The overall goal of this methodology is to identify the synergistic molecules, which amplify each others activity, when combined through High-throughput screening. Synergistic drug pairs are a promising strategy, for combating drug resistant infections but they are difficult to identify. This method can identify molecules that interact with existing antimicrobial drugs used to treat infectious diseases.
In the long term these combinations, can enter preclinical assessment and eventually improve patient care. The main advantage of this technique is that it can be done with any organism of interest and with any with any library of small molecules. The implications of this technique, extend towards therapy of multiple infectious diseases as it works with any organism of interest.
Use a standard spreadsheet to calculate the mean growth score and standard deviation for all the mutants, in the presence of a particular concentration of the small molecule added. Then, calculate the Z scores for each of the small molecule concentration. Then, highlight the gene mutants with significant Z scores.
First, grow an E.coli culture in M9 minimal medium, for overnight at 37 degrees Celsius. Then, dissolve the small molecules in dimethyl sulphoxide at a high concentration above 10 milligrams per milliliter. Next, add 100 microliters of M9 medium to each well in a 96 well plate.
Pipette an additional 100 microliters of M9 medium in column one such that the volume in that column is 200 microliters. Then, add approximately 10 microliters of the concentrated small molecule solution, in the column one. After adding the small molecules, start diluting it across the X axis of the plate.
Continue diluting till there is, 100 microliters in column 11. After mixing column 11, discard 100 microliters from that column. Let there be only medium in column 12 to normalize the data.
Next, record the optical density value at 600 nanometers of the overnight culture grown in M9 minimal medium. Then, add two microliters of the culture into each well. Such that there are 1000 cells per well.
Then, shake the plate gently. And note the optical density reading at 600 nanometers for zero hour time point. Once the reading is recorded, incubate the plate at 37 degrees Celsius for a day.
Again, measure the optical density value at 600 nanometers for 24 hour time point. To perform the checkerboard assay, grow an E.coli culture in M9 minimal medium for overnight at 37 degrees Celsius. Then, add 100 microliters of M9 medium to each well and add an extra 100 microliters of the same medium in column one of a 96 well plate.
Next, add the tests small molecules, in each well in column one. And add double the concentration in well A1.Then, create a dilution gradient along the X axis by transferring 100 microliters, from column one to column two. Continue the dilution process, until 100 microliters is added in column 11.
After mixing column 11, discard 100 microliters from that column and leave column 12 with only medium without any drugs. Create another gradient for the second drug dilution. To do this, add 100 microliters of the medium containing two X, minimal inhibitory concentration of the input molecule in row A.Then, transfer 100 microliters from row A to B.Continue the dilution process, until 100 microliters is added in row G.After mixing row G, discard 100 microliters from the well.
Then inoculate the plate with two microliters of the culture into each well. Such that there are 1000 cells per well. Then, measure the optical density reading at 600 nanometers for zero hour time point.
Once the reading is recorded, incubate the plate at 37 degrees Celsius for 24 hours. And then, measure the optical density value at 600 nanometers for 24 hour time point. To conduct High-throughput screening, for synergistic drugs, culture both wild type and synergy prediction mutant cells in M9 minimal medium.
Then, add the drugs from the library to the medium. Next, inoculate one plate of library drugs with the wild type and one other plate with synergy prediction mutant bacteria. This plate represents the checkerboard assay used to investigate synergistic drug combinations.
In this plate, well F9 with the lowest FICI score of 0.07 indicates that the small molecule pair is synergistic. This plate represents the checkerboard assay to investigate non interacting drug combination. In this plate, the well C3 with the lowest FICI score of 1.0 indicates that the small molecule pair, does not interact with each other.
This plate represent the checkerboard assay used to investigate antagonistic drug combinations. In this plate, well A1 with the lowest FICI score of 8.0 indicates that the interaction between, the drug molecules is antagonistic. Next Befeldin A, a small molecule drug in Fluconazole, is used to study the growth of C and U Formins.
The plot shows that the growth of both the wild type and the synergy prediction mutant is inhibited slightly and to the same extent. This indicates that Befeldin A and Fluconazole are non-synergistic drug partners. On the contrary, the growth of synergy prediction mutant of C and U Formins is inhibited significantly, in the presence of Refromisyn and Fluconazole.
Indicating that the drugs are synergistic partners. Interestingly, the wild type growth is not inhibited at all. Here, the greatest growth difference is observed, between 32 and 49 hours post inoculation.
Once mastered, this technique can shorten the time to identify synergistic drug interactions. While carrying out this procedure, it's important to remember not to combine drugs in the last column in the last row of the checkerboard assay. Following this procedure, other methods of synergistic molecule identification, can be performed in order to identify additional synergistic drug interactions.
After watching this video, you should have a good understanding of how to use the O2M methodology to identify putative synergy markers in order to find synergistic drug interactions with your organism of interest. Don't forget that while working with some small molecules can be hazardous and any precautions in the MSDS, should be followed while performing this protocol.
