1. Consultation
Potential users are invited to discuss a screening idea with any member of the SMDC team. Options around types of compounds (e.g. non-covalent vs. covalent, diversity vs. target class-focused) are discussed and decided. Once assay development is underway, the project will be matched with an HTS liaison, who will consult during assay development and help run the HTS.
2. Assay development, miniaturization and optimization
The assay is developed by the initiating lab in their laboratory. Clients are welcome to use SMDC instrumentation to test their assay performance. Miniaturizing the assay format from 96-well microplates to 384-well microplates is recommended (as the SMDC's small molecule libraries and collections are in 384-well plates). A typical use of instrumentation is included in the recharge, and there are hourly rates for extensive development or other project-related use.
3. Dry-run
The proposed HTS assay is tested using positive and negative controls to determine the Z’ for the assay. The dry run should use the HTS protocol, including SMDC robotics and plate readers where appropriate. When Z’ is consistently > 0.5, pilot screening can proceed.
4. Pilot screen
Approximately 2,000 compounds are run using the HTS protocol. All aspects of the screen, including data analysis, are tested. When the Z’ and Z-factors are >0.5, HTS can proceed. The pilot set can be a focused library or the first few plates of the desired screen set. A typical screen is run at a volume of 50 µl, at a final concentration of 10 µM compound, in 0.1% DMSO.
5. High-throughput screening (HTS)
Screening begins in earnest, working at the fastest pace appropriate for the assay. See Available Compounds for a list of the SMDC's libraries and collections.
6. Data analysis
Each day of screening, results are uploaded to the SMDC database and are accessible via the HiTS platform. Collaborators are encouraged to sign up for a free account to HiTS. Data will be kept confidential and each project is accessible only to approved users. Z’ and Z-factors determine whether a run is interpretable. “Active” wells are those with signals > 3 standard deviations from the mean of the baseline signal.
7. Hit confirmation
Actives are cherry-picked for rescreening, usually at multiple doses. Close analogues of confirmed hits present in SMDC libraries and collections may also be tested at this stage.
8. Preliminary SAR
HTS data are analyzed by substructure or other similarity algorithm to determine preliminary structure-activity relationships (SAR). SAR can be used to identify compounds for repurchase and/or for generating hypotheses about the binding site. At this stage, the SMDC will also provide available information about which actives are nonselective, cytotoxic, and/or covalent modifiers. Data for screens, cherry-picks, and chemical structures are available to the client via our web-based user interface, called HiTS.
9. Repurchase or resynthesis
The SMDC will provide information on repurchasing the active compounds. The initiating lab then purchase the compound for their own use. Sometimes, it is not possible to purchase the compound from commercial sources; in these cases, the SMDC will suggest alternatives, such as resynthesis or purchasing close analogs that are commercially available.
After repurchase, the screening services are complete, but there are avenues to continue working with the SMDC. If equipment is needed, we will provide the instrument recharge policy. Further collaboration is often encouraged, and begins with a discussion of long-term goals and strategy. See also: hit-to-lead chemistry.
10. HIts-to-Leads (H2L) medicinal chemistry
After a hit series is selected, we can move to initiate the synthesis of structural analogs. In general, the number of scaffolds taken forward into hit-to-lead chemistry depends on their properties and chemistry resources available to make analogs.
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