Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair

Abstract

The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment.

Keywords: DNA double-strand break (DSB) repair; DNA repair and DNA damage response (DDR); homology directed repair (HDR); non-PIKKs inhibitors; non-homologous end joining (NHEJ); polymerase theta-mediated end joining (TMEJ); single-strand annealing (SSA); synthetic lethality.

Figure 1

The two major pathways of DNA double-strand break repair: During NHEJ, the DNA double strand sites are initially recognized by heterodimeric Ku70/80. This is followed by recruitment of DNA-PKcs and Artemis, DNA end processing by Artemis, polymerase λ and μ, TDP and PNKP and finally ligation of DSB breaks by Ligase IV/XRCC4/XLF complex for completion of the repair pathway. The other accessory proteins like APLF, PAXX and XLF also participate in the repair functions. During HDR/HR, DSBs are recognized and resected by the MRN complex to generate a 3’ overhang. BRCA2/RAD51, along with other RAD51 paralogs, binds to the RPA coated ssDNA tails after which RAD51 replaces RPA in a BRCA1-and BRCA2-dependent process, forming a presynaptic filament. Upon strand invasion, D-loop formation and DNA repair synthesis can be resolved through Holliday junction, after which distinct independent pathways can operate to complete the HDR repair pathway. NHEJ is available throughout interphase while HDR is restricted to S/G2 phases of the cell cycle.

Figure 2

Schematic representation showing non-PIKKs DSB repair inhibitors that target key/core and accessory proteins involved in DSB repair pathways.

Figure 3

Small molecule inhibitors of Ku70/80 and their respective IC50 values for disruption of DNA-binding by Ku70/80 and DNA-PK activity.

Figure 4

Molecular interactions of (A) compound 149 and (B) 245 (all in green carbon) with Ku70/80 heterodimer (key amino acids are shown in yellow carbon (Ku70), blue carbon (Ku80) and cartoon is shown in cyan color). Interaction with amino acid side chains is indicated with the dashed magenta lines and π – π stacking interactions are shown in solid magenta dumbbell. The DNA helical structure is depicted in greenish blue sticks and light orange cartoon. Interaction distances indicated in Å.

Figure 5

Small molecule inhibitors targeting Artemis and their respective IC50 values for disruption of endonuclease activity.

Figure 6

Small molecule inhibitors targeting DNA Ligase IV and their IC50 values for either inhibition of Ligase IV adenylation or Ligase IV end-joining.

Figure 7

Structures of XRCC4 inhibitors.

Figure 8

Small molecule inhibitors targeting MRE11 with their respective IC50 values for inhibition of nuclease activity.

Figure 9

Small molecule inhibitors targeting RPA N-terminal protein-protein interactions and RPA-DNA interactions with their respective Kd/IC50 values.

Figure 10

Small molecule inhibitors targeting RAD51 with their respective Kd/IC50 values for either disruption of RAD51 binding or RAD51 mediated D-loop formation.

Figure 11

Small molecule inhibitors targeting RAD52 with their respective Kd/IC50 values for either RAD52 binding or ssDNA annealing activity.

Figure 12

Small molecule inhibitors targeting ERCC1-XPF with their respective Kd/IC50 values for inhibition of ERCC1-XPF endonuclease activity.

Figure 13

Small molecule inhibitors targeting Pol θ with their respective IC50 values for inhibition of polymerase activity.

Figure 14

Small molecule inhibitors targeting WRN, BLM and MCM helicases with their respective IC₅₀ values.