Propelling GPCR drug discovery through innovative products and services Be the change the market seeks With the cost of bringing a new therapeutic agent to market now estimated at around $2 billion and the disappointing pace of approvals for new therapeutics, the drug discovery community is under increasing pressure to develop smarter and safer drugs. To do this, improvements need to be made in the overall effectiveness of the marketed drugs while minimizing potential deleterious side effects. Now more than ever, the industry is poised to take on this challenge as new, innovative drug discovery technologies have emerged that are revolutionizing the way new GPCR drugs are developed. G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), are the largest and most druggable class of cell surface receptors. It is now well understood that activation of GPCRs results in signaling by both G-proteins and β-arrestin, as well as receptor desensitization and internalization. The complex relationship that exists between G-proteins and β-arrestin signaling determines both the efficacy and potential side effects of GPCR-targeted drugs. Therefore, quantitatively examining these pathways can aid in defining compound function and can lead to the discovery of novel biased ligands with unique attributes. Since our founding in 2000, DiscoveRx has been at the forefront of GPCR drug discovery by identifying and developing high quality products and services that mitigate development bottlenecks and meet the complex needs of researchers across the drug discovery continuum. The company s innovative approach to GPCR signaling assay development, using a simple, homogeneous, chemiluminescent, read-out based on -galactosidase-based enzyme fragment complementation (EFC), is core to the success of many innovative drug discovery programs. Analysis of GPCRs can now be addressed using multiple assay approaches. From the traditional second messenger signaling assays to the novel, universal β-arrestin recruitment assays for known and orphan GPCRs to the next generation platforms for studying GPCR internalization and heterodimerization, DiscoveRx is addressing current and emerging drug discovery needs through innovative GPCR solutions. Past, Present and Future of GPCR Drug Development Technology Innovation That Drives Discovery As recently as 5 years ago, the understanding of GPCR biology was limited. Although researchers had identified hundreds of GPCR receptors using genomic DNA approaches, they had little to no understanding of the receptor function or the signaling molecules that activated 1
these receptors. The race was on to pair these receptors with their natural ligands and to seek out new technologies that could lead to advancements in GPCR discovery and de-orphanization. Between 1986 and 2006, more than 300 Orphan GPCRs were paired with their natural ligands. Despite these efforts, approximately 100 Orphan GPCR receptors remain for which endogenous ligands have not been identified. This combined with the fact that only 30% of the 800 to 1000 known GPCRs are actual drug targets demonstrates that great investments are still being made by the pharmaceutical industry to discover new drugs that modulate GPCR activity. Past - Limited Screening Options One needs to look back only a few years to realize that from the time of its inception, the GPCR drug discovery market has always had a palpable thirst for new and innovative technologies. Initially, orphan GPCR receptors were screened using lengthy and labor intensive procedures that included exposure of the orphan receptor to tissue extracts that researchers hoped contained the natural ligand. Second messenger assays were used during this time to make best guesses at the appropriate ligand:receptor pairings. But progress was not fast enough. Next, binding technologies were introduced and proved to be the early work horse in the GPCR drug discovery process, resulting in the first FDA-approved GPCR targeted drugs. Although these assays provided an initial assessment of compound efficacy based on direct interaction of the ligand with the receptor, these assays were limited by the availability of radiolabelled ligands, could not be used to study Orphan GPCR receptors which by definition lack known ligands, and because they were not run in whole cells, these assays provided very limited information on the activity of the compound. The GPCR drug discovery community needed a better understanding of the GPCR drugs that they were developing. As a result, cell-based technologies were developed that allowed the interrogation of the GPCR in intact cells rather than cell membranes. The HTS community quickly began to favor the use of functional assays that measured GPCR receptor activation in whole cells via the direct interaction of G-proteins with the GPCR. The primary readout of these assays was the accumulation of second messenger levels including camp, IP3, and Calcium. These assays were simpler to run, easier to automate and highly quantifiable. 2
Figure 1. Evolution of GPCR drug discovery. By the mid-1990s, functional assays that allowed detection of intracellular Calcium levels were extremely popular due to their scalability and the availability of low-cost, calcium-sensitive fluorescent dyes together with automated real-time fluorescence plate readers such as FLIPR (Molecular Devices) and FDSS (Hamamatsu Corp). Today, Calcium-based functional assays still account for over 20% of assays used to study GPCR activation. Calcium assays, however, could only be used to interrogate Gq-coupled receptors unless the Gi or Gs-coupled GPCR receptor was artificially forced down the Gq-coupled pathway. As a result, additional HTS-compatible second messenger technology platforms were developed that could be used to interrogate native Gi or Gs-coupled GPCR receptors in live cells by detecting the accumulation of intracellular camp using a competitive immunoassay approach. Many different camp assay technologies emerged including radiometric, fluorescence polarization (FP)-based camp detection as well as non-radioactive, homogeneous time resolved fluorescence (HTRF). And then in 2001, DiscoveRx revolutionized the GPCR drug discovery community by introducing the innovative HitHunter technology. High-throughput screeners could now take advantage of a highly sensitive, highly scalable, non-radiometric cell based screening platform that was compatible with multiple plate readers and could minimize the impact of compound interference based on its fluorescent or chemiluminescent detection system. Present Explosion of Technology Options DiscoveRx s ground breaking camp assay technology, not only led to an explosion of similar technology options, but paved the way for what was to become the defining GPCR assay 3
technology; the measurement of GPCR functionalization through the binding of -Arrestin. This technology emerged when it was demonstrated that a majority of GPCRs bind a second class of intracellular proteins called Arrestins. This immediately provided an opportunity to use an alternative primary screening platform that was independent of G-protein signaling. With this finding came the emergence of a universal, confocal microscopy-based translocation assay that allowed real-time analysis of Arrestin recruitment to activated GPCRs. However, this approach was limited by low throughput and expensive instrumentation. In 2007, DiscoveRx once again advanced the field of GPCR drug discovery with the introduction of the highly innovative PathHunter -Arrestin screening platform. This system was a major advancement over the existing microscopy or BRET-based approaches because the assay platform was enzymatic, highly quantitative, extremely sensitive, easy to use and readily amenable to high-throughput screening. With the introduction of the PathHunter technology, researchers could now integrate a G-protein independent orthogonal screening approach into their drug discovery program. This PathHunter-based orthogonal screen could be applied to any GPCR of interest regardless of coupling status and could be used to better understand the mechanism of action of lead compounds thus enabling the acceleration of only the most qualified and best understood lead compounds. Even more impressive was that the availability of the PathHunter Arrestin platform led to the reactivation of multiple de-orphanization campaigns resulting in the discovery of novel compounds against formerly intractable targets. Today, DiscoveRx has launched yet another major GPCR screening and profiling platform that directly measures GPCR receptor recycling and desensitization. The introduction of the PathHunter Activated GPCR Internalization technology, a non-imaging, non-antibody based chemiluminescent platform, provides a direct and quantitative measurement of internalized GPCR localized to intracellular endosomes allowing the fate of unlabeled, activated GPCRs to be monitored in live cells without need of expensive microscopy. Future Biased ligands represents the future of GPCR drug discovery Our goal has always been centered on the idea that we will create technologies that enable the highest levels of innovation and development to enrich GPCR drug discovery. And by focusing on this goal, we have become a well-known and trusted supplier and are becoming an essential ingredient in high quality assay development for GPCR drug discovery. It is now well appreciated that GPCR receptors are activated through both G-protein dependent and independent pathways, and hence possible to discover compounds that are biased towards one pathway or the other. With this understanding, DiscoveRx is committed to the development of additional novel platforms that will enable the industry to develop designer therapeutic drugs that increase efficacy while decreasing unwanted side effects. The comprehensive GPCR offering from DiscoveRx that covers greater than 90% of the druggable GPCRome allows for advanced lead optimization and SAR work while gaining a greater understanding of receptor biology. 4
From the traditional HitHunter camp second messenger signaling assays to the novel, universal PathHunter β-arrestin assay for known and orphan GPCRs, DiscoveRx is your partner for GPCR drug discovery. We have now embarked on an era of GPCR drug development that will deliver on the promise of smarter and more effective drugs. With the largest and most comprehensive menu of GPCRs for screening and profiling, we can deliver on the promise of high content imaging using a high content compound analysis approach. Figure 2. Multiple GPCR signaling platforms from DiscoveRx. For more information on DiscoveRx, please contact: DiscoveRx Corporation Sailaja Kuchibhatla Sr. VP Business Development tel 510.979.1415 x104 skuchibhatla@discoverx.com www.discoverx.com 5