Dose / Concentration Response 

After hits have been validated it is important to generate an EC50 or IC50 for each compound and rank them by their potency.

In general we want lower EC50/IC50s, meaning more potent compounds, moving forward through drug discovery and development. More potent compounds typically have better on target activity and reduced off target effects.

How does the ACDD perform dose response studies?

  • Compounds are cherry picked from the compound library and plated a special format in a 96 well pcr plate in row A.
  • Then the plate moves to the Biomek FX robot where DMSO will be added to the rest of the plate and serial dilution.
  • First a fixed volume of DMSO is added to rows B-H. The robot then serially dilutes the compounds from row A vertically down the plate in either 1:2 or 1:3 dilution steps creating 8 concentrations for each compound.
  • The plate is either immediately used in assays or foil sealed and stored @-20C. Each dose response source plate has a custom plate map uploaded to CDD Vault with compound ID and concentration information.

96 well source plates can be directly pin transferred to a 96 well assay plate OR 96 well source can be quad pinned into a 384 well assay plate.

In the 384 well scenario each dose of each compound is tested 4 times (n=4) resulting in high quality dose response curves.

We like the 96 to 384 quad pin protocol because it keeps all replicates necessary for the dose response curve on a single plate thus eliminating plate to plate variation.

In order to manually pipette a dose response curve and keep the DMSO down below 0.5%, intermediate dilutions are necessary. In this example, 10mM compound stock is diluted into a volume of media, then a fraction of that volume is transferred to the assay plate.

Example Case in 384 Well Plate with Cells

Manual pipetting method
Cells in 46ul of media

  1. Dilute 1ul of 10mM stock in 80ul of media in a 384 well plate. This results in a 125uM intermediate concentration with an intermediate DMSO % of 1.25%.
  2. Transfer 4ul of the 125uM intermediate into 46ul of cells. This results in a final concentration of 10uM and final DMSO % of 0.1%.

Direct pipetting method
Cells in 49ul of media

  1. Predilute compounds from 10mM down to 500uM in DMSO.
  2. Pipette 1ul of compound into 49ul of cells. This results in DMSO % of 2% and final of 10uM.

Direct pintool method
Cells in 50ul of media

  1. Pintool transfer 50nl of compound into 50ul of cells in 384 well plate. This results in a final concentration of 10uM and DMSO % of 0.1%.

In most cases edge effect cannot be completely eliminated but can be minimized using a few different alterations to the lid.

  1. Breathe easy seals replace the plastic lid with a gas permeable adhesive seal that allows each well to breath similarly to each other.
  2. Custom metal lids with drilled holes and rubber gasket can replace the plastic lids and provide significantly better breathing and temperature consistency.

Benefits of Pinning Versus Manual Pipetting

In order to manually pipette a dose response curve and keep the DMSO down below 0.5%, intermediate dilutions are necessary. In this example, 10mM compound stock is diluted into a volume of media, then a fraction of that volume is transferred to the assay plate.

Example Case in 384 Well Plate with Cells 

Manual Pipetting Method (Cells in 46ul of Media)

  1. Dilute 1ul of 10mM stock in 80ul of media in a 384 well plate. This results in a 125uM intermediate concentration with an intermediate DMSO % of 1.25%.
  2. Transfer 4ul of the 125uM intermediate into 46ul of cells. This results in a final concentration of 10uM and final DMSO % of 0.1%.

Direct Pipetting Method (Cells in 49ul of Media)

  1. Predilute compounds from 10mM down to 500uM in DMSO.
  2. Pipette 1ul of compound into 49ul of cells. This results in DMSO % of 2% and final of 10uM.

Direct Pintool Method (Cells in 50ul of Media)

  1. Pintool transfer 50nl of compound into 50ul of cells in 384 well plate. This results in a final concentration of 10uM and DMSO % of 0.1%.

Why do we dilute compounds in DMSO and not media / buffer?

Small molecules / drugs typically have limited solubility in water/aqueous solutions compared to organic solvents like DMSO. When screening commercial collections, most compounds are soluble to 10mM DMSO while water solubility for many will only be 10uM or lower. If a compound with low water solubility of 10uM is prediluted from 10mM (DMSO) to 500uM in media / buffer, the compound will crash out / precipitate out of solution. If the compound precipitates out, it’s very difficult to control how much compound is transferred in each pipetting step resulting in noisy data.