Glucocorticoid receptor (GR) is a member of the nuclear receptor (NR) superfamily that is instrumental in regulating genes involved in inflammation and immunosuppression and represents a major therapeutic target for asthma, arthritis, lupus, and other chronic disorders. A major challenge in GR drug design is the necessity to enhance GR-mediated anti-inflammatory and immunosuppressive action, while down-regulating deleterious corticosteroid side effects including diabetes, weight gain, hypertension, and osteoporosis. The most critical interaction for GR-mediated gene transcription occurs at its glucocorticoid response element (GRE) DNA site. Variations of the GRE dictate differential gene transcription, yet a comprehensive analysis of GR interaction exploring every permutation of the GRE has not been done. We have developed Cognate Site Identifier (CSI) DNA microarrays containing every permutation of a 12 base pair DNA sequence within duplex DNA. Our proposal will focus on understanding how interactions with ligands and heterodimeric proteins affect GR recognition of DNA, expanding the CSI technology for discovery of novel targets for drug design, small-molecule screening, and development of artificial transcription factors.
The specific aims of this Phase I proposal are: 1. DNA binding preferences of GR in response to receptor interactions with ligand and with a heterodimeric partner AP-1 will be characterized using CSI analysis. These studies will identify potential new drug targets and key changes in DNA binding specificity. 2. DNA-binding small-molecule polyamides, comprised of N-methylpyrrole and N-methylimidazole pairs that can precisely target DNA sites, will be designed to disrupt GR-GRE and GR-AP1-DNA interactions. These molecules will be useful as therapeutics to down-regulate GR-mediated transcription at corticosteroid-regulated genes involved in negative side effects. 3. A high throughput screening platform using CSI microarrays to differentiate test compounds as disruptors or enhancers of GR-GRE interactions and GR-AP1 binding at composite sites will be developed. In this proposal, we develop a microarray platform to examine the DNA binding specificity of important drug targets for anti-inflammatory therapeutic development. Targeted effectors of protein-DNA interactions and DNA-mediated allosteric effects on protein-ligand interactions are under-studied areas of drug design that have enormous therapeutic potential. CSI information from GR-DNA interactions will be used to meaningfully annotate the human genome, to identify differences due to critical single nucleotide polymorphisms in patient populations, and to predict whether a drug target may affect undesirable genes or pathways. The CSI methodology can be applied for any DNA-binding proteins and molecules, many of which are linked to cancer, diabetes, obesity and other disorders.
A major challenge in glucocorticoid receptor (GR) drug design is the necessity to identify """"""""dissociated ligands"""""""" that enhance anti-inflammatory and immunosuppressive action for treatment of asthma, arthritis, lupus and other disorders, while down-regulating corticosteroid-mediated deleterious side effects such as weight gain, diabetes, hypertension, and osteoporosis. The most critical interaction for GR-mediated gene transcription occurs at its glucocorticoid response element (GRE) DNA site. Variations of the GRE dictate differential gene transcription, yet a comprehensive analysis of GR interaction exploring every permutation of the GRE has not been done. Using Cognate Site Identifier (CSI) DNA microarrays displaying every permutation of a 12 base pair DNA sequence, our proposal will focus on understanding how interactions with ligands and heterodimeric proteins affect GR recognition of DNA for discovery of novel targets for drug design, small-molecule screening, and development of directed transcription factors.
