Wednesday, March 12, 2025
3/12/2025
Project Summary/Abstract Our research program involves the use of DNA-encoded chemical libraries (DELs) and DNA-linked enzyme activity probes, which are new approaches for biomedical research that capitalize on the power of DNA analysis techniques. Specifically, this work involves development of in vitro selection assays for both DELs and enzyme activity probes. It is the in vitro selection that encodes transduces information (drug molecule activity or biochemical activity of a sample) into DNA sequences to facilitate analysis. This work advances these techniques into new areas, particularly for medicinal chemistry applications, to provide tools for biological discovery and development of new therapeutics. We are using DELs in a directed, targeted way (on-DNA medicinal chemistry) to produce inhibitors to the chromodomains in the CBX family and to several bromodomains. The homology of the chromodomains in the eight chromobox (CBX) proteins and of the family of bromodomains (61 in humans) makes selective inhibition challenging. Inhibitor probes generated will be used to the decipher roles of these proteins in transcriptional regulation and in disease states. DELs are now routinely used for de novo discovery of compounds that bind to a drug target to initiate a drug development campaign. The selection assay used for this discovery is a simple affinity purification with a purified protein on a solid support. The requirement of a pure and active protein for this assay severely limits the target scope of DELs, particularly for membrane bound protein targets, which constitute a large portion of drug target space. We are developing selection assays to enable use of DELs to protein targets both on and within live cells. These assays rely on bioluminescence resonance energy transfer (BRET), which is a common modality for detection of interacting molecules in cells. We will apply these unique assays with highly diverse (>109), commercially available DELs to challenging protein targets including Nrf2 (potential cancer target) and adenylyl cyclase 1 (a potential target for pain). In addition, we are developing selection assays to identify molecules that not only bind to a protein receptor but activate downstream signaling pathways. We are applying this selection to the opioid family of GPCRs to identify novel agonists. We will advance to use DNA-linked enzyme activity probes for the proteomic profiling of tyrosine kinase activities and for drug binding assays amenable to high throughput screening of traditional (off-DNA) compound collections. We are implementing our DNA-based kinase activity profiling to further understand the mechanism of drug resistance to tyrosine kinase inhibitors in cancer therapy. Also, the high sensitivity of the approach (enabled by DNA amplification) will be used to assay kinase activities in single cells, which will provide a greater understanding of cellular complexity within tumors.
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