Chemically induced dimerization (CID) is a biological mechanism in which two proteins bind only in the presence of a certain small molecule, enzyme or other dimerizing agent. [1] Genetically engineered CID systems are used in biological research to control protein localization, to manipulate signalling pathways and to induce protein activation. [2]
The first small molecule CID system was developed in 1993 and used FK1012, a derivative of the drug tacrolimus (FK506), to induce homo-dimerization of FKBP. [2] This system was used in vivo to induce binding between cell surface receptors which could not bind in the normal way because they lacked the transmembrane and extracellular domain. Addition of FK1012 to the cells caused signal transduction.
| Target proteins | Dimerizing agent | References | |
|---|---|---|---|
| FKBP | FKBP | FK1012 | [3] |
| FKBP | Calcineurin A (CNA) | FK506 | [4] |
| FKBP | CyP-Fas | FKCsA | [5] |
| FKBP | FRB (FKBP-rapamycin-binding) domain of mTOR | Rapamycin | [6] |
| GyrB | GyrB | Coumermycin | [7] |
| GAI | GID1 (gibberellin insensitive dwarf 1) | Gibberellin | [8] |
| ABI | PYL | Abscisic acid | [9] |
| ABI | PYRMandi | Mandipropamid | [10] |
| SNAP-tag | HaloTag | HaXS | [11] |
| eDHFR | HaloTag | TMP-HTag | [12] |
| Bcl-xL | Fab (AZ1) | ABT-737 | [13] |
| Anti caffeine camelid nanobody | Camelid nanobody (homodimer) | Caffeine | [14] |
| VH-anti nicotine | VL-anti nicotine | Nicotine | [15] |
| Anti RR120 camelid nanobody | Camelid nanobody (homodimer) | RR120 (Azo dye) | [15] |
CID has been used for a number of applications in biomedical research. In most applications each dimerizing protein is expressed as part of a fusion construct with other proteins of interest. Adding the chemical dimerizing agent brings both constructs into proximity with each other and induces interactions between the proteins of interest. CID has been used to regulate and monitor gene transcription, signal transduction and post translational modifications in proteins.
Recently, CID has also been used to create a basic component of biocomputers, logic gates, from genetically manipulated cells. [8] In this application, two independent CID systems, one based on plant proteins and one based on bacterial proteins are expressed in the same cell. Each set of proteins can be induced to dimerize by the addition of a separate chemical. By creating fusion proteins with the dimerizing proteins, membrane bound proteins and proteins that activate cell ruffling an AND gate and OR gate can be created that take chemical dimerizing agents as inputs and returns a ruffled or unruffled state as output.
G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily related proteins that are cell surface receptors that detect molecules outside the cell and activate cellular responses. They are coupled with G proteins. They pass through the cell membrane seven times in the form of six loops of amino acid residues, which is why they are sometimes referred to as seven-transmembrane receptors. Ligands can bind either to the extracellular N-terminus and loops or to the binding site within transmembrane helices. They are all activated by agonists, although a spontaneous auto-activation of an empty receptor has also been observed.
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events. Most commonly, protein phosphorylation is catalyzed by protein kinases, ultimately resulting in a cellular response. Proteins responsible for detecting stimuli are generally termed receptors, although in some cases the term sensor is used. The changes elicited by ligand binding in a receptor give rise to a biochemical cascade, which is a chain of biochemical events known as a signaling pathway.
In cellular biology, paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance, as opposed to cell signaling by endocrine factors, hormones which travel considerably longer distances via the circulatory system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain.
In biology, cell signaling is the process by which a cell interacts with itself, other cells and the environment. Cell signaling is a fundamental property of all cellular life in prokaryotes and eukaryotes.
Stuart Schreiber, Ph.D. is the Morris Loeb Research Professor at Harvard University, a co-Founder of the Broad Institute, Howard Hughes Medical Institute Investigator, Emeritus, and a member of the National Academy of Sciences and National Academy of Medicine. His work integrates chemical biology and human biology to advance the science of therapeutics. Key advances include the discovery that small molecules can function as “molecular glues” that promote protein–protein interactions, the co-discovery of mTOR and its role in nutrient-response signaling, the discovery of histone deacetylases and the demonstration that chromatin marks regulate gene expression, the development and application of diversity-oriented synthesis to microbial therapeutics, and the discovery of vulnerabilities of cancer cells linked to genetic, lineage and cell-state features, including ferroptotic vulnerabilities. His notable awards include the Wolf Prize in Chemistry and the Arthur Cope Award. His approach to discovering new therapeutics guided many biotechnology companies that he founded, including Vertex Pharmaceuticals and Ariad Pharmaceuticals. He has founded or co-founded 14 biotechnology companies, which have developed 16 first-in-human approved drugs or advanced clinical candidates.
Receptor tyrosine kinases (RTKs) are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Of the 90 unique tyrosine kinase genes identified in the human genome, 58 encode receptor tyrosine kinase proteins. Receptor tyrosine kinases have been shown not only to be key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer. Mutations in receptor tyrosine kinases lead to activation of a series of signalling cascades which have numerous effects on protein expression. Receptor tyrosine kinases are part of the larger family of protein tyrosine kinases, encompassing the receptor tyrosine kinase proteins which contain a transmembrane domain, as well as the non-receptor tyrosine kinases which do not possess transmembrane domains.
Platelet-derived growth factor receptors (PDGF-R) are cell surface tyrosine kinase receptors for members of the platelet-derived growth factor (PDGF) family. PDGF subunits -A and -B are important factors regulating cell proliferation, cellular differentiation, cell growth, development and many diseases including cancer. There are two forms of the PDGF-R, alpha and beta each encoded by a different gene. Depending on which growth factor is bound, PDGF-R homo- or heterodimerizes.
The FKBPs, or FK506 binding proteins, constitute a family of proteins that have prolyl isomerase activity and are related to the cyclophilins in function, though not in amino acid sequence. FKBPs have been identified in many eukaryotes, ranging from yeast to humans, and function as protein folding chaperones for proteins containing proline residues. Along with cyclophilin, FKBPs belong to the immunophilin family.
Signal transducer and activator of transcription 2 is a protein that in humans is encoded by the STAT2 gene. It is a member of the STAT protein family. This protein is critical to the biological response of type I interferons (IFNs). STAT2 sequence identity between mouse and human is only 68%.
Receptor tyrosine-protein kinase erbB-3, also known as HER3, is a membrane bound protein that in humans is encoded by the ERBB3 gene.
Nuclear factor of activated T-cells, cytoplasmic 2 is a protein that in humans is encoded by the NFATC2 gene.
G-protein coupled receptor 3 is a protein that in humans is encoded by the GPR3 gene. The protein encoded by this gene is a member of the G protein-coupled receptor family of transmembrane receptors and is involved in signal transduction.
Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is a signal transduction enzyme that in humans is encoded by the autosomal MAP3K1 gene.
Cell surface receptors are receptors that are embedded in the plasma membrane of cells. They act in cell signaling by receiving extracellular molecules. They are specialized integral membrane proteins that allow communication between the cell and the extracellular space. The extracellular molecules may be hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients; they react with the receptor to induce changes in the metabolism and activity of a cell. In the process of signal transduction, ligand binding affects a cascading chemical change through the cell membrane.
Gerald R. Crabtree is the David Korn Professor at Stanford University and an Investigator in the Howard Hughes Medical Institute. He is known for defining the Ca2+-calcineurin-NFAT signaling pathway, pioneering the development of synthetic ligands for regulation of biologic processes and discovering chromatin regulatory mechanisms involved in cancer and brain development. He is a founder of Ariad Pharmaceuticals, Amplyx Pharmaceuticals, and Foghorn Therapeutics.
The STAT3-Ser/Hes3 signaling axis is a specific type of intracellular signaling pathway that regulates several fundamental properties of cells.
FK1012 is a dimer consisting of two molecules of tacrolimus (FK506) linked via their vinyl groups. It is used as a research tool in chemically induced dimerization applications. FK1012 is a chemical inducer of dimerization (CID) which makes the protein capable of dimerization or oligomerization of fusion proteins containing one or more FKBP12 domains. It is used in pharmacology to act as a mediator in the formation of FK506 dimer. FK506 binding proteins (FKBPs) do not normally form dimers but can be caused to dimerize in the presence of FK1012. Genetically engineered proteins based on FKBPs can be used to manipulate protein localization, signaling pathways and protein activation.
The bump-and-hole method is a tool in chemical genetics for studying a specific isoform in a protein family without perturbing the other members of the family. The unattainability of isoform-selective inhibition due to structural homology in protein families is a major challenge of chemical genetics. With the bump-and-hole approach, a protein–ligand interface is engineered to achieve selectivity through steric complementarity while maintaining biochemical competence and orthogonality to the wild type pair. Typically, a "bumped" ligand/inhibitor analog is designed to bind a corresponding "hole-modified" protein. Bumped ligands are commonly bulkier derivatives of a cofactor of the target protein. Hole-modified proteins are recombinantly expressed with an amino acid substitution from a larger to smaller residue, e.g. glycine or alanine, at the cofactor binding site. The designed ligand/inhibitor has specificity for the engineered protein due to steric complementarity, but not the native counterpart due to steric interference.
James Allen Wells is a Professor of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology at the University of California, San Francisco (UCSF) and a member of the National Academy of Sciences. He received his B.A. degrees in biochemistry and psychology from University of California, Berkeley in 1973 and a PhD in biochemistry from Washington State University with Ralph Yount, PhD in 1979. He completed his postdoctoral studies at Stanford University School of Medicine with George Stark in 1982. He is a pioneer in protein engineering, phage display, fragment-based lead discovery, cellular apoptosis, and the cell surface proteome.
Trimethoprim-Halotag (TMP-HTag) is a small molecule chemical linker developed for the rapid and reversible control of protein localization in living cells (Ballister). TMP is an dihydrofolate reductase (DHFR) inhibitor chosen for its specificity in binding to the bacterial form of DHFR. The other half of the linker is a Halotag, a self labelling bacterial globular protein ligand that can bind covalently and irreversibly to the chloroalkane group of a Haloenzyme. Positioned between the TMP group and HaloTag is a flexible linker that can be modified to optimize protein linking efficiency. The modular structure of TMP-HaloTag makes it an ideal heterobifunctional tool for use in chemically induced dimerization (CID). Additionally, TMP- HTag can be modified to include photo-cleavable groups that allow for the control of CID using light.
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