NIH – Assay development and screening for discovery of chemical probes or therapeutic agents (R01)

July 31, 2017 by School of Medicine Webmaster

Participating Institutes:

  • National Cancer Institute
  • National Institute of Allergy and Infectious Diseases
  • National Institute on Deafness and Other Communication Disorders
  • National Institute on Drug Abuse

Through this FOA, NIH wishes to stimulate research in 1) discovery and development of novel, small molecules for their potential use in studying disease treatment relevant to the missions of the participating NIH Institutes, and 2) generation of new insight into the biology of relevant diseases and processes that have yet to be validated as important drug targets.

Assessment of the reasons for failures of small molecule therapeutic agents in the clinic points to several areas for improvement of the drug discovery and development process that are pertinent to this FOA:

  • First, increased rigor in target identification is necessary. For instance, whether prior studies of the selected target were adequately controlled and powered are important considerations. Were cell lines verified, plasmids sequenced, and protein reagents tested for contaminants?
  • Second, reproducibility of the proposed primary assay should be carefully considered because this assay is often the basis for assessing not only initial hits, but also for iteratively assessing optimized hits during structure-activity relationship (SAR) studies. Development of primary screening assays that test a key biological function of the target of interest are likely to yield hits of increased relevance. In this respect, phenotypic screens have had a resurgence.
  • Third, a hit validation scheme or “critical path” that includes orthogonal assay(s) to eliminate false positives, as well as a series of assays in diverse biological systems with diverse read-outs, particularly including assays that model human disease, is likely to yield hits of increased relevance.
  • Fourth, inclusion of skilled synthetic and/or medicinal chemists to assess the validity of the hit chemotypes to eliminate PAINS (pan-assay interference compounds) or other undesirable chemotypes.
  • A final area for improvement is the optimization of hits to yield high quality probes. Technological innovations in high throughput screening, chemical synthesis, and cheminformatics have allowed rapid discovery of novel, small-molecule probes for the study of disease related biological processes and mechanisms in academic environments (see Academic Screening Facilities DirectoryAcademic Drug Discovery Consortium). The Chemical Probes Portal, established in July 2015, provides a list of credentialed probes. It is noted that probes may be the predecessors of drugs, but drugs with known off-target effects are seldom useful as probes of specific biological activities. Importantly, identification of chemical probe(s) for a given target provides an unprecedented opportunity for investigators to translate knowledge about diseases into tangible tools for translational research and opens the door to validation of the target prior to clinical testing.
Research Scope:

Projects for this FOA may span up to three stages of discovery research:

  1. assay development;
  2. primary screen implementation; and
  3. hit validation.

For applications requesting support for more than one stage, demonstration of feasibility is needed, including strong justification and supporting preliminary data for the stages proposed. Areas to be considered for each stage are described below and given in greater detail in Section IV (Application and Submission Information, PHS 398 Research Plan).

1. Assay Development

This FOA seeks to apply new knowledge and screening technologies to develop assays for novel targets and pathways. Projects for assay development should emphasize the design and validation of creative approaches to assay biological and disease processes that have potential to be used for chemical probe or drug discovery. Assays focusing on areas and approaches that have been extensively studied should be avoided unless a strong rationale is provided for additional studies in the projected area. Targets associated with rare and neglected diseases are encouraged.

Proposed primary screening assays should be relevant to the scope of the research within at least one of the participating NIH ICs focusing on specific diseases or on relevant basic physiology, cell biology, or developmental processes that provide insight into a disease.

Primary screening assays may be target-, pathway-, or phenotype-based. Some examples include:

  • target-based biochemical or cellular assays that measure activities of enzymes, receptor-ligand binding, protein-protein interactions, ion channels, transporters, nuclear receptors and other transcription factors, and other new targets emerging from genetic and proteomic research in model systems and in human diseases;
  • cell- or organism-based assays that detect phenotypic changes that may involve unidentified molecular targets; and
  • non-traditional targets of interest such as nucleic acids, protein folding, polymorphic gene products, post-transcriptional editing or gene splicing factors, and protein or RNA stabilizers.

Assay detection methods may include, but are not limited to:

  • fluorescence, luminescence, absorbance;
  • fluorescence resonance energy transfer (FRET);
  • time-resolved fluorescence resonance energy transfer (TR-FRET);
  • fluorescence polarization;
  • flow cytometric measurements;
  • fluorescence imaging;
  • bioluminescence resonance energy transfer (BRET);
  • AlphaScreen, scintillation proximity assay (SPA);
  • electrophysiological assays, and;
  • biophysical assays.

In general, assays should adopt an adequate detection principle that results in a sensitive detection of even weak binders with expected low rates of false positives and false negatives. Complementary research including virtual screening may also be conducted to improve the likelihood of success and cost effectiveness.

Additional considerations for the design and testing of a primary assay may be found in Section IV (Application and Submission Information, PHS 398 Research Plan).

2. Primary Screen Implementation

Applicants are encouraged to collaborate with an experienced screening facility, particularly if high throughput screening (HTS) is planned. The screening facility may provide advice such as identification and selection of commercial HTS assay reagents, and suitable HTS assay format and readout. In addition, the screening facility may be able to provide assistance in adapting assays to an HTS format (e.g., 1536-well or 384-well microplate) and performing a pilot screen of a small library of compounds. Further, the researchers might seek advice from the screening laboratory about orthogonal assays to validate the screening hits, and about chemical improvement of the initial hits via a structure-activity relationship (SAR) study following hit validation.

Projects focusing on screen implementation are encouraged to provide preliminary data demonstrating that a primary screen has been developed, fully characterized, and tested in a pilot format. See Section IV for further details on expected information prior to the Primary Screen Implementation stage.

Other technical resources about assay development and screening include the online comprehensive guidebook (Assay Guidance Manual), assay protocols deposited on the PubChem BioAssay data base, ASSAY and Drug Development Technologies, a peer-reviewed bimonthly journal, and the Journal of Biomolecular Screening.

3. Hit validation

Hits from a primary screen may be systematically assessed using a cascade of follow-up assays to efficiently and effectively remove false positives. Primary HTS assays typically generate hundreds to thousands of hits, many of which are false positives or are chemically intractable. Hits from smaller scale primary screens are also likely to generate false positives or chemically intractable molecules that require additional screens. Post-primary screening assays may include:

  • an assay that is essentially identical to the primary assay but with an orthogonal detection scheme (e.g., switching light detection mode or wavelength to avoid intrinsic compound interference);
  • a target-minus assay (e.g., coupling enzymes in the absence of the assay target enzyme, parental cell line without the assay target protein, etc.);
  • an assay that is different in biological context and process (e.g., protein functional assay vs. protein binding assay; RT-PCR and Western assay vs. reporter gene assay; cell-based assay vs. biochemical assay);
  • cytotoxicity assay;
  • target selectivity assay(s);
  • specificity assays to distinguish biological activities of chemical entities among orthologous targets across organism species through kingdoms (e.g., yeast vs. mammalian cell targets; parasite vs. host targets);
  • mode of action assays (e.g., allosteric vs. orthosteric; competitive vs. noncompetitive or uncompetitive); and
  • target identification assays. The assays farther downstream may also include cellular and tissue models pertaining to the relevant physiology or pathophysiology.
  • assays farther downstream may include cellular and tissue models pertaining to relevant physiology or pathophysiology, or to mode and mechanism of action of the validated hits. In vivo assays (e.g. whole animal models) should only be proposed if they are needed to demonstrate the biological or physiological effects of lead compound(s).

In addition, investigators should conduct advanced cheminformatics analysis and medicinal chemistry inspection to prioritize the hit set. It is expected that the investigators will test powder samples of hit compounds and commercially available analog compounds during the hit validation stage. Investigators should verify the structure of hits using a combination of analytical methods and, if possible, re-synthesis of select hits.

Screening campaigns may yield validated hits that can be further optimized via medicinal chemistry. Extensive medicinal chemistry optimization is not appropriate for this FOA. Investigators of a successful screening project are encouraged to consider further funding opportunities to support medicinal chemistry optimization of the validated hits (e.g. see “Institute Interests” below).

Institute Interests

  • NCI: Assays and screens pertinent to the mission of NCI should be justified in the application as relevant to cancer. Of interest to NCI are assays and screens to identify or evaluate small molecules for use in elucidating molecular, cellular, or in vivo mechanisms or processes of probable or known importance in cancer biology, and for use in developing strategies for cancer prevention, diagnosis, treatment or clinical monitoring of treatment. Discovery of small molecule probes, preventive or therapeutic drug leads, or imaging agent leads are of interest. NCI encourages projects focusing on small molecules that specifically target pediatric fusion oncoproteins or that address novel targets in small cell lung cancer or pancreatic cancer. Projects for discovery of small molecules directly involved in cancer immunomodulation should apply to PAR-17-331. Validated hits identified through this FOA may be appropriate for entry in the NCI Experimental Therapeutic Program (NExT). Applicants may also find the NCI Developmental Therapeutics Program resources to be helpful.
  • NIAID: NIAID is particularly interested in applications directly addressing infectious or immune-mediated disease or basic immunology. Applications may focus on assays targeting etiologic agents of human infectious disease including, but not limited to, potential agents of biodefense (categories A, B, and C), as well as other newly emerging infectious agents NIAID Emerging Infectious Diseases/Pathogens with high global burdens of disease, such as tuberculosis and malaria, as well as toxins or arthropod vectors of infectious agents as targets will also be included, along with viral pathogens of public health consequence that establish intracellular reservoirs and are uniquely challenging drug targets (i.e., HIV and HBV). Additionally, applications may focus on augmenting immune responses towards infectious pathogens or vaccines.  Such studies may include, identification of molecules that target immune molecules to enhance early innate immune responses and/or induce or prolong immunological memory to vaccination (e.g., adjuvants, immunomodulators). Also of prime interest is the prevention, amelioration, or reversal of immune-mediated diseases by small molecules. Targets relevant to asthma, allergy, inflammatory diseases, transplant rejection, and all types of autoimmune diseases may be proposed. Assays that facilitate studies on basic immunological mechanisms are also appropriate, including the development of small molecules to track/visualize immune cells in vivo or in vitro (e.g., nanobodies, imaging probes).
  • NIDA:  NIDA is interested in the discovery and development of HTS assays that can be used to identify small molecules that elucidate underlying molecular, cellular, or in vivo mechanisms of probable processes or known importance to addiction biology. NIDA is particularly interested in assays that can be applied to future opioid use disorder, chronic pain pharmacotherapeutic development or treatment of overdose. Assays can be biochemically, cellularly or behaviorally based. Related chemical discovery opportunities include: Chemical Discovery Program (PAR-16-384), Drug Discovery For Nervous System Disorders (PAR-16-041), and the Chemistry Science Track Award for Rapid Transition (C/START) (PAR-16-383). Investigators considering applying to this FOA are strongly encouraged to consult the NIDA Scientific/Research staff member listed below prior to submitting an application.
  • NIDCD:  The NIDCD is interested in the development of high throughput screens (HTS) to assess and validate putative novel, or small molecules that have potential therapeutic value in the treatment, protection or prevention of communication disorders, including hearing, balance, smell/taste, voice, speech and language.  Applications could include, but are not limited to, improved HTS towards the identification of clinically-relevant candidate therapeutics or biological targets that might lead to clinical relevancy in hearing/balance areas of otoprotection, regeneration, otitis media, tinnitus, and normal/abnormal development; chemosensory abnormalities such as they relate to serious diseases of obesity, diabetes, Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis; disorders involving voice speech, language, including swallowing, aphasia or dysarthria, and laryngeal replacement.  Potential applicants are encouraged to review the NIDCD mission  prior to submitting an application.

Deadlines:  standard dates apply.


Filed Under: Funding Opportunities