Recent advances in GPCR research have provided additional opportunities for drug discovery beyond the single conventional binding site approach used to date for all currently marketed drugs that target liganded GPCRs. The discoveries of many yet-to-be defined orphan GPCRs, the finding that GPCRs can form homo- and/or hetero-oligomeric complexes with other GPCRs and the ever-growing number of accessory proteins found to interact with and influence the GPCR signaling complex mean that the number of potential sites at which to target drug action continue to grow and perhaps provide new ways to increase the specificity of action. Advances in drug screening technologies that now allow for screening of orphan GPCRs in the absence of endogenous ligands, more facile determination of effects on protein-protein interactions, continued miniaturization of conventional screening assays, and optimization of homogenous assay systems will undoubtedly speed the rate of discovery at these novel GPCR targets.
Orphan GPCRs represent one area of ongoing drug discovery research targeting approximately 160 receptors across three GPCR families (Figure 3.3, top) in the search for novel therapeutic agents. Several strategies have been used to identify ligands for orphan receptors including reverse pharmacology, the first methodology applied that involves the screening of a panel of known transmitters on recombinantly expressed orphan GPCRs and the measuring of signaling responses. Examples of GPCRs discovered by this approach include the H3 histamine21 and LTB4 leukotriene64 receptors. A second approach, termed the orphan receptor strategy, also involves the expression of recombinant GPCRs, but the cells expressing the orphan GPCR are exposed to a series of tissue extracts and a variety of signaling responses are monitored for activation responses.65
Once a particular extract elicits a positive response, it is fractionated and assayed iteratively until the cognate ligand and, consequently, the GPCR is identified. Noci-ceptin or orphanin FQ, the ligand for ORL1,66 was the first ligand identified in this manner. Another approach known as constitutively activating receptor technology (CARTTM) genetically modifies the orphan GPCR to stabilize the receptor in an active conformation so that it can constitutively activate a number of signaling pathways.17 Compound libraries can then be screened for both agonists and antagonists.
Since 1995, ligands have been identified for approximately 50 orphan GPCRs (Figure 3.3, bottom) using reverse pharmacology and orphan receptor strategies. However, as previously described, approximately 160 orphan GPCRs for which the endogenous ligands and functions are unknown still remain, and the rate of discovery appears to be decreasing.67 This suggests that a number of orphan GPCRs may not be "classical" liganded GPCRs and may have other biological functions. These include oligomerization partners with liganded GPCRs, accessory trafficking proteins such as the GABAb(2) receptor with the GABAb(1) receptor,68-70 signaling through heterotrimeric G protein independent mechanisms, discrete temporal or developmental expression, and highly constitutive "ligandless" GPCRs.67 Determining the biological functions and therapeutic potentials of the remaining orphan GPCRs will be a challenging endeavor. Orphan receptors are described in more detail in Chapter 16.
Chapter 15 covers GPCR oligomerization in great depth. The potential implications of this phenomenon on GPCR drug discovery efforts will only be briefly discussed here. An ever-increasing body of evidence accumulated in the past several years demonstrates the existence and function of homo- and hetero-oligomeric complexes of GPCRs in recombinant expression systems and in native tissues. The homo- and
200 175 150 125 100 75 50 25 0
^M Liganded I I Orphans
Family A Family B Family C
2003 2002 2001 2000 1999 1998 1997 1996 1995 1994 1993
GPRS, GPR40, GPM1, GPR43, TGR5, HM74A
GPR7, TG1019, LGR7, LGRS, BG37, C5L2
NAA1, BX36, HX37, H3G8, OGR-1, CyS-LTR2, BLT2, FM-3, FM-4, H4R, H.WAR77, CRTH2
GHS-R, GPR14, GPR38, B3G3, BX34, Cy»-LT1R, H3R, M CHI
Orexln-1, Orexln-2, GPR10, APJ, CRLR, EXS1, BX32
0 1 23456789 10 11 12 Number of De-orphaned Receptors
FIGURE 3.3 Orphan GPCR discovery. Top: Numbers of liganded and orphan GPCRs in GPCR families. Bottom: Elucidation timeline of orphan GPCRs since 1995.
hetero-oligomers could account for the complex and subtle changes in activation of different signaling pathways, receptor desensitization and sensitization, and modulation of GPCRs by a variety of agonists.7172 This could significantly impact drug discovery efforts by greatly expanding the number of potential drug targets (theoretically over 129,000 possible oligomeric pairs67) and produce important implications for the screening and development of new drugs. Additionally, a different mindset for GPCR drug discovery would have to be implemented to take into consideration the possibility of designing drugs that may act at additional sites beyond the conventional monomeric GPCR binding sites.
George et al.73 proposed several different types of novel drug designs based upon GPCR oligomerization. One design encompasses dimeric ligands, compounds that contain two covalently linked monovalent ligands that may more readily induce or stabilize dimeric conformations of receptors. It may also be possible to design drugs that enhance and/or disrupt oligomerization, potentially by interfering with protein-protein interactions between the two receptors composing the oligomeric GPCR unit. Minimal drug discovery efforts in this area have been reported to date, but the concept appears to have great potential for producing useful therapeutics.
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