Alternative methods to identify hit material in the drug discovery process always catch my eye. When a very recent publication using a Mass spectrometry (MS) detection system as a primary assay was released, I took the time to read it through. A link to the publication is shown below:
In this publication the authors were looking to identify inhibitors of the obesity target monoacylglycerol acyltransferase, which is responsible for acylation of monoacylglycerol (MAG) to diacylglycerol (DAG) in certain tissues. Diacylglycerol is then further metabolised by diacylglycerol acyltransferases resulting in triacylgycerol (TAG). Triacylglycerol is then stored in tissues as an energy source. Interrupting this metabolic pathway could then be used as a method to assist in diseases such as type 2 diabetes, which are influenced by excess storage of Triacylglycerol
Historic assay formats for this target previously included a scintillation proximity assay and thin layer chromatography, both of which have specific drawbacks. The authors took a different path for this target and developed a Mass spectrometry readout utilising the rapid-fire system.
In this assay format, crude human intestinal microsomes were allowed to react with substrate in the presence of test compounds using 384 well plates. The reaction was then quenched and transferred to the rapid-fire system (a solid phase extraction system). The samples where then measured on a triple quad mass spectrometer. The specific products of the reaction were then identified and a % inhibition determined for each test compound.
Enzyme activity monitored on a Mass spectrometry system. Adachi, R., Ishii, T., Matsumoto, S., Satou, T., Sakamoto, J., and Kawamoto, T. (2016). Discovery of human intestinal MGAT inhibitors using high-throughput mass spectrometry. Journal of biomolecular screening (in press).
One of the advantages of using the Mass spec based detection method was that as crude human intestinal microsomes were being used, both the production of DAG and TAG could be monitored in one reaction sample. This allows the ability to identify different enzyme inhibitors from one screen.
Remarkably the screen was carried out on 500,000 compounds at a screening concentration of 1μM. Given the cycle time of 10 seconds per sample, this suggests about 2 month’s work, assuming 100% up time for the Mass spec system. This is probably longer than a standard plate based biochemical assay could take to screen those number of compounds, however I would assume further improvements to this cycle time could occur with further technical development. The results showed the screen had an average Z prime of 0.7 and 0.83 for the different enzymes measured. Hit compounds were further classified with concentration response curves against a variety of different Monoacylglycerol acyltransferase subtypes (MGAT2, MGAT3), and they were able to release a structure of a selective compound in the paper and highlighted a number of other compounds which were identified.
The use of mass spectrum based screening has been highlighted in other publications, although mostly used at a hit confirmation stage of a screening cascade. This is the first time, I personally have seen it used as a primary screen with compound numbers of this size. The technique may open up primary screening of large compound collections on targets that have unable to be fully explored due to the failure to develop a robust assay format.
I do envision more targets using mass spectrometry as a primary screen and I think this publication is a step forward in that direction.
Blog written by Gareth Williams