SOP for High-Throughput Screening (HTS) in Drug Discovery

Standard Operating Procedure (SOP) for High-Throughput Screening (HTS) in Drug Discovery

1) Purpose

The purpose of this Standard Operating Procedure (SOP) is to describe the process for conducting High-Throughput Screening (HTS) in drug discovery. HTS is a crucial technique used to rapidly test large numbers of compounds against a biological target to identify those that exhibit desirable biological activity. This SOP ensures that HTS is conducted efficiently, reproducibly, and in compliance with regulatory guidelines, leading to the identification of promising lead compounds for further drug development.

2) Scope

This SOP covers the entire process of HTS, from compound library preparation and assay design to data collection, analysis, and hit identification. It is applicable to all teams involved in HTS within the drug discovery process, including screening scientists, laboratory technicians, data analysts, and project managers. This SOP applies to HTS conducted in both academic and commercial settings for the identification of potential drug candidates across various therapeutic areas, such as oncology, infectious diseases, and neurodegenerative disorders.

3) Responsibilities

Screening Scientists: Responsible for designing the HTS assays, selecting appropriate biological targets, and ensuring that the assays are optimized for high-throughput applications. They also ensure that the screening process is conducted with

precision and accuracy, and they analyze the screening data for hit identification.

Laboratory Technicians: Assist in setting up and conducting the HTS assays, preparing compound plates, maintaining equipment, and ensuring that the laboratory environment meets necessary standards for high-throughput screening.

Data Analysts: Responsible for analyzing the HTS data to identify potential hits. They use statistical tools and software to assess the activity of compounds and prioritize them for further validation.

Project Managers: Oversee the HTS process, ensuring that milestones are met and resources are appropriately allocated. They also facilitate communication between different teams and ensure that the process remains on schedule.

Quality Assurance (QA): QA ensures that all HTS processes are conducted in accordance with regulatory guidelines, industry standards, and internal protocols. They verify that the data generated is reliable and reproducible, and they review documentation to ensure compliance.

4) Procedure

The following steps outline the detailed procedure for conducting HTS in drug discovery:

Step 1: Compound Library Preparation
1. Prepare a compound library that includes a diverse set of small molecules. The library can include commercially available compounds, in-house collections, or natural product libraries.
2. Ensure that each compound in the library is well-characterized, including information on chemical structure, purity, and concentration. Maintain a database for easy tracking of compounds during the screening process.
3. Prepare compound plates or solutions in the required concentrations for screening, ensuring that the compounds are aliquoted correctly to prevent cross-contamination between samples.
Step 2: Assay Design and Optimization
1. Design assays that are suitable for high-throughput screening. This may involve selecting an appropriate biological target (e.g., enzyme, receptor, or protein) and determining the assay format (e.g., fluorescence-based, luminescence-based, or cell-based assays).
2. Optimize the assay conditions to ensure that the biological target is active and responsive to compound treatment. This includes determining the optimal assay concentration, incubation time, temperature, and buffer conditions.
3. Ensure that the assay is robust and reproducible, with a low coefficient of variation (CV) and high Z-factor, which indicates the assay’s ability to discriminate between positive and negative controls.
Step 3: HTS Setup
1. Set up automated screening systems to conduct the HTS efficiently. This may include robotic liquid handling systems, plate readers, and other instrumentation that can handle large numbers of samples simultaneously.
2. Load the compound library into the automated screening system, ensuring proper plate formatting and adherence to screening protocols.
3. Run positive and negative controls in parallel with the compound library to ensure the accuracy and reliability of the screening results.
Step 4: Screening and Data Collection
1. Run the HTS assays with the compound library and controls. Monitor assay reactions in real-time, depending on the assay format used (e.g., fluorescence intensity, luminescence, or cellular changes).
2. Ensure that data collection is automated and that the results are recorded and stored in a central database for subsequent analysis.
3. Perform quality control checks during the screening process to identify and address any issues with assay performance or compound contamination.
Step 5: Data Analysis and Hit Identification
1. Use statistical tools and software to analyze the screening data. Identify “hits” based on criteria such as dose-response relationships, statistical significance, and consistency of activity.
2. Use algorithms to prioritize compounds with the most promising activity, taking into account their potency, selectivity, and chemical properties.
3. Generate dose-response curves for the identified hits to calculate EC50 or IC50 values and assess their potential for further development.
Step 6: Hit Validation and Secondary Screening
1. Validate the hits identified in the initial HTS by performing secondary assays to confirm their activity and specificity. This may include orthogonal assays or assays using different biological systems to confirm the target engagement.
2. Screen the hits for off-target effects and cytotoxicity to ensure that the compounds do not have unintended biological effects.
3. Prioritize the most promising hits for structure-activity relationship (SAR) analysis and optimization.
Step 7: Documentation and Reporting
1. Document all HTS procedures, including assay protocols, screening results, data analysis methods, and hit identification criteria.
2. Prepare an HTS Report that includes detailed information on the screening process, data analysis, and a list of validated hits for further development.
3. Ensure that all data is recorded accurately and maintained for future reference and regulatory compliance.

5) Abbreviations

HTS: High-Throughput Screening
EC50: Half-Maximal Effective Concentration
IC50: Half-Maximal Inhibitory Concentration
Z-factor: A statistical parameter for assay quality
Z’-factor: A variation of the Z-factor used in HTS

6) Documents

The following documents should be maintained throughout the HTS process:

HTS Screening Report
Assay Protocols
Screening Data Sheets
Secondary Screening Results

7) Reference

References to regulatory guidelines and scientific literature that support this SOP:

FDA Guidance for Industry on Drug Discovery
ICH E6: Good Clinical Practice
PubMed and PubChem for compound and assay information

8) SOP Version

Version 1.0: Initial version of the SOP.