The Crich Domain

For mGluR3, there is now an available X - ray structure of the EC region including both the VFT and C-rich domains. The extensive cysteine bonds in this small domain render it fairly rigid. This suggests [75] that, to the extent that structural/conformational changes in the VFT are propagated via the C-rich region, the latter probably functions like a rigid connecting rod. In the related, Class C, sweet taste receptors, there is evidence suggesting that receptor activation can be achieved by interaction with the C-rich region.

16.4.4. Computational Studies

Prior to the publication of the X- ray structures of the mGluR EC regions described above, the leucine/isoleucine/valine binding protein (LIVBP) was used as a template for homology modeling (see, e.g., Reference 77 and references therein). LIVBP is a member of the bacterial periplasmic binding proteins (PBP), which are involved in ligand recognition and transport, and which share significant homology [78] with the EC domains of the mGluRs. Homology models were developed, generally in conjunction with mutational data, to help understand the binding of known ligands. For example, using LIVBP as a template for homology modeling, Costantino explored [ 77] the binding of 4MCPG [74] to mGluR1. Similarly, Hampson et al. developed [79] a model of L-serine O-phosphate (L-SOP) binding to mGluR4. With the X-ray publication of the EC domain, the mGluR1 structure has been used in studying subclass II (mGluR2 and 3) receptors. Malherbe et al. developed [80] models for the selective agonist LY354740, as did Monn et al. [81, 82], who also considered LY404040. Bertrand et al. [83] docked eight different ligands into the X-ray structure of mGluR1 and homology models of mGluR2 and mGluR4. Yao et al. studied [84] DCG-IV binding to an mGluR3 homology model. The subsequent publication of the X-ray structure of the mGluR3/DCG-IV complex revealed that the proposed [84] direct interaction between Arg277 and the ligand is not present. These findings underscore the value of the availability of X-ray structures spanning the various mGluR subclasses. Moreover, the full potential of the suite of EC X-ray structures for mGluRs in a drug-discovery environment has yet to be evaluated.

16.5. CONCLUSIONS

The importance of GPCR proteins in signal transduction in almost all physiological processes lead to their predominance as therapeutic targets. The long-awaited emergence of GPCR X-ray structures in the last 9 years is affording a better understanding of the structure/function relationships of GPCRs, including the mechanism of activation, and more effective drug design for these proteins.

For the transmembrane region, starting in 2000, crystallographic structural determinations were reported for rhodopsin, a light-activated Class A GPCR, and after 7 years, it was followed by the 2007 publication of the first non-rhodopsin/opsin (p2AR) Class A GPCR X-ray structure. Since the reporting of the first p2AR X-ray structure, several X-ray structures of ligand-mediated

Class A GPCRs and related publications have appeared. Due to significantly different ligand- binding cavities, for example, as observed in the p1AR and p2AR versus A2a adenosine X-ray structures, it is clear that additional X-ray structures will be necessary for efficient structure-based drug design at many targets. Also, reliable homology models for remotely related GPCRs, such as those of Class B or Class C, will be difficult to construct if the models are based on Class A GPCR X-ray structures. However, the reported adaptation of different approaches to facilitate purification and crystallization suggests that other GPCR X-ray structures will be determined at an accelerated rate. The value of GPCR X-ray structures for use in drug discovery already has been demonstrated and validated, for example, by quickly identifying nanomolar p2AR inhibitors by using a p2AR X-ray structure. In addition, promising results from homology modeling have been reported. Other anticipated advancements in structure-based drug design driven by X-ray crystallography include an understanding of the structural differences of other GPCR classes and subclasses, ligands of other character (e.g., agonists, allosteric modulators, etc.), other protein states, and dimeric structures. Based on the progress observed in GPCR X-ray crystallography since 2007, one would expect an acceleration in the rate of structure determination, advances in understanding of GPCR structure/function relationships, and more successful GPCR structure-based drug design.

For the VFT domain of Class C GPCRs, a broad palette of X-ray structures are now available to be used directly or as templates for homology models. Some structure-based studies have already appeared, but extensive evaluation of the potential impact in a drug-discovery environment is still needed.

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