A. Basics of high throughput screening (HTS): Bridging chemistry and biology

(James Inglese)

HTS equation: chemical libraries + assays (automated) = informatics, followed up with the identification and prioritization of hits

  • Quantitative HTS (qHTS) performed by looking at concentration response profiles.

There are multiple types of chemical libraries to choose from.

  • Small to moderate in size:
    • synthetic bioactives and natural products libraries; includes approved drugs, chemogenomic collections, and natural product extracts
    • privileged scaffold-based libraries; includes untested analogs of synthetic drugs or natural products
  • Moderate to very large in size:
    • biologically uncharacterized libraries (with low diversity, high density); includes combinatorial chemistry-derived libraries
    • consolidated samples/collections (with extensive structural diversity); includes Molecular Libraries Small Molecule Repository (see PubChem)
  • Biological libraries (e.g. siRNA, antibodies, etc)
  • See TABLE 1 of example chemical libraries used in drug discovery.

Libraries with more complex compounds could allow for increase selectivity and specificity

  • Example: Boston University Chemical Methodology & Library Development Center (BU-CMLD): natural product inspired, with more ring systems and stereogenic centers (compared to commercially available libraries)

Re-purposing and re-indication of approved drugs are time and money efficient.

  • There are libraries of approved drugs (~3000 compounds).
  • A significant benefit is that existing drugs have already been optimized for human use and have known profiles.
  • This strategy may reduce preclinical and regulatory hurdles.

HTS assay - it is critically important to ensure the use of an appropriate assay that measures a biological process of interest in the disease

  • Assay development is an iterative process.
  • There are important factors to consider when moving from a “bench top” assay to a high throughput assay (see TABLE 2).
    • There is an amplification of variation when moving from “bench top” to HTS, which requires careful optimization

HTS assays are becoming increasingly diverse, with many assay types available based on:

  • Purified molecular targets (e.g. pro-fluorescent substrates, enzyme cascades, coupled-enzyme reporters.
  • Cell extracts (e.g. membrane preparations)
  • Cellular/organism phenotypes (e.g. reporter gene, cellular sensors, multiparametric imaging, model organisms)

HTS can be based on isolated molecular targets and cultured cells (monolayers or suspensions). Automated microscopy can be used for medium-throughput screening of complex phenotypes.

Operational efficiency can be increased with miniaturization.

  • In a 1536 well microtiter plate format, the same percentage of plate space as a 96 well will allow for full control titrations (for quantitative HTS).
  • Parallel processing of miniaturized assays will greatly decrease the amount of screening time needed (dependent on plate type/well density and assay design).

HTS assay formats:

  • ligand binding (e.g. competitive)
  • enzymatic activity (e.g. biochemical, cellular)
  • ion/ligand transport (e.g. ion-sensitive dyes, membrane potential dyes)
  • protein-protein interactions (e.g. biochemical, cellular)
  • cellular signal transduction (e.g. reporter gene, second messenger)
  • phenotype (e.g. cell viability, protein redistribution, multiparametric imaging)

HTS detection methods:

  • absorbance
  • radioactivity (e.g. scintillation proximity assay)
  • luminescence (e.g. chemiluminescence, bioluminescence, bioluminescence resonance energy transfer (BRET), Amplified Luminescent Proximity Homogeneous Assay (ALPHA)
  • fluorescence (e.g. fluorescence intensity (FI), fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF), fluorescence polarization (FP), fluorescence correlation spectroscopy (FCS), fluorescence lifetime (FLT)

Artifacts can occur with HTS – apparent activity in a HTS can be false positives resulting from various non-specific effects that interfere with the assay (see TABLE 3)

  • Use assays with built-in orthogonal readouts (directly or inversely proportional) for the same biological process.
  • It’s recommended to use two different reporters that complement each other for orthogonal assays (e.g. luciferase + fluorescence).

EXAMPLES (see refs below):

  • Dual bioluminescent reporter cell-based assay (Davis 2007)
  • HTS for epigenetic modulators (Hager 2006)
  • Microplate laser scanning cytometry analysis of HTS LDR assay (Auld 2006)
  • HTS assay with built-in orthogonal confirmation assay (Johnson 2008)
  • Construction and treatment of concentration response curves (CRCs) in qHTS (Inglese 2006)

Multiple components joined together for a HTS robotics platform: reagent and compound delivery systems + signal detection modalities.

qHTS process allows pharmacology at the primary screening stage.

Intro >