Abstract
BackgroundDNA polymerase beta (pol beta), the error-prone DNA polymerase of single-stranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. Several inhibitors have been identified, but none of them presents a sufficient affinity and specificity to become a drug. The fragment-based inhibitor design allows an important improvement in affinity of small molecules. The initial and critical step for setting up the fragment-based strategy consists in the identification and structural characterization of the first fragment bound to the target.ResultsWe have performed docking studies of pamoic acid, a 9 micromolar pol beta inhibitor, and found that it binds in a single pocket at the surface of the 8 kDa domain of pol beta. However, docking studies provided five possible conformations for pamoic acid in this site. NMR experiments were performed on the complex to select a single conformation among the five retained. Chemical Shift Mapping data confirmed pamoic acid binding site found by docking while NOESY and saturation transfer experiments provided distances between pairs of protons from the pamoic acid and those of the 8 kDa domain that allowed the identification of the correct conformation.ConclusionCombining NMR experiments on the complex with docking results allowed us to build a three-dimensional structural model. This model serves as the starting point for further structural studies aimed at improving the affinity of pamoic acid for binding to DNA polymerase beta.
Highlights
DNA polymerase beta, the error-prone DNA polymerase of singlestranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis
Systematic analysis of the 100 best docked structures revealed that all of them were located in a single site, http://www.biomedcentral.com/1472-6807/8/22 pamoic acid could move freely around the 8 kDa domain during docking
A NOE correlation helps in determining the correct conformation In order to further discriminate the right position, we looked for intermolecular NOEs. 3D 15N-TOCSY-HSQC (TOtal Correlated SpectroscopY) and 3D 15N-NOESYHSQC (Nuclear Overhauser Enhancement SpectroscopY) spectra of the free 15N-labeled 8 kDa domain were acquired and 3D 15N-NOESY-HSQC spectrum was recorded in the presence of equimolar pamoic acid concentration
Summary
DNA polymerase beta (pol beta), the error-prone DNA polymerase of singlestranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. The first is termed the 8 kDa domain and is located at the N-terminal position It binds to single-stranded and double stranded DNA, recognizes the 5'-phosphate group in gapped DNA and possesses dRP lyase activity [2]. The second, the C-terminal 31 kDa domain, displays the catalytic activity of polymerisation [3]
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