Aqueous Solubility from MLSMR Stock Solutions
Solubility is one of the most fundamental physicochemical properties of drug candidates or chemical probes and its measurement is an essential component in the in vitro profiling of drug-like properties. ..more
BioActive Compounds: 40282
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: SBCCG and ANALIZA, Inc. Cleveland, OH.
Solubility is one of the most fundamental physicochemical properties of drug candidates or chemical probes and its measurement is an essential component in the in vitro profiling 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.
Compounds with low aqueous solubility can often 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 probe candidate.
Solubility is widely recognized as a critical parameter in both early and late stages of probe and drug discovery. Solubility is defined as the concentration of a dissolved compound in equilibrium with a solvent. Early HTS campaigns are primarily interested in solubility data to assess binding data, essentially asking the questions "is the compound precipitating in the well and is it tested at the presumed concentration(s)?" These simple questions are only answerable by measuring solubility under the conditions which the biological screen is conducted. Chemical probe libraries suffer from the same fundamental ambiguity. In a direct study of this issue, Popa-Burke and coworkers have demonstrated that a high percentage of library compounds have low aqueous solubility. Other unpublished studies in the industry have noted almost 10-fold increase in hit rate from libraries with soluble compounds vs. libraries with conventional mix of early discovery compounds - with all other screening parameters remaining the same.
Measuring solubility under biological assay conditions early in the discovery process can identify potentially ambiguous activity data such as false negatives due to poor solubility therefore improving the overall efficiency of activity screening and hit identification.
1. Kearns, E.H.; Di, L. (2008). Drug-like Properties: Concepts, Structure Design and Methods. New York: Elsevier Inc; p. 56.
2. Popa-Burke et al. (2004). Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results. Analytical Chemistry, 76, 7278-7287.
3. Bhattachar, S. et al. (2005). Evaluation of the chemiluminescent nitrogen detector for solubility determinations to support drug discovery. Journal of Pharmaceutical and Biomedical Analysis, 41, 52-157.
4. Lechner, P. et al. (2007). Improved Accuracy of Solubility and Dosage Measurements by Simultaneous Measurement of Library Concentration Using Chemiluminescent Nitrogen Detection AAPS Annual Meeting and Exposition.
Measure the solubility of MLSMR compounds, provided as DMSO stocks, in aqueous buffer using quantitative chemiluminescent nitrogen detection.
1. Aqueous buffer solution: Phosphate Buffered Saline, pH 7.4. (Fisher Bioreagent part number BP399-500)
2. 96-well filter plates with 0.45uM polycarbonate membrane (Millipore, Inc).
3. 96-deep well plates (Abgene)
4. EZ-Peal Heat Seals (Abgene)
5. Control compounds: 10mM Stock solutions in DMSO (Imipramine HCl, Sulfamethizole and Astemizole) final concentration in assay: 200 uM.
6. Test compounds: 10mM Stock solutions in DMSO, final concentration in assay: 200 uM
7. The Hamilton STARlet automated liquid handling instrument (Hamilton) is used in this assay
8. Automated Discovery Workstation (ADW) (Analiza, Inc.) is used for analysis
1. The compound must contain nitrogen in its molecular formula, and must be provided as a DMSO stock (10mM preferred) which is otherwise free of nitrogen containing solvents or modifiers.
2. The compound ID, well location, formula weight, and number of nitrogen atoms per molecule must be provided in an Excel spreadsheet for each compound.
3. Samples will be submitted in Abgene 96 well deepwell polypropylene storage plates containing at least 10uL of 10 mM DMSO stock per buffer to be used. Samples will be shipped frozen on dry ice and will be sealed with a polypropylene heat seal (Abgene). Other labware may be used if pre-certified for lack of nitrogen background.
Sample Preparation, Incubation and Filtering:
1. Plates will be allowed to thaw at room temperature and then will be placed in a 40 oC sonic water bath for 10 minutes to facilitate resuspension. Any wells containing insoluble residue will be noted in the report.
2. Hamilton STARlet robotic liquid handling will be used to prepare 50-fold dilutions of stocks by combining 6 ul aliquots with 294 ul buffer directly into a Millipore solubility filter plate. The final DMSO concentration will be 2%, and the theoretical maximum concentration will be 0.2mM if source concentration is 10 mM. The same process will be used to prepare reagent blanks.
3. Plates will be heat sealed, and incubation will be carried out at ambient room temperature (ca. 22.5-24.5 C) for 18-24 hours with shaking at 200 rpm.
4. Plates will be vacuum filtered to prepare the filtrates for injection into the nitrogen detector. All wells will be inspected for sufficient volume of filtrate for assay, with exceptions to be reported in the Solubility Comments column of report.
1. Filtrates will be injected into the nitrogen detector using Automated Discovery Workstation (ADW). The concentrations of filtrates will be determined by chemiluminescent nitrogen detection.
2. Since the nitrogen detector is equimolar in response to total nitrogen content, a universal detector calibration curve is used to quantify compounds rather than compound-specific standard curves, as required by other detectors. The only exceptions are compounds having adjacent nitrogen atoms.
3. Results will be reported in uM, and ug/mL using the formula weights and nitrogen molar content provided by the customer.
4. Consideration of the reported concentration in comparison to the theoretical maximum concentration if fully dissolved must be made by the customer, particularly in light of compounds having adjacent nitrogen atoms which may exhibit submaximal response.
In this assay compounds were tested one or more times and those with a mean solubility in aqueous buffer > 10 ug/mL at pH 7.4 are considered as active.
Solubility Classification (in ug/mL):
<10 ug/mL = low solubility (Inactive)
10-60 ug/mL = moderate solubility (Active)
>60 ug/mL = high solubility (Active)
Scoring for single-concentration screening:
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics employs a 3-tiered system:
1) First tier (0-40 range) is reserved for primary screening data.
Compounds that are inactive in this assay receive a score of 0
Compounds that are active in this assay receive a score of 40
2) Second tier (41-80 range) is reserved for dose-response confirmation data and thus does not apply here.
3) Third tier (81-100 range) is reserved for dry powder SAR compounds and thus does not apply here.
Data Table (Concise)