Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Abstract

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

“…We also examined the effect of M3814 treatment on signaling events mediated by other kinases related to DNAPKcs: ATR (CHK1-S345p), ATM (ATM-S1981p), and MTOR (ribosomal protein S6-S235/236p) 40 – 42 . We found that these phosphorylation events were not obviously affected at the doses used in the above experiments (i.e., 250 nM, 500 nM, and 1000 nM, Supplementary Fig 2a ), which is consistent with the notion that M3814 is a specific inhibitor of DNAPKcs 38 , 39 , and supports the use of the intermediate dose of 500 nM for the below experiments. Finally, we also examined cellular localization of XLF-WT vs. XLF-K160D by immunofluorescence, and found similar staining patterns (i.e., staining in both the nucleus and cytoplasm, Supplementary Fig.…”

“…We then performed similar experiments with inhibition of DNAPKcs kinase activity, which appears to block its dissociation from DNA ends 7 , 8 , 35 – 37 . To begin with, we interrogated the effect of inhibiting DNAPKcs kinase activity with the small molecule inhibitor M3814 (i.e., Nedisertib) 38 , 39 , and found a concentration dependent decrease in No Indel EJ events in HEK293 (Fig. 2a ).…”

Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM

“…Lastly, single particles are boxed out for iterative classification to sort out suitable particles for the final three-dimensional (3D) reconstruction (Cheng et al, 2015;Lyumkis, 2019). The obtained structural details of biological macromolecules at atomic resolution provide valuable information for not only unravelling the fundamental mechanisms of myriad biological processes but also driving the development of drugs for treating diseases caused by the dysfunctional biological macromolecules (Zhu et al, 2018;Han et al, 2022;Liang et al, 2022). Recent technical breakthroughs in single-particle cryo-EM have created a "resolution revolution" in structural biology by circumventing the major challenges faced when using the traditional X-ray crystallography methods (Cheng, 2015;Cheng, 2018).…”

Perspective on the Use of DNA Repair Inhibitors as a Tool for Imaging and Radionuclide Therapy of Glioblastoma

“…The BBB penetration capabilities are still under investigation (CilinicalTrials.gov Identifier: NCT04555577). Based on the structural interactions between nedisertib and the active site of the DNA-PK, both the quinazoline and morpholino moieties bind into the hydrophobic pocket, while the pyridazine ring rotates to have π-π interactions with the quinazoline plane [ 284 ]. The chloro-fluorobenzene ring in the active site is directed towards the N-lobe, thus potentially allowing radiohalogenation at positions one and three of the ring.…”