Small Compound Screening Overview

Introduction

This report is designed to be an introduction to small compound high throughput screening (HTS) at the Target Discovery Institute, Nuffield Department of Medicine, University of Oxford. The TDI benefits from a broad range of experience across a wide range of screens and our scientists have extensive knowledge of high throughput screening. We are available for consultation at the earliest stages of assay conception and we will work closely with each investigator through all stages of assay development, optimization, automation and screening.

HTS Transfer Protocols

All assays being transferred to the Janus for HTS will be subjected to a thorough validation process before the production screening beings.

This validation process will consist of:

1. Initial Consultation
2. Stability and Process study
3. Liquid Handling Validation
4. Plate Uniformity Assessment
1. Control Validation, Z’calculation, Assessment of edge effects and drift within the body of the screen
2. Replicate Experiment
1. We require as a minimum a 2 replicate study over 2 different days for biological reproducibility and robustness
2. Pilot Screen
3. a small number of plates containing compounds of varied pharmacologically activity and the chosen controls
4. Production Runs

1. Initial Consultation

The initial consultation with the HTS facility manager is an opportunity to discuss your ideas for a potential screen during the conceptual stage or as you begin the bench-top development.

We have experience across a diverse range of screening platforms and formats and have run a wide variety of screens in our facility. We are able to advise you on what might be the most effective approach (both in terms of time and cost) for the question you would like to investigate.

We have also developed a number of novel screening approaches designed to answer specific criteria and we welcome the opportunity to develop additional novel screens.   We would encourage you to discuss your proposed assay at the earliest stages of development as we can assist you with the following issues:

1. There are several types and many approaches to HTS. Each type of screen has merits and demerits (i.e. - easy to set up but requires more consumables), we will discuss these in full to determine the most appropriate screening regime for you.
2. Your choice of reagents - Some reagents have limited stability, may be very expensive compared to alternatives, susceptible to pH or temperature changes, etc. make them unsuitable for HTS.
3. Specific Phenotype Optimization - Optimizing the phenotype under investigation is the single most crucial step to successful screening. We can help you devise a specific protocol to target the best cell density, cell cycle stage, target gene knock-down, minimise cytotoxicity and optimise the most appropriate assay reagents
4. We can begin to discuss logistics and timings of your assay as well as the projected cost.

An initial consultation at the earliest stage of assay development will ensure a smooth transition from bench-top to HTS.

2. Stability and Process

The stability and process study is the initial step of the HTS assay validation. It will be necessary to examine all reagents used in the assay for the following properties:

1. Stability during production.
2. Storage stability - for any un-used reagents you may wish to store for future assays.
3. Emergency stability - the stability of assay reagents at room temperature in the unlikely event of a robotics failure
4. Time-course experiments to determine the range of acceptable times for each reagent in process. This is of particular importance for cell health which may be subject to changes over time as plates are seeded throughout the course of the experiment.

This study may be run concurrently with the final steps of the liquid handling validation process and will be the responsibility of the assay developer.

3. Liquid Handling Validation

Once your proposed screen assay has met the criteria for moving from the bench-top to the Janus Liquid Handling Work Station, the programming of all assay steps on the liquid handling robotics will become the responsibility of the high throughput developer (TDI operator).

1. Initially, we will meet to thoroughly discuss the processing steps in the protocol. Any questions or clarification should be addressed here so that adjustments may be made to the protocol at the earliest opportunity.
2. Within a couple of days a written time-line for the development of the screen will be produced and must be agreed by the assay scientist(s) and ourselves.
3. Once the High Throughput liquid handling protocol writing is complete, we will validate all steps of the protocol together using coloured dyes to track all liquid handling steps. You will thoroughly evaluate the protocol at this point. There will invariably be alterations from your protocol and any variations will be approved by you.

4. Plate Uniformity Assessment

Once your proposed screen assay has passed the Liquid Handling Validation step, a Plate Uniformity Assessment will be conducted (Detailed below in this report). This is a series of experiments designed to address:

1. Drift – this usually seen as a left right shift across the body of the screen.
2. Edge Effect – this is usually seen along the perimeters of the plate. As a standard approach in a 384 well plate we leave the outer row and columns empty of compounds. For 96 well plates, edge effects are generally minimised by a larger volume in each well.

* In general, Drift or Edge Effects <20% are considered acceptable and random effects seen on <20% are also considered acceptable. If HTS protocol adjustments are necessary to address unacceptable Drift or Edge Effects, they will be made by the HTS developer and approved by you.

5. Replicate Experiment - Final Assay Validation

This is a set of experiments designed to validate the HTS procedure immediately before the Production runs.  Along with the Plate Uniformity Assessment, the Replicate Experiment will ensure:

1. A set of experiments, a "dry run", using all components of the assay will be performed. We will seed plates and carry out all steps of the assay to produce a Z' Factor [1], to determine if the screen produces data with sufficient sensitivity and specificity to accurately differentiate between positive "hits" and negative samples.
2. Once an acceptable Z’ has been found (this is usually in excess of 0.3 for cell based HTS) a minimum of two concentration pilot studies will be used to validate the procedure using Janus Liquid Handling robots (detailed below in this report).
3. Inter- and Intra-Plate Variability will be assessed using a standard coefficient of variance calculation (CV) for the body of the screen - an acceptable CV range will fall within 10%.
4. All Plate and Assay controls will be assessed.

If all aspects of the HTS assay are within acceptable range for the above criteria, we will proceed to the Production run.

6. Production Run

1. Run the HTS assay. This is where your HTS assay produces reams of data to be written up in high impact scientific journals.
2. Post-Production evaluation and report. See appendix 1 for HTS Final Report layout.

Types of Small Compound High Throughput Screens

High throughput screening assays can be divided into two main types:

1. Protein based biochemical screens - These screens use purified proteins, substrates and small compound inhibitors in buffered solutions to produce an optical (fluorescent, luminescent, etc.) readout that monitors enzymatic or binding activity using high throughput plate-readers. Small compound inhibitors are ranked on their ability to reduce the protein function, and by extension the optical signal, in this type of screen.

Advantage: Very high throughput, small volume reactions reduce reagent costs and simple readouts. Target of the inhibitor is defined.

Disadvantage: Small compound may not be water soluble, membrane permeable and may be promiscuous or toxic. Single protein inhibition may not cause desired phenotypic change in cells or tissues due to redundant pathways.

2. Cell based screens- These small compound screens utilize cells plated in 96 or 384 well plates to produce a visual phenotypic change in the cells which can be quanitified. The three general measurement types (although others exist) of a cell based screen are:

Uniform well readouts - These include cell viability assays such as Resazurin turn over and may include simple reporter gene assays. These assays usually employ high throughput platereaders for measurement nd are generally luminescence, absorbance or fluorescence based.

High-Content Imaging Screens - These screens are designed to probe changes to a cellular phenotype (i.e. foci formation screens, nuclear and cellular morphology, localization of proteins, etc).  HCI screens employ specialized high content imagers to produce high content images which can be used to measure phenotypic changes. These assays are content (and data) rich and can provide in-depth analysis of cellular changes. In addition, they can also be "multi-plexed" with multiple probes for DNA, membrane and specific protein staining to provide large-scale information on several changes to a cellular phenotype. They can also provide cell cycle information over the course of the assay.

The high-content images generated for these types of screen are held indefinitely and may be re-interrogated at a later stage for more in-depth analysis as information becomes available. Plates which contain fixed cells are also stored where possible in case plates are required for further staining and analysis.

Reporter gene systems - These may take the form of high throughput FACS based assays producing readouts of GFP, luciferase, etc. internalized signal; or a more simplified high throughput approach utilising plate-readers for measurement generally in the form of luminescence, absorbance or fluorescent signal

Advantages: Cell based assays produce phenotypic changes affecting pathways directly associated with disease states in which the target protein or pathway may not beknown. Small compound cell based screens ensure compound solubility, membrane permeability, non-toxic and effectiveness at low, therapeutically relevant concentrations.

Disadvantages: Lower throughput than protein based assays, much more technically difficult and much longer duration (days/weeks vs. minutes). Target of inhibitors are not definitively known.

Criteria for Developing Robust Small Compound HTS