Summary And Future Directions

The diverse range of allosteric modulators now being identified is challenging for those involved in drug discovery. This chapter has sought to explore some of the issues faced in screening for allosteric modulators of GPCRs, ranging from the choice of assay format to the methods for data analysis.

The Section 12.1 discussed the benefits and drawbacks of radioligand binding. While it is clear that radioligand binding does not represent a good approach for primary screening for novel allosteric modulators, the impor tance of the technique should not be underestimated, as it is the only method that can be used to detect allosteric effects on affinity without potentially confounding effects of modulation of efficacy. Given the prevalence of high-throughput functional screening assays, the wider effects of allosteric modulators were considered: Efficacy modulation and allosteric agonism, in addition to affinity modulation, were explored. There are now a range of newer analytical models, incorporating the operational model of agonist action, which has been shown to be highly useful in dissecting the mechanism of action of allosteric modulators exhibiting this wide range of behaviors. The availability of such analytical protocols will greatly aid the understanding of allosteric modulator function.

Also reviewed were the consequences of using different types of functional assays. This is a relatively new consideration for screening allosteric ligands, but as shown above, using transient read assays (such as intracellular calcium flux) as the screening assay compared to an accumulation format (such as cAMP or p5S]-GTPyS binding) can markedly change the output for compounds that display intrinsic allosteric agonism. This may have implications for an SAR program and should be carefully considered when designing a screening program for allosteric modulators.

To increase throughput and avoid running multiple concentration-response curves for a single compound, the use of modulator titration curves as a screening paradigm is now popular. It is apparent that such curves can yield significant information about an allosteric ligand's mechanism of action, and a method of analysis for titration curves is presented and discussed. However, there are limitations to the amount of mechanistic information that can be obtained from a single concentration-response curve. This highlights an interesting conundrum for screening—)s it worth reducing the amount of data points per compound to achieve a higher throughput and consequently reduce the level of information obtained? Or, is it better to incur the time and costs, and have more data points per compound and get a greater degree of understanding of a compound's profile? Clearly, there is a finite amount that a single concentration-response will yield; this will not change. However, there have been great improvements in the number of data points that can be screened in a single assay; 1536-well plate screening is commonplace in most HTS facilities. Therefore, it is not difficult to imagine that in the future, it may be possible to perform more quantitative screens, even screens that elucidate full allosteric modulator mechanism of action. This quantitative approach has already been attempted for enzyme targets [52] and could well represent the future of high-throughput compound screening for allosteric modulators of GPCRs.

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