Huntington’s disease is a debilitating disease for which there is no known cure. It is a genetically inherited disease. Individuals with a mutation in the huntingtin (HTT) gene will develop Huntington’s disease. The HTT gene codes for a 350-kDa protein which has a polyqlutamine (polyQ) tail. Previous research has shown that if expansion of the polyQ tail occurs then the protein will not fold correctly, which leads to the development of Huntington’s disease. As a potential treatment option, research has shown that phosphorylation of serine 421 on the HTT protein can reduce the toxicity (Kratter et al., 2016).Therefore this makes it a good target for the development of drugs for Huntington’s disease treatment (Kratter et al., 2016). After determining a target and validating its effects, the next step is to design compounds that are able to modulate the target of interest. After compounds and/or antibodies are developed they need to be tested to see if they are able to produce the desired effects without any off target side effects (Blass, 2015).
In order to test the compounds of interest there are two main types of assays that can be used, in vitro cell assay, and in vivo animal model studies (Blass, 2015). In this particular situation it is assume that a compound which increases the phosphorylation of serine 421 has been determined.
When finding compounds for a particular target, biochemical assays are often used. This provides information on if the compound can affect the target, but does not determine how the compound will react inside cells. As compounds often need to cross cell membranes in vitro cell assays must be conducted to identify biologically relevant compounds (Blass, 2015).
In this particular case the goal is to increase phosphorylation of serine 421 to reduce the negative effects of HTT. Therefore, for an in vitro cellular assay a cell-based enzyme-linked immunosorbent assay (ELISA) well be used (Michelini, Cevenini, Mezzanotte, Coppa, & Roda, 2010). In this assay an antibody which can specifically bind to the serine 421 phosphorylation and produces a fluorescence signal is required. As well a cell line, which has good expression of the defective HTT, should be used (Michelini et al., 2010). These cells can be plated on a cell culture plate and then the cells can be treated with the compounds of interest. The cells can then be fixed and treated with the antibody. The amount of fluorescence will correspond to the amount of serine 421 phosphorylation. If a compound causes an increase in fluorescence this means that this compound causes an increase in the amount of serine 421 phosphorylation (Michelini et al., 2010). However, this does not tell us if the compound is selectively phosphorylating only serine 421. To test this, compounds which have increased fluorescence will undergo additional testing. The in cell assay will be repeated, using a radioactively labelled ATP. After the assay, protein will be extracted and undergo Edman sequencing to determine where phosphorylation has occurred. The control samples will be compared to the samples with compound to ensure that only phosphorylation of serine 421 is increased (Delom & Chevet, 2006).
While in vitro assays provide useful information about compounds getting into cells, they do not provide any information on how compounds will react once inside an organism. In order to determine the efficacy of the compounds identified in the in vitro tests an in vivo assay is required.
For studying Huntington’s disease there are several mouse models which have been developed. These models are a good choice for in vivo testing (Ehrnhoefer, Butland, Pouladi, & Hayden, 2009). While there are many models available for this experiment two separate models should be used. For example the YAC128 mouse which contains the full length human HTT, and displays behavioural defects could be used. This particular mouse has already been used to in trails of several Huntington’s candidate therapeutic compounds (Ehrnhoefer et al., 2009). Another mouse model which could be used is that of the BACHD mice, which also expresses the human mHTT but has progressive motor defects and behavioural defects could be used (Ehrnhoefer et al., 2009). By using these two separate models this will provide a better idea as to the efficacy of the compounds. As there is no one perfect model of Huntington’s disease, using two separate models increases the chance of successfully identify useful therapeutic compounds (Ehrnhoefer et al., 2009).
In these mouse models, the compounds of interest could be given to the animals and then the behaviour of the mouse could be compared to that of a group of control mice. Specifically with the BACHD mice, as they display motor defects, compounds which delay or prevent the onset of these symptoms are drug therapy candidates. As well analysis of the levels of the toxic form of HTT protein can be compared to that of control mice. A decrease in this protein would indicate the compound is an ideal choice for drug therapy (Kratter et al., 2016).
Overall, in vitro cell assays are a quick and relatively easy way to narrow down a list of potential compounds. Once this has been done, the next step is to take these compounds and conduct in vivo animal studies to see how the compounds react inside a living organism. Although mouse models are not equivalent to studies in humans they can provide useful information and narrow down the list of potential compounds.