Practical Guidance for Small Molecule Screening

Summary: The goal of this document is to communicate practical guidance that can be applied during development of small molecule high throughput screens, and their presentation in papers and grants. The sources of guidance are literature papers, federal grant evaluation criteria, and conversations with scientific contacts overseeing grants from multiple NIH institutes.

Assay Development and Screening

“A well-defined phenotype is the single most important consideration in designing a screen.” Robustness and reproducibility of the phenotype are essential (Eggert, 2013).

Describe how positive and negative controls are used to set assay window and comment on the level of biological response expected relative to them. Describe calculations for data normalization based on controls. Describe your hit scoring strategy (Inglese et al., 2007).
Indicate sources of all reagents, including batch numbers, in methods (Inglese et al., 2007).
Experimental procedure should include specifics instruments and settings used for liquid handling and reading assay. Add information about the number of assay plates screened and duration of screen (Inglese et al., 2007).
If you are going to use siRNA approaches of pathway targets with known small molecule treatments of same, making assays as similar as possible will help prevent confounding factors (Eggert, 2013). At the same time, “results with small molecule inhibition and siRNA knock down will not necessarily align at the phenotype level, for example, when a small molecule inhibits an enzyme but protein-protein interactions are intact, or that effect stoichiometry in downstream processes” (Weiss et al., 2007).

Add statements about library quality, solubility, stability. Describe analysis methods to exclude ‘frequent hitters’ molecules. Medicinal chemistry triage following screening can address compound stability and solubility, and solubility measurements can be undertaken in the laboratory for highest priority leads (editorial, 2009).

Additional considerations for image-based assays

User should review images from a least three full plates to get a sense of expected and unexpected phenotypic changes to monitor (Eggert, 2013).
Include description of image analysis algorithm in terms of what metrics of the phenotype are being quantified.

Hit selection, ranking and validation

Include statements about how compounds were ranked, and criteria for exclusion of screen hits (Inglese et al., 2007).
Orthogonal detection but similar assay as primary screen, to rule out compound interference (NIH guidance in PAR-13-364 and PAR-12-058)
Target-minus assay, such as coupling enzymes lacking target, to rule out compounds acting on other steps in signal generation (NIH guidance in PAR-13-364 and PAR-12-058)
Different biological context and process (e.g. functional assay vs. binding assay, biochemical vs. cell-based, reporter vs. Western) (NIH guidance in PAR-13-364 and PAR-12-058)
From phenotypic assays, methods to identify the target (NIH guidance in PAR-13-364 and PAR-12-058)
Cell-based or animal studies (NIH guidance in PAR-13-364 and PAR-12-058)
Describe potency (ideally below 1 uM), selectivity from paralogs (proteins with a shared ancestry) and cellular activity (Frye, 2010; NIH guidance in PAR-13-364 and PAR-12-058).
Structure Activity Relationship (SAR) studies should be done and multiple scaffolds identified per target. Ideally, co-crystal structure can be solved to rationalize SAR results. Use of close analogs that lack activity from SAR can be used to rule out off-target effects (Frye, 2010; Bunnage et al., 2013; Weiss et al., 2007).
SAR profiles should correlate between in vitro biochemical affinity and effective cellular concentration (Weiss et al., 2007).
Using three different scaffolds that elicit biological activity is suggestive that results are not due to off-target effects (Kodadek, 2010; Bunnage et al., 2013).
Mode of action assays to identify allosteric vs orthosteric, competitive vs noncompetitive or uncompetitive modes (NIH guidance in PAR-13-364 and PAR-12-058)
Target protein can be mutagenized to find mutations that render protein insensitive to compound (Kodadek, 2010, Weiss et al., 2007).
Activity response and toxicity response should be evaluated. It can be helpful to monitor cell/compound ratio in addition to standard concentration-dependence (Eggert, 2013).
siRNA or shRNA can be used to demonstrate target selectivity (Frye, 2010).
Target protein expression levels can be modified with KO/KD toevaluate if DR shifts in expected directions (Kodadek, 2010).
Irreversible inhibitors (covalent modifiers) could be used to find binding partners of small molecules (Kodadek, 2010).
Databases, such as PubChem, should be queried to find out what else may be known about scaffolds from clusters of potential hit compounds, and to see if closely related compounds have scored in other assays (Eggert, 2013).

Freshly-sourced powders should be analyzed for 1H NMR, 13C NMR, High Resolution MS for chemical structure and HPLC for purity. If it is not available, molecule should be resynthesized. “Illogical” SAR may point to errors in structural assignment from vendor. 1 mg of powder is typically needed for these evaluations (Editorial, 2009; Inglese et al., 2007; Eggert, 2013).

Advice for seeking federal funding through NIH institutes

NIH offers two variations of the standard PA: a Program Announcement with Set-Aside Funds (PAS) and a Program Announcement Reviewed by an Institution (PAR) With PAR mechanisms special review sections are assigned to evaluate the grants, and they have a very different perspective than standard study sections. For-profit companies can apply for these grants and often do very well, particularly small biotechnology companies. PAR does not mean that money is set aside for the grant. Pay lines follow the standards of that institute. PAS announcements have specific funds set aside for them.
There may be a preference for screening at government sponsored Centers with some funding announcements. You should discuss with the Scientific Contact. Demonstrating abilities of Center and format/quality of screening collections is necessary. Include track record with bibliography of awarded projects.
NIGMS is dropping participation in R21s for screening because they felt review sections were unrealistic in expectations and none were ever scored at levels sufficient for funding. NIGMS does not fund work that would fall to another institute (e.g. eye disease if NEI is not participating) if that institute is not participating.
You should definitely call the Scientific Institute contact to discuss feasibility and fundability before you start work on the grant. You should send then your Specific Aims early in the writing process for feedback and improvement.
About half of assay development applications go to disease specific study section and about half go to special emphasis study section. One NIH contact thought chances were better if application goes to disease specific study panel. He recommends reviewing standing study sections and panels with people you’d want to review the application and recommend in your cover letter. Generally those recommendations are met. You can request a change of study section if you don’t like where it has been assigned.

References

Eggert US. The why and how of phenotypic small-molecule screens. Nat Chem Biol. 2013 Apr;9(4):206-9.

Inglese J, Shamu CE, Guy RK. Reporting data from high-throughput screening of small-molecule libraries. Nat Chem Biol. 2007 Aug;3(8):438-41.

Weiss WA, Taylor SS, Shokat KM. Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat Chem Biol. 2007 Dec;3(12):739-44.

Editorial. Screening we can believe in. Nat Chem Biol. 2009 Mar;5(3):127.

Frye SV. The art of the chemical probe. Nat Chem Biol. 2010 Mar;6(3):159-161.

Bunnage ME, Chekler EL, Jones LH. Target validation using chemical probes. Nat Chem Biol. 2013 Apr;9(4):195-9.

Kodadek T. Rethinking screening. Nat Chem Biol. 2010 Mar;6(3):162-165.