ARIAD's cell-signaling regulation technologies allow specific cell-signaling, gene transcription and protein secretion events to be activated at will, in cultured cells or in whole animals. These technologies can be used to develop gene and cell therapy products featuring small-molecule regulation. They also represent versatile tools for exploring and identifying gene and protein function, for use in cell biology, functional genomics, proteomics, and drug discovery research.
Research groups throughout the world are using ARIAD Regulation Kits in diverse areas of research, and over 300 scientific publications have described their use. ARIAD Regulation Kits are distributed free of charge to academic researchers; (although we may request that investigators wishing to perform follow-on experiments requiring additional, very large quantities of dimerizers, such as studies in large animals, seek grant funding to cover our compound preparation costs). For researchers in commercial entities, we have established an alternative commercial licensing program.
The links below provide a brief description of the technologies that underlie the Regulation Kits and how they can be applied in research and drug discovery. The overview concludes with links to sources of more detailed information and to forms for requesting access to these kits.
Our Regulation Technologies
Research Applications
ARIAD’s ARGENT cell-signaling regulation technology involves the use of small molecule drugs, called “dimerizers”, that are able to bring together (or “dimerize”) two proteins simultaneously. Because protein-protein interactions are such a common mechanism in cell signaling, ARGENT Signaling Technology provides a generic method for generating conditionally-active versions, or alleles, of signaling proteins (see figure below).
Controlling cell signaling using ARGENT homodimerization
Over 100 different proteins have been controlled using the ARGENT system, allowing detailed functional analysis of signaling pathways in vitro or in vivo. Examples include growth factor receptors such as the EGFR family (see reference) and numerous intracellular signaling proteins, such as Src family tyrosine kinases, Raf, IKKs and caspases. The technology has also been used to control the activity of many non-signaling proteins that participate in regulated protein-protein interactions, such as cell adhesion molecules. We offer two Regulation Kits for promoting either homodimerization or heterodimerization, allowing specific combinations of proteins to be uniquely induced.
Control of gene transcription with the ARGENT Transcription Technology provides a general means to study the function of any gene. The ability to vary expression levels at will and to monitor effects on cells or in whole animals allows detailed questions to be asked about the physiological role of the gene and the protein it encodes. The tight regulation afforded by the ARGENT system is particularly important when studying potentially toxic genes, such as those that promote cell death or block the cell cycle. For example, we have generated stable cell lines inducibly expressing the highly toxic diphtheria toxin A chain gene (see reference). Two Regulation Kits are provided in which ARGENT components are provided either on plasmids, or retroviral vectors for one-step delivery of the entire system (including target gene) to any mitotic cell type for rapid analysis of gene function.
RPD Technology is a “reverse dimerization system” that complements the ARGENT dimerization system. It is especially useful for studying the function of proteins that can be activated or inactivated by self-aggregation. The ability to rapidly create and destroy large protein aggregates in discrete subcellular locations has particular utility in cell biology and protein trafficking research. For example, this approach has been used to discover the existence of "mega-vesicles" transporting cargo across the Golgi stack (see reference). We provide reagents for RPD in our RPD Regulated Secretion/Aggregation Kit.
Functional Genomics and Drug Discovery Applications
The ability to turn specific genes and proteins on and off precisely with small molecules has broad application in functional genomics, proteomics and drug discovery research. Three broad areas of utility can be defined: target validation, inducible animal models of disease, and drug screening and drug discovery.
Target Validation
Cell Signaling
A key challenge in the post-genomic era is the identification of genes encoding proteins that are good drug targets. This process of "target validation" can be approached at many levels, from in vitro studies on cultured cells through to studies of the putative target in whole animal models of disease. Many potential targets function through protein-protein interactions and dimerization—in particular those involved in cell signaling, many of which are centrally involved in disease states such as cancer. Our ARGENT Technology represents a powerful and generally applicable way to study these interactions and targets precisely: individual signaling proteins can be activated in isolation, allowing the consequences of activation, and hence the functional role of the protein, to be assessed in vitro and then in vivo. An example is the analysis of gene expression or proteome profile changes that occur following the precisely timed activation or inhibition of a single signaling protein.
Once a cell-signaling protein or pathway has been validated, the same ARGENT technology can provide useful tools for the next stages of drug development. The inducible gene can be engineered into experimental animals to provide an ARGENT model of the associated disease. ARGENT cell lines in which the validated signaling complex can be inducibly activated also can provide the basis for highly targeted cell-based screening for small-molecule drug candidates.
Gene Transcription
Varying the expression level of a gene is an effective way to study its function. The tight, dose-dependent control of expression afforded by our ARGENT Transcription Technology allows precise correlation of gene expression levels with their physiological consequences. Our technology also can be used to inducibly express inhibitors of supposed targets, such as dominant-negative mutants, gene-specific inhibitory RNA molecules or gene-specific inhibitory DNA binding proteins, for validation purposes.
Inducible Animal Models of Disease
An important feature of the ARGENT and RPD approaches is that they are fully compatible with in vivo studies; the inducing ligands have been validated and optimized in mice and (for ARGENT inducers) also in large animals such as primates. The technologies can therefore be used to reversibly activate the activity of a particular gene or protein in vivo in order to validate its physiological role. Activation can be restricted to cells or tissues of interest using tissue-specific promoters. If these studies indicate that a disease state is linked to activity of a particular gene or protein, these engineered animals provide an inducible model of disease that can allow further study of disease pathology, or serve as specific models for in vivo screening of drug candidates.
An example is the study of cancer. Transgenic mice have been engineered that express conditionally active alleles of individual FGF receptors exclusively in prostate tissue. Administration of the homodimerizer AP20187 to these mice dimerizes the receptors, leading to activation of signaling. Such mice have been used to identify a unique role for FGFR1, but not FGFR2, in particular stages of prostate cancer development (see reference; and figure below). The inducible FGFR1 mice in turn could potentially be used to screen for drugs that block these FGFR1-specific proliferative changes.
In vivo validation of FGFR1 as a target in prostate cancer using ARGENT
Another general use of ARGENT technology is the development of inducible mouse models of degenerative disease, in tissues such as liver and heart (see reference). These models rely on the general approach of using dimerizable caspase proteins as conditional cell death switches. Expression of such proteins in a tissue-specific manner leads to cell or tissue-specific ablation upon administration of homodimerizer drugs such as AP20187.
Drug Screening and Drug Discovery
The ability to induce a specific cell-signaling or gene activation event in a cell allows the configuration of "targeted" cell-based screens in which the unique cell context of interest for drug design can be chemically induced. These screens very specifically search for drugs affecting cells in which a particular signal transduction or gene activation event has occurred. The tight regulation afforded by our ARGENT technology means that exquisitely specific screens can be set up, using the uninduced cell line as a highly stringent counterscreen. Because the cellular event of interest can be induced chemically, the induction step can readily be configured into high-throughput screens.
ARGENT also forms the basis for three-hybrid assays and screens, in which ARGENT Transcription Technology is configured to include a novel ligand as part of the dimerizer, or a library of proteins as part of the regulated transcription factor. Three-hybrid approaches are powerful methods for discovering and characterizing novel target proteins for existing small molecules, or novel small molecules that bind to known target proteins. We have entered into licensing agreements with GPC Biotech AG and ALTANA Pharma AG for such applications.
Further Information
- For detailed information on individual Regulation Kits and their specific applications, click here.
- For an annotated bibliography of scientific publications describing use of our gene regulation technologies, click here.
Getting Access to Our Cell-signaling Regulation Technology
- If you are an academic researcher, click here to proceed directly to Requesting a Regulation Kit.
- If you are a researcher in a commercial or for-profit entity, click here to learn about our commercial licensing program.
