Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, Lake Nona, FL)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number:
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute

The solubility is one of the most fundamental physicochemical properties of drug candidates, and its measurement is essential in the in vitro evaluation of drug-like properties.

The early assessment of this property during the discovery stage provides important information for better interpretation of the screening results and designing of new molecules.

Drug candidates with low aqueous solubility can produce erroneous results during functional assays, thus increasing the risk of obtaining false hits or leads. Extra time and resources are required to develop a poorly soluble drug candidate.

Classical approaches for measuring solubility are based on the so-called saturation shake-flask method. The techniques of these approaches are slow and not well adapted to high-throughput needs of drug discovery research.

uSOL is our method of choice for determining the solubility of a compound. It is based on a 96-well microtitre plate format and a high speed detection UV detection system. Compound-dependent method optimization is not required. The concentration of a compound is determined by measuring the UV spectrum of a solution of the compound at equilibrium (saturated), and comparing it to the UV spectrum of a precipitation-free solution of the compound.

References:
1. Zhengyin Yan and Gary W. Caldwell. Optimization in Drug Discovery, In Vitro Methods.
2. Instruction Manual for uSOL Evolution Plus. pION Inc.
3. Avdeef, A. 2001. Physicochemical profiling (solubility, permeability and charge state). Curr Top Med Chem 1:277-351.

Assay Materials:

1) Aqueous buffer solution (System Solution, pION Inc) pH 5.0, 6.2, and 7.4.
2) 96-well filter plates (pION Inc).
3) Spectroscopically pure 1-Propanol (Sigma).
4) 96-well microplates and 96-deep well plates.
5) Control compounds: Diclofenac Na and Dipyridamole, final concentration in assay: 500 uM.
6) Test compounds, final concentration in assay: desirably as high as the control compounds.
7) The Tecan Freedom Evo 150 automated liquid handling instrument (Tecan US) is used in this assay.

Assay Protocol:

1) Samples are incubated in aqueous buffer solution at room temperature for a minimum of 18 hrs to achieve equilibrium (assay DMSO final concentration: 1%).
2) The samples are then filtered to remove any precipitate formed.
3) The concentration of the compound in the filtrate is measured by UV absorbance (250-498 nm) using the Infinite M200 (Tecan US) and compared to the spectra of the precipitation-free reference solutions.
4) Spectroscopically pure 1-Propanol is used as a cosolvent to suppress precipitation in the reference solutions.
5) The solubility of each compound is determined using uSOL Evolution Plus software v3.2 (pION Inc) and is expressed as the concentration (ug/mL) of a solute in a saturated solution.

Solubility Classification:
<10 = low solubility
10-60 = moderate solubility
>60 = high solubility

In this assay compounds with a solubility in aqueous buffer >= 10 ug/mL at pH 7.4 are considered as active.

Scoring for single-concentration screening:

Activity scoring rules developed at San Diego Center for Chemical Genomics employs a 3-tiered system:

1) The first tier (0-40 range) is reserved for primary or single-concentration screening data and is not applicable to this assay.

2) The second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits that are cherry picked from the HTS mother plates and is not applicable to this assay.

3) The third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues.

Compounds that are inactive in this assay receive a score of 81.
Compounds that are active in this assay receive a score of 90.