A CRISPR method for rapid in-cell validation of epigenetic targets

Lead Research Organisation: University of Oxford
Department Name: SABS IDC

Abstract

A key bottleneck in the drug discovery process is target validation. This process, in which a given cellular target is shown to be linked to the desired therapeutic outcome, frequently prolongs the drug discovery process. Incomplete or incorrect target validation can lead to costly failures at later stages of the drug discovery process, once more time and money has been invested. Consequently, techniques and approaches that enable rapid and robust target validation are of great value to those seeking to discover new medicines, and ultimately, the heath and wealth of the UK.

Here we aim to combine synthetic organic chemistry and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR- Cas9) technology to develop a method that allows rapid cellular target validation of a given protein. Using computational techniques to guide our work, we will first identify a combination of cysteine protein mutants and covalent ligands that interact selectively over other nucleophilic residues in the protein, and other endogenous cellular nucleophiles (e.g. glutathione [GSH]). The protein mutant will retain similar function to the wildtype (WT) protein. Once a selective combination has been identified, we will use CRISPR- Cas9 to engineer the same mutation into the protein in human cell lines. In the absence of the covalent ligand the protein will function as normal. Addition of the covalent ligand will act like a conditional knock-out, ablating the activity of the protein and resulting in a cellular phenotype, and information on the cellular function of the target protein. The advantage of using small molecules is that individual domains, rather than the whole protein, can be "knocked-down". The advantage of using covalent ligands is that significant optimisation of the ligand's affinity will not be required, provided that it shows sufficient selectivity for the target protein. To achieve proof-of-concept we have chosen the first bromodomain of BRD4 [BRD4(1)]. Bromodomains are epigenetic reader protein modules that bind to acetylated lysine residues (KAc) found in >3000 cellular sites in >1700 proteins throughout cells. Most work in this area has focused on the binding of BRD4 to KAc on histone proteins - the hub proteins around which DNA is wound in chromatin. BRD4 has proved an exciting new drug target, in part because it affects the expression level of the oncogene myc, and there are now >10 compounds in >20 clinical trials, mainly in the oncology space. We have picked BRD4(1) as our target as we have experience in the expression and purification of the protein, we have expertise in the development of BRD4 bromodomain ligands, and inhibition of BRD4 function has a clear phenotype, which will act as our cellular read-out. This work falls into the EPSRC areas of "chemical biology and biological chemistry" and "synthetic organic chemistry" and the BBSRC area of "systems approaches to the biosciences".

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R512333/1 01/10/2017 30/09/2021
1941466 Studentship EP/R512333/1 01/10/2017 30/09/2021 Adam Thomas