At Perlara, we work to find therapeutics for rare genetic diseases. We develop and use immunoassays, phase contrast imaging, as well as enzymatic assays, in order to screen thousands of compounds and obtain hits from cell readouts to complement our model organism screens. For two diseases we are currently working on, Gaucher and Phosphomannomutase 2 (PMM2), we have found that enzymatic assays will help us determine if our model organism screening hits are altering enzymatic activity as way of rescuing the disease phenotype.
Enzymatic assays
We use enzymatic assays when a genetic mutation is known to affect an enzyme and cause the enzyme activity levels to drop. A basic understanding of the enzymatic reaction taking place is key to developing any enzymatic assay. Some of the common factors to be considered while developing an enzymatic assay are pH, temperature, coenzymes, activators, substrate and enzyme concentrations. By altering one or more of these common factors, we can drive/control the enzymatic reaction and optimize it for our specific purpose. Most of the enzymatic assays are performed near the physiological pH of 7.5 and body temperature of 37 °C.
Intact cell enzymatic assays are physiologically more relevant than enzymatic assays performed with cell lysates. Depending on whether appropriate substrates are available for an intact cell assay, these can be developed for any disease.
High Throughput enzymatic assay on Gaucher fibroblasts
In the case of Gaucher disease, mutation in GBA1 gene causes glucocerebrosidase enzyme activity levels to drop. Glucocerebrosidase is the enzyme that is responsible for the breakdown of glucosylceramide into glucose and ceramide. A deficiency in Glucocerebrosidase results in the accumulation of glucosylceramide in the lysosomes of cells. The most common patient mutations are L444P and N370S. We have fibroblast cell lines from Coriell with both the mutations in-house. I adapted a high throughput (HT) intact cell enzymatic assay mentioned in the literature for our purpose of characterizing screening hits. In the HT Gaucher intact cell enzymatic assay, cells are seeded on destination plates that contain drugs and incubated for a certain period of time. Subsequently, an assay buffer containing substrate is added, and the plate is read after allowing the reaction to take place for 70 minutes (Figure 1). In this case, the assay buffer has a pH of 4.5 since the reaction we are interested in takes place inside lysosomes (acidic). The blue fluorogenic substrate fluoresces upon the activity of glucocerebrosidase and its consequent hydrolysis into glucose and the blue fluorophore. A stop solution with pH 10 is added after 70 minutes to raise the pH and consequently, stop the reaction. The fluorescence is then read with a plate reader at 365 nm excitation and 440 nm emission. There are wells with only assay buffer and this reading is subtracted from the other readings as background fluorescence adjustment. The readings are normalized to the number of cells per well.
Figure 1. Gaucher enzymatic assay schematic diagram
Developing a novel HT PMM2 enzymatic assay
The second disease we developed an enzymatic assay for is Phosphomannomutase 2 deficiency (PMM2). Unlike the Gaucher assay, there was no published way to conduct PMM2 enzymatic assay in high-throughput, so we ultimately set out to accomplish that task!
Phosphomannomutase 2 deficiency is caused by a defect in the PMM2 gene and the most common patient mutations are R141H and F119L. We obtained a patient fibroblast cell line (GM20942) which is a compound het with R141H and F119L alleles. There were no suitable substrates available in the market to perform an intact cell assay. We found a paper from 1995 (Jaak Jaeken, 1995, pp. 318-320) with a PMM2 enzymatic assay protocol, which people have been using up until this day with a few modifications.
Reaction:
In the above reaction, mannose-1-phosphate behaves as the substrate and each of the consequent products are formed because of the enzymes we add (1 – Phosphomannomutase from cell extract, 2 – phosphomannose isomerase, 3 – phosphoglucose isomerase and 4 – glucose-6-phosphate dehydrogenase which is an activator of PMM2). We also add NADP which is reduced to NADPH+, and the absorbance is read by the plate reader at 340nm.
Initially, I tried the conventional way of using cell lysates from a few 10 cm Petri dishes for each condition, but we needed to have an assay that worked in a higher throughput manner. When we develop assays, we aim to develop an assay that can be used in a screen of thousands of compounds. Even to use an assay as a secondary screen, we need it to be high throughput in order to test tens of hits that come out of our large library model organism screens. So we, as a team, started to think of ways this could be done. In the Jaeken paper, there were a number of steps to obtain the cell extract which included obtaining cell pellets by scraping the cells off the 10 cm plate, adding homogenization buffer, freeze-thaw cycle, and then centrifuging the homogenized pellet to obtain the supernatant (cell extract). In order to make the assay more high-throughput, we decided to seed cells in a 96-well plate, homogenizing them right in the well, then adding the assay buffer and consequently taking readings.
I tried two different concentrations of substrate to see if that would give me better readings. I found that it worked but the readings were not high enough above the background (Figure 2), to be able to have a nice difference between our positive controls and diseased cells. In one of our cell team meetings, we came up with the idea of testing 2x and 3x the amount of protein, by homogenizing one well, transferring the homogenized cell contents along with the buffer to the next well, and repeating this again. So, essentially, we had 3x the amount of protein as compared to that in one well. Another thing that we thought was worth testing was incubating the assay plate at a higher temperature, i.e. at 37 °C instead of 30 °C which was suggested in the Jaeken paper. By making these optimizations, we found that we got high readings as well as a good separation between WT and PMM2 fibroblasts (Figure 3). We are going to normalize to total protein amount using the Qubit since it requires only 1 mL of cell extract.
Figure 2. High throughput PMM2 enzymatic assay with WT fibroblasts, tested with 2x and 4x the amount of substrate
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b.
Figure 3. High throughput PMM2 enzymatic assay with WT and PMM2 fibroblasts (GM20942), 2x the amount of substrate and a) plate incubated at 37 °C, b) plate incubated at 30 °C
Once this assay is completely optimized, it can be used as a secondary assay to characterize hits coming out of our yeast, worm and fly PMM2 model organism pipelines. Stay tuned for our updates!
