Delineation of key XRCC4/Ligase IV interfaces for targeted disruption of non-homologous end joining DNA repair

Abstract

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair.

Chemotherapeutic Compounds Targeting the DNA Double-Strand Break Repair Pathways: The Good, the Bad, and the Promising

“…The minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of abolishing XRCC4/XIR complex was recently identified (181). The key interfaces of ligase IV necessary for interaction with XRCC4 were identified by the development a competitive displacement assay using ESI-MS/MS.…”

“…The results suggest that by targeting the interface of helix 2 of DNA ligase IV, modulators that inhibit the XRCC4/DNA ligase IV complex may be identified. In addition, adjuvant compounds to further block the XRCC4/DNA ligase IV complex may be discovered by further targeting helix 1 and the loop regions of the helix–loop–helix clamp, which offer a secondary target surface (181). While this study has the potential to identify inhibitors of XRCC4, to date, inhibitors that have been tested in vivo and in clinical trials have not been described.…”

Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

“…However, additional studies using ectopic expression of truncated Lig4 constructs have also shown that the sole BRCT2 domain was not sufficient to prevent formation of the endogenous XRCC4/Lig4 complex 16 . Moreover, a recent biophysical study with fragments from the XRCC4 Interacting Region (XIR) reported that a competitive displacement of the XIR from XRCC4 was only significant with either fragments of Lig4 containing Helix2 or with the Helix2 alone 26 . Accordingly, our MD simulation revealed that in addition to the BRCT2 domain, the small Lig4 Helix2 fragment had also several strong contacts while only Phe778 in Helix1 reached a percentage of contact over 80% ( Fig.…”

“…XRCC4/Lig4 interface spans over ~2900 Å 2 16 . The XRCC4/Lig4 interaction resists high salt or urea 9 10 22 and organic solvents 26 . Indeed, the binding constant between the Lig4 XIR fragment and XRCC4 is in the nanomolar range 21 26 likely precluding disruption of the preformed complex by small molecules.…”

“…The XRCC4/Lig4 interaction resists high salt or urea 9 10 22 and organic solvents 26 . Indeed, the binding constant between the Lig4 XIR fragment and XRCC4 is in the nanomolar range 21 26 likely precluding disruption of the preformed complex by small molecules. Nevertheless, interference with the assembly between XRCC4 and Lig4 in human cells has proven to be a feasible radio-sensitization strategy through over-expression of XRCC4 27 or Lig4 fragments (our group, 16 ).…”