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Improving the drug development process

Assay Development & Screening

One of the first steps in drug development and toxicity testing is creating test systems (assays) on which to evaluate the effects of chemical compounds on cellular, molecular or biochemical processes of interest. Investigators from the biomedical research community submit ideas for assays to NCATS scientists, who help enhance them for high-throughput small molecule screening. The results of these screens, called probes, can help researchers further explore protein and cell functions, biological processes, and effects of environmental chemicals that are relevant to human health and disease. In addition, these probes can become potential therapeutic candidates in the drug development pipeline.

NCATS Capabilities

NCATS experts develop innovative assay and screening methods to enable a wide range of pre-clinical activities. Specific capabilities include assay design, optimization, validation and miniaturization; high-content and phenotypic screening; and gene expression analysis. Assay guidance criteria are available below.

Quantitative High-Throughput Screening (qHTS)

For the Center’s screening purposes, NCATS experts use qHTS, a process in which each compound of a large chemical library is tested at multiple concentrations. Through automated robotic screening, the technology integrates the pharmacologic dose-response relationship with the speed and accuracy of automated HTS. The qHTS method has enhanced the productivity of multiple NCATS activities, and it also has provided the most detailed picture to date of how assay artifacts and library diversity affect interpretation of HTS results.

Specific features of the NCATS qHTS platform include:

  • Automated robotic screening
  • 1,536-well format
  • Screening of approximately 40 plates per hour and about 1.5 million compounds per day
  • Pin-tool dispensing
  • Acoustic dispensing
  • Plate incubation and chemical library storage
  • Compounds assayed at 15 concentrations ranging over 4 logs (up to 100 µM)
  • Miniaturization of assay volumes from 2 µL to 6 µL in 1,536-well plate
  • Data processing, curve-fitting and classification through an informatics pipeline
  • Generation of pharmacological actives rather than statistical “hits” (increases reliability and reduces false-positives and -negatives)

Matrix Combination Screening

Matrix combination screening is an unbiased, high-throughput means to explore hundreds and even thousands of drug-drug pairs for their synergistic, additive and/or antagonistic activities. NCATS scientists use this platform to define potential therapeutic combinations for further testing.

Chemical, Pharmaceutical and Natural Product Library Exploration

NCATS scientists use qHTS to explore the structure and activity of compounds from the Center’s chemical, pharmaceutical and natural product libraries. The information that emerges from these studies can provide clues to agents’ potential as therapeutics or chemical probes.

Chemical Biology

Biomedical research functions most efficiently when investigators use chemistry and biology together to address common problems and goals. In service of NCATS’ assay development and screening efforts, chemistry technology projects address fundamental problems and insufficiencies in molecular biology and drug discovery. NCATS supports chemical technologies ranging from novel library design to inventive bioanalytical techniques.

Phenotypic Screening and Drug Repurposing

Scientists carry out phenotypic screening in early-stage disease research. The results of these efforts inform subsequent target identification and validation studies.

High-Throughput Screening Assay Guidance Criteria

 Criteria

Biochemical

Cell Based

Plate format*

96-well or higher density plate

1,536-well format

Assay volume 2-6 μl

96-well or higher density plate

1,536-well format

Assay volume 4-6 μl

Assay steps

= 10 steps with 96-well plate

Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc.

= 10 steps with 96-well plate

Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc.

Minimum time increments and maximum assay duration

Minimum assay window is 5 min. (i.e., earliest time point after last reagent addition)

< 24 hour is ideal; max is 48 hours

Minimum assay window is 5 minutes

Reagent addition steps

4 maximum (4 unique reagents max, more if pre-mixed)

4 maximum (4 unique reagents including cells max, more if pre-mixed)

Reagent removal steps*

No plate coating steps

Aspiration steps*

Temperature

Between RT and 37°C

Between RT and 37°C

Demonstrated DMSO tolerance*

0.5 - 1% DMSO

0.5 - 1% DMSO

Signal: background ratio

= 3-fold

= 3-fold

Day-to-day variation of control (e.g., IC50, EC50)

< 3-fold

< 3-fold

Reagent stability at final working concentration

= 8 hours @ RT or on ice bath

No online thawing

= 8 hours @ RT or on ice bath

No online thawing

Validation run reagent supply

10 - 96-well plate equivalents

10 - 96-well plate equivalents

Protocol

Complete detailed protocol

All steps, equipment used, all vendor and catalog numbers for reagents

Data from 96-well or high-density plate tests

Complete detailed protocol

All steps, equipment used, all vendor and catalog numbers for reagents

Detailed cell culture procedure, passage number

Data from 96-well or high-density plate tests

Detectors

PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence)

PE Envision (bottom reading FI, ALPHA)

Acumen Explorer (fluorescent laser cytometry)

PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence)

PE Envision (bottom reading FI, ALPHA)

Acumen Explorer (fluorescent laser cytometry)

Special

For unique reagents, either investigator prepares sufficient quantity for screening or identifies a reliable third-party vendor.

Cells must be certified micoplasma-free by direct culture assay and cell-DNA fluorochrome staining.

*Table Notes

Plate Formats: 96-well plates contain 8 rows x 12 columns with volumes ranging between 50-200 μl; 384-well plates contain 16 rows x 24 columns with volumes ranging between 30-50 μl; 1,536-well plates contain 32 rows by 48 columns with volumes ranging between 2-8 μl. NCATS will convert assays to 1,536-well from 96- or 384-well format.

Reagent removal steps: Are any step that requires the removal of material from the well of a microtiter plate. Although such steps may be routine with 96-well plates, they are not recommended on robotic systems using 1,536-well plates.

Demonstrated DMSO tolerance: Because all compounds screened are stored in ~100% DMSO and delivered as a 1 to 100 dilution to the assay, the assay sensitivity to between 0.5 percent and 1 percent DMSO must be determined.

Last updated: 07-17-2017
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