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  • Review Article
  • Published:

Universal strategies in research and drug discovery based on protein-fragment complementation assays

Nature Reviews Drug Discovery volume 6, pages 569–582 (2007)Cite this article

Key Points

  • Protein-fragment complementation assays (PCAs) provide a general methodology to detect and study spatial and temporal dynamics of protein–protein interactions in intact living cells and have many applications, which are listed below.

  • PCAs have been used to engineer novel binding proteins for research and potential therapeutic applications including antibodies and alternative binding proteins.

  • To study mechanisms of membrane receptor-mediated and other signal transduction processes.

  • To probe actions of small molecules on cellular pathways that regulate cell-fate decisions.

  • To predict on- and off-target effects and potential therapeutic applications of small molecules.

  • To link potential disease drug-target genes to specific cellular functions by a general expression-cloning strategy

  • To visualize spatial and temporal dynamics of protein complexes in cultured cells and whole animals.

  • To visualize pharmacodynamics of drugs and effects of drugs on tumour growth in whole animals

Abstract

Changes in the interactions among proteins that participate in a biochemical pathway can reflect the immediate regulatory responses to intrinsic or extrinsic perturbations of the pathway. Thus, methods that allow for the direct detection of the dynamics of protein–protein interactions can be used to probe the effects of any perturbation on any pathway of interest. Here we describe experimental strategies — based on protein-fragment complementation assays (PCAs) — that can achieve this. PCA-based strategies can be used with or instead of traditional target-based drug discovery strategies to identify novel pathway-component proteins of therapeutic interest, to increase the quantity and quality of information about the actions of potential drugs, and to gain insight into the intricate networks that make up the molecular machinery of living cells.

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Figure 1: Linking genes to function.
Figure 2: Principles of PCA.
Figure 3: Solving the expression-cloning problem.
Figure 4: Linking small molecules to specific pathways using PCA screens.
Figure 5: Unique subcellular locations of two different functional states of the pro-apoptotic protein BAD revealed by two PCA sentinels.
Figure 6: Spatial and temporal dynamics of PCAs.
Figure 7: Alternative ways to extract information about drug actions on different pathways and cellular processes.

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Acknowledgements

The work published from our group was supported by the CIHR, NSERC, NFSP and NIH. S.W.M. holds the Canada Research Chair in integrative genomics.

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  1. Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, H3C 3J7, Québec, Canada

    Stephen W. Michnick, Po Hien Ear, Emily N. Manderson, Ingrid Remy & Eduard Stefan

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  1. Stephen W. Michnick
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  2. Po Hien Ear
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Correspondence to Stephen W. Michnick.

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S.W.M. is a shareholder in Odyssey Thera Inc.

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FURTHER INFORMATION

Stephen Michnick's Laboratory

Glossary

Perturbation

Any treatment of a cell, including chemicals, gene deletion or ablation with a short-interfering RNA that may produce an observable effect on an assay that reports activity of a pathway.

Module

A group of genes that are shown to interact with each other to produce a common phenotype, or proteins that are functionally linked and have more physical interactions between themselves than with other proteins.

Cross-talk

Interactions between two pathways that are thought to mediate common or different cellular processes.

Allosteric

A change in the spatial orientation of subunits within a protein complex that is shown to regulate the function of the individual subunits. For example, a change in the affinity of haemoglobin subunits for molecular oxygen on changing from one configuration to another.

Reversibility

In the context of protein-fragment complementation assays, the complete unfolding and separation of protein-reporter fragments when the proteins to which they are attached dissociate.

Phage-display

A method to identify or design proteins with the specific ability to bind to a molecule by expressing proteins as fusions to filamentous bacterial viruses, testing binding to a molecule arrayed on a solid surface, identifying those phage particles that bind and then replicating them in bacteria.

Expression-cloning

The identification of a gene that performs a specific cellular function by expressing a library of cDNAs, and screening for resulting proteins that perform the function.

Sentinel

An assay that provides a direct readout for activity in a specific biochemical pathway.

Off-pathway effect

Effects of a perturbation on pathways that are not predicted to be affected by the perturbation.

High-content imaging

Generally a quantitative analysis of morphological changes or spatial and temporal changes of proteins or protein complexes. Usually performed with fluorescent molecules (for example, antibodies covalently coupled to fluorescent molecules) or fluorescent proteins genetically fused to proteins of interest. These can be imaged by epifluorescence or confocal microscopy.

Hidden phenotypes

A potential outcome to treatment of a cell with some perturbation such as death, differentiation state or morphology that is predicted by pathway sentinel assays, but not necessarily observed in the cells in which the assays were performed.

Hierarchical clustering

A general term for many mathematical strategies used to establish relationships between objects according to common features.

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Michnick, S., Ear, P., Manderson, E. et al. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6, 569–582 (2007). https://doi.org/10.1038/nrd2311

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