In the previous assignment on target identification and validation a target which could help to regulate type II diabetes was chosen. This target was the G-protein coupled receptor (GPCR) leukotriene B4 receptor (Ltb4r1) and it’s naturally occurring ligand LTB4. As mentioned previously Ltb4r1, like other GPCRs in this family, has been shown to be involved in the regulation of type II diabetes. Specifically Ltb4r1 has been shown that when present in increased levels, which occurs in obesity, and then this leads to insulin resistance and increased inflammation. In contrasts, as shown in the previous assignment, if Ltb4r1 is inhibited then insulin sensitivity is improved and there is decreased inflammation (Spite et al., 2011). Currently, there are no known drugs for Ltb4r1 undergoing clinical trials. However there are other members of this family, such as GLP1, which have drugs which have been approved for used (Olefsky, 2016).
As this receptor appears to only have 1 natural ligand, Ltb4r1, it is an ideal candidate for small molecule drug design. Besides its natural ligand, there is a specific inhibitor for this receptor which is commercially available. This agonist is known as CP-105696 (Li et al., 2015). As there is already an agonist and a natural substrate known, this makes this receptor an ideal candidate for high-throughput screening.
High-throughput screening is a technique in which a library of compounds is screen for activity against the known target (Mayr & Fuerst, 2008). In this case, since decreased activity of the GPCR is the desired outcome, compounds will be screened to see if they decrease the activity of Ltb4r1. As the natural ligand, LTB4, is known and there is also an agonist, CP-105696, available, the library that is screened should be developed around these two compounds. For example modifications to these compounds can be made, through the addition of other groups, to help improve the binding of the compound over the natural ligand.
With any high-throughput approach, an assay is required in order to determine which compounds are active against the identified target. In this particular case, there are antibodies available which can be used to obtain purified Ltb4r1. With this purified Ltb4r1 a biochemical assay can be run in vitro (Flanagan, 2016). In order to detect a signal there are two main methods which can be used, radioligand and fluorescent ligand binding assays. In radioligand binding assays the ligand is radiolabeled with a tag such as 3H, 14C or 33P. In this case the radioligand would be LTB4 and it would be incubated with Ltb4r1 and the compound of interest in the well of a plate (Blass, 2015). The plate would then be washed to remove unbound compounds and the amount of signal determined. A reduction in signal would indicate that there compound is a potential agonist of Ltb4r1 (Blass, 2015).
Fluorescent ligand binding, functions in the same why as radioligand assays, however no radioactive compounds are used (Blass, 2015). Instead the ligand is bound to a fluorescent molecule. In this case the fluorescent compound would be LTB4 and again the compounds in the library would be incubated with this LTB4 and Ltb4r1. The LTB4 will also have a quencher so that only when it is cleaved is there a fluorescent signal. The plate would be washed to remove unbound compounds and the amount of fluorescence would be determined. A reduction in the amount of fluorescence indicates a potential agonist (Blass, 2015).
One of the reasons for only labeling the natural ligand is that labels can sometimes alter the properties of the compound of interest. By keeping it consistent and always labeling just the natural ligand this can be reduced. As well this can also reduce cost as only one compound must undergo additional labeling.
Although biochemical assays, such as the two listed above, are useful for determining binding of potential compounds to the target they provide no information on the bioavailability of the compound. For example, a compound may be able to tightly bind the receptor but if it is unable to make it through the plasma membrane then it will not be useful as a therapeutic drug. A way to evaluate a compounds ability to be used in a cell is to perform a cellular assay (Blass, 2015).
Like biochemical assays, cellular assays are done in vivo. However, whole cells are used. In a cellular assay, cell lines are often used as the source of cells. As most cell lines are immortal they provide a cheap and readily available source of cells (Siehler, 2008). Similar to the biochemical assays, the fluorescent labelled LTB4 will be used. After determining the baseline signal the compounds in the library which showed potential agonist activity in the biochemical assay will be tested. Again a reduction in the amount of signal will indicate that the compound is a potential agonist to Ltb4r1. Although this will provide information on if the compound can enter the cell it will not provide information on how the compound will act in a whole organism. For this information the positively identified potential targets will need to undergo further testing (Siehler, 2008).
Overall there is variety of different ways to screen for potential lead compounds. The methods shown here are designed to maximize the likelihood of finding leads, while reducing time and cost. However, as mentioned, once identified further testing will be required to ensure that these compounds are in fact suitable for therapeutic use.