins resulting in increased intracellular Ca2+ and activation of the protein kinase C (PKC) signaling cascade.  Additionally, FFA4-S is rapidly phosphorylated by G protein-receptor kinase 6 (GRK6), leading to the β-arrestin-2 association.

In contrast, little is known about FFA4-L signaling.  Early research suggests that FFA4-L activation does not mediate Ca2+ signaling but does maintain the ability to associate with β-arrestin-2, thereby hinting at a potentially native β-arrestin-biased receptor.

In early 2020, Senatorov et al. reported CRISPR-derived data that revealed the mechanism of FFA4-L β-arrestin recruitment and ERK1/2 signaling.15  Their studies demonstrate that phosphorylation of the C terminus of FFA4-L is directly responsible for β-arrestin-2 recruitment and that unlike FFA4-S, FFA4-L requires β-arrestin-1/2 for the phosphorylation of ERK1/2.  These data directly confirm earlier findings of the β-arrestin-biased nature of FFA4-L and present an opportunity for further investigation of the mechanisms underlying biased-agonism.

Powering the pipeline

CRISPR has the potential to accelerate GPCR-targeted drug discovery not only through expanding our knowledge of GPCR biology but also by enabling a clearer definition of candidates’ structure-activity relationship and mechanism of action as well as transforming the available catalog of in vitro and in vivo tools.

For both GPCR- and non-GPCR-targeted drugs, CRISPR has been applied across the drug development pipeline from early target identification and candidate screening through lead optimization and pre-clinical animal studies (Figure 2).

Examples within GPCR drug discovery include:

  • Genome-wide CRISPR-Cas9 screening24, 25
  • Target identification and disease mechanism of action26–28
  • Elucidating drug candidate mechanism of action29
  • Early-stage animal models30–32
  • Cell-based functional assays via luciferase and BRET fusions33, 34
  • Construction of GPCR fusions as tools35–38

Two areas CRISPR has significantly impacted within the drug development pipeline include genome-wide sgRNA screening and the generation of isogenic cell lines.

No target left behind

Genome-wide screening using short hairpin RNA (shRNA)-mediated gene knockdown has been widely used to identify the genetic contributors to specific cellular phenotypes.  In drug discovery, shRNA screening is a powerful tool to identify and validate new therapeutic targets.

Figure 2

While powerful, shRNA screens can be limited by the partial nature of protein expression knockdown and high off-target effects.  CRISPR-Cas9 technology provides the ability to perform genome-wide systematic gene KO using sgRNA libraries that result in the complete elimination of target protein expression and have lower off-target effects.

Moreover, CRISPR a/i screens based on dCas9 provide the ability to systematically examine the effects of altered gene expression on cellular phenotypes.

CRISPR In Action:

Treatment of non-small cell lung cancer (NSCLC) patients whose tumors contain mutated epidermal growth factor receptors (EGFRs) with EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, has achieved some clinical success; however the heterogeneity of patient responsiveness and high frequency of acquired drug resistance results in cancer progression in most patients. Early studies to define the mechanism of TKI resistance through loss- or gain-of-function synthetic lethality screens were based on short interfering RNA (siRNA) or shRNA and/or focused on subsets of genes such as tumor suppressors and oncogenes.

In late 2019 Zeng et al. reported the use of a genome-wide CRISPR-Cas9 screening strategy to systematically identify the full complement of genes that play a role in EGFR TKI responsiveness.24  Within the identified proteins that altered the sensitivity of HCC827 cells, a human lung cancer cell line with high sensitivity to EGFR TKI to erlotinib was several that suggested reliance on GPCR signaling.  Among these was RIC8A, a molecular chaperone required for Gαq/i/13 protein folding and membrane association that also functions as a Gαq/i/13 guanine nucleotide exchange factor (GEF). Furthermore, their studies demonstrate that RIC8A acts as a positive regulator of yes-associated protein (YAP) signaling, which, when independently activated, could rescue erlotinib sensitivity in RIC8A knockout cells.

Thus, RIC8A, as well as a variety of other previously unidentified regulators of EGFR TKI sensitivity that were discovered in this study, represent putative targets for new drug candidates that may provide improved efficacy and durability of responsiveness.

Tiny change, big impact

Isogenic cell lines are a compelling means of clearly delineating the relationship between genotype and cellular phenotype, but before the introduction of CRIPSR technology, it could be difficult to construct.  CRISPR enables the streamlined generation of isogenic cells with well-defined genomic alterations that are instrumental for directly testing functional hypotheses, establishing causative roles of genetic mutations, accurately modeling disease, and evaluating candidate specificity—all of which provide insightful knowledge leading to improved candidate development.

Finding success

The diversity of skill sets needed for CRISPR implementation is daunting and can include guide RNA design and optimization, delivery of CRISPR tools (transduction or transfection), bioinformatics, molecular biology, and cell culture. For many GPCR-targeted CRISPR initiatives, it is ultimately the creation of quality stable cell lines from which CRISPR’s power is harnessed.

As with many areas of drug development, outsource providers are available to drive the success of your CRISPR initiatives. Not all CRISPR implementation and stable cell line generation providers are the same, so take the time to find a partner whose expertise you can count on with an unmatched track record of success to entrust with these critical pieces of the CRISPR workflow.

Read Multispan’s follow-on article entitled “A New Break in the Pipeline: CRISPR Stable Cell Lines as Drug Discovery Tools” to better understand the need for quality, clonal-derived stable cell lines during your GPCR-focused CRISPR research and how to identify the right outsource provider.

Multispan, Inc. is a premier cell-engineering, assay development, and compound screening company focusing on drug discovery targeting GPCRs and beyond. [email protected]



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