Screening Core Project flow

HTS2

The typical project work-flow consists of four steps:

Project design

An investigator  brings a project idea to the Screening Core staff and discusses their screening needs, hypotheses and desired outcomes. Together, the Core and the investigator develop a project plan and sets project plans, goals and milestones.

Screening design

The Screening Core scientific team assists the investigator’s research team in designing and validating high throughput screens, with the bulk of the assay development and validation work performed by a researcher from the Investigator’s research team. This will be validated by performing a small subset screen (~2k compounds). Data is initially generated using a positive and negative control, generating 100-400 assay points for each. The mean and standard deviations for the two controls are calculated, and a z factor (a measure of assay robustness) is generated.  Based on this, the Screening Core can assess if the assay performance is suitable for screening, and the hit criteria are established.

Screening execution

The screen is executed by the HTS Core at single compound concentration (~1uM) per the design plans, screening against the compound screening set (~35k compounds) obtained from the BU Compound Repository. Screening data for each compound is deposited in a database, which is mined for compounds passing the hit criteria. Those that meet these criteria will be retested at a single concentration in order to confirm the hit. In the function-based screen, cell health is assayed at the end of the assay using Promega’s Cell Titer-Blue reagent. Compounds that impair cell viability are eliminated based on the fluorescence of alamar blue, a measure of cell viability that can be assessed following determination of function and on the same cells.  Confirmed screening hits are assayed at varying concentrations of compound to establish the ED50 (dose that generates half of the max effect).   Screening hits that produce a sigmoidal dose response curve are re-assayed to confirm the ED50.

Hit triage

Working with the cheminformaticist, the director of the MCCSF-BU assesses the hit matter obtained in the high-throughput screen on the basis of several criteria, including potency, chemical tractability and attractiveness, physical properties, and prior art in the area. Active hit compounds are re-procured by purchase or synthesis and the purity and identity checked (LCMS, NMR). Those that are confirmed pure and active are designated as “confirmed hits”, and rank ordered on the basis of efficacy and potency in the screening assay. These compounds will be provided to the investigator and flagged for potential collaborative follow-up within the BU-CMLD.