Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku–DNA interaction

Abstract

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku–DNA interaction. We have developed a series of highly potent and specific Ku–DNA binding inhibitors (Ku-DBi’s) that block the Ku–DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi’s directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi’s cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi’s. The utility of Ku-DBi’s was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi’s, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

The Multifaceted Roles of Ku70/80

“…Weterings et al identified several potential inhibitors by computational screening and characterized a low micro-molar inhibitor that disrupts the binding of Ku70/80 to DNA substrates [ 55 ]. Recently, Gavande et al described the discovery of highly potent and selective DNA-PK inhibitors that block the Ku70/80 heterodimer interaction with DNA [ 56 ] ( Figure 3 f).…”

“…The loops of Ku in contact with DNA superimpose well, indicating that Ku has the same inner ring conformation with or without DNA-PKcs. ( f ) Inhibitors of Ku-DNA interactions reported by [ 55 ] (compound L) and by [ 56 ] (compound 245).…”

DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation

“…Small molecule inhibitors have been developed that target the first step in the NHEJ pathway, namely binding of Ku70/80 dimer to DNA ( Figure 3B ; Weterings et al, 2016 ; Gavande et al, 2020 ). The most promising inhibitors target a ligand binding pocket in close proximity to the DNA-binding region, interfacing with both the Ku70 and Ku80 subunit.…”

“…The most promising inhibitors target a ligand binding pocket in close proximity to the DNA-binding region, interfacing with both the Ku70 and Ku80 subunit. By blocking the DNA binding capacity of the Ku heterodimer, the downstream catalytic activity of DNA-PKcs is inhibited, hereby preventing DSB repair ( Weterings et al, 2016 ; Gavande et al, 2020 ). The inhibitors induce increased sensitivity to DSB inducing agents, such as IR, in in vitro experiments.…”

Improved and Flexible HDR Editing by Targeting Introns in iPSCs

“…NHEJ is the predominant and fast-acting pathway to repair CRISPR-mediated DSBs and outcompetes other editings [24,33]. Therefore, NHEJ inhibitors improve HDR efficiency [34][35][36]. One of the most effective NHEJ inhibitors is M3814, which strikingly improves HDR by blocking NHEJ [24,37].…”