Relaxed complex scheme suggests novel inhibitors for the lyase activity of DNA polymerase beta
Graphical abstract
Research highlights
▶ DNA polymerase β is a promising therapeutic target for cancer treatment. ▶ We filtered the NCI diversity set, DrugBank and fragment compounds for novel pol β inhibitors. ▶ We screened the set of ∼12,500 compounds against an ensemble of 11 structures of the 8 kDa domain. ▶ Our results predicted new compounds that can bind with high affinity to the lyase active site.
Introduction
After several decades of cancer research, the scientific community is faced with an astounding fact. That is, DNA repair pathways serve as an important determinant of cancer cells’ sensitivity to anticancer agents and a major means of acquiring antitumor drug resistance [1], [2]. This is mainly because, most anticancer treatments in clinical use today, directly or indirectly damage DNA by causing DNA single- or double-stranded breaks or by interfering with the functions of crucial DNA interacting proteins [3]. Following the detection of the damaged DNA, a number of multiple and overlapping DNA repair pathways attempt to restore the genome, allowing the cell to survive or, if the damage is too heavy, induce apoptosis, forcing the cell to die.
In the middle of these pathways, base excision repair (BER) is the major cellular pathway that is responsible for the recovery of single strand breaks (SSB) and removal of damaged bases such as oxidized-reduced, alkylated and de-aminated bases [4]. These DNA modifications can occur spontaneously by exposing cells to environmental mutagens [2], or synthetically as a result of anticancer treatments using alkylating agents or ionizing radiation. Although, in the former case, BER protects normal cells from lethal mutations, in the latter, it acts as a dominant way for tumor cells to reduce the efficacy of a growing list of DNA damaging agents including bleomycin [5] monofunctional alkylating agents [4], cisplatin [6] and other platinum-based compounds. Therefore, it has been broadly proposed that regulating the BER pathway via small molecule inhibitors can reduce the required dosage of such DNA damaging agents while potentiating their efficacy in eradicating cancer cells [7].
Fortunately, most of the proteins involved in the BER process have been identified, cloned and crystallized allowing the rational design of small molecule inhibitors for their activity. One of these proteins, DNA pol β, plays an important role in chemotherapeutic agent resistance, as its over-expression reduces the efficacy of anticancer drug therapies including bleomycin and cisplatin [8], [9]. Furthermore, small-scale studies on different types of cancer showed that pol β is mutated in approximately 30% of tumors, which in turn reduces pol β fidelity in DNA synthesis exposing the genome to serious and often deleterious mutations [10], [11]. The uncomplicated small structure of the protein (39 kDa) made it a standard model that helped in understanding the functional mechanisms of other DNA polymerases. In addition, there is a large body of evidence that pol β plays an important role throughout cell's life. For example, a “knock-out” of the gene that encodes for pol β in mice results in embryonic lethality, confirming the importance of the protein during fetal development [12]. Based on these findings, pol β, the error-prone polymerase of BER, has been seriously considered as a promising therapeutic target for cancer treatment. Many inhibitors of DNA pol β have been identified during the last two decades (reviewed in Refs. [13], [14]). To name but a few, this list includes polypeptides [15], fatty acids [16], triterpenoids [17], sulfolipids [18], polar lipids [19], secondary bile acids [20], phenalenone-derivatives [21], anacardic acid [22], harbinatic acid [23], flavanoid derivatives [24], and pamoic acid [25]. However, most of these inhibitors are not potent enough or lack sufficient specificity to eventually become approved drugs.
Receptor-based virtual screening (VS) is a well-established technique to uncover novel molecular inhibitors that complement a target protein in terms of shape, charge and several additional biophysical or biochemical properties [26]. Although VS applied against a crystal or relaxed receptor structure is a commonly used approach in structure-based drug design, the dynamic changes due to receptor flexibility are always neglected to the detriment of the predictive success of these methods. For instance, a ligand can induce significant conformational changes to its target, ranging from local reorganization of side-chains to hinge movement of domains [27]. Sampling these conformational changes during docking is impractical, as they involve too large a number of degrees of freedom. One successful approach that has been reported by McCammon et al. is the relaxed complex scheme (RCS) [28], [29]. In this method, molecular dynamics (MD) simulations are applied to explore the conformational space of the protein receptor, while docking is subsequently used for the fast screening of drug libraries against an ensemble of receptor conformations. This methodology has been successfully applied to a number of cases [28], [30], [31]. An excellent example is an HIV inhibitor, raltegravir [32], which became the first FDA approved drug targeting HIV integrase. MD simulations played a significant role in discovering a novel binding site, and compounds that can exchange between the two binding sites have formed a new generation of HIV integrase inhibitors.
Here, we applied the RCS technique to account for the dominant backbone dynamics in screening for inhibitors of the lyase activity of DNA pol β. Using pamoic acid as a positive control, an inhibitor that targets pol β [25], we employed AUTODOCK version 4.0 [33], to filter a library of ∼12,500 small molecules for their activity against the lyase domain of pol β. This library was comprised of the National Cancer Institute (NCI) diversity set, DrugBank small molecules and a set of ∼9000 small fragments with drug-like properties. The full set of compounds has been screened against an ensemble of pol β structures. The ensemble incorporated the NMR structure of the 8kDa domain of the protein (PDB code 1DK3) [34], in addition to the 10 principal conformations as extracted from MD simulations. In this way, AUTODOCK was used to place the compounds within the specified binding site and to search for their minimal energy conformations. Finally, the irredundant top 300 hits from AUTODOCK screening were rescored using the molecular mechanics Poisson–Boltzmann surface area (MM–PBSA) method [35]. It is hoped that our results will eventually be used in the design of more potent and particularly pol β-specific inhibitors, aimed at improving existing cancer therapies including ionizing radiation, bleomycin, monofunctional alkylating agents and cisplatin.
Section snippets
Results and discussion
The faithfulness of BER is dependent on the polymerization step, where the major BER DNA polymerase, pol β, must incorporate the correct Watson–Crick base paired nucleotide into the one-nucleotide repair gap. The enzyme has been identified as a 39-kDa protein with 335 amino acids in its sequence (see Fig. 1) [36]. Its small size compared to other polymerases, made it the smallest and simplest cellular DNA polymerase found. Although pol β lacks the proof-reading 3′- or 5′-exonuclease activities,
Molecular dynamics simulations
The 8-kDa domain of pol β (residues 1–87) was taken from the PDB entry 1DK3 [34]. MD simulations were carried out using the NAMD program [54], at a mean temperature of 310 K and physiological pH (pH 7) using the all-hydrogen AMBER99SB force field [55]. Protonation states of all ionizable residues were calculated using the program PDB2PQR [56]. Following parameterization, the protein was immersed in the center of a TIP3P water cube after adding hydrogen atoms to the initial protein structure. The
Selection of ligand database
The National Cancer Institute Diversity Set (NCDIS) [65], DrugBank small molecules [66], and a set of 9135 fragment structures were used as our test libraries of compounds. The NCIDS is a collection of approximately 2000 compounds that are structurally representative of a wide range of molecules, representing almost 140,000 compounds that are available for testing at the NCI. Unfortunately, a number of ligands containing rare earth elements could not be properly parameterized and were excluded,
Conclusions
Family-X of DNA polymerases in general and pol β in particular are the foremost elements of the BER pathway [37b]. Fortunately, a lot of structural data and biological information about pol β are currently available, making it the first DNA polymerase enzyme whose structural description is complete [41], [68]. These observations stimulated research aimed at finding regulators of BER through the discovery of inhibitors of the polymerization step of the pathway. The fundamental principle behind
Acknowledgments
All of the molecular dynamics simulations and virtual screening experiments were produced using the SHARCNET, AICT (University of Alberta cluster) and WESTGRID computational facilities. Funding for this work was obtained from NSERC, Alberta Cancer Foundation, Alberta Cancer Research Institute, Canadian Breast Cancer Foundation, Alberta Advanced Education and Technology and the Allard Foundation.
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