Design, Synthesis, and Biological Evaluation of Compounds for DNA Targeted Therapy

• Author / Creator

  Ahmed Hamdy Mahmoud Elmenoufy

• At present, the number of cancer patients who develop resistance to conventional cancer therapeutics, such as DNA damaging chemotherapeutics, or radiotherapy, is increasing. A major reason for developing such resistance is that cancer cells have the ability to repair their DNA damage caused by these therapies through various DNA repair pathways. The most important of these damages are photo adducts produced by the UV component of sunlight, such as cyclobutane pyrimidine dimers (CPD) and cisplatin-DNA products, which cause intra-strand crosslinks (ICL). These bulky, helix distorting lesions are repaired mainly by nucleotide excision repair (NER), a highly versatile repair pathway that can recognize, verify, remove, and correct these damages. ERCC1–XPF is a 5´-3´ structure-specific endonuclease that is involved in NER and ICL repair pathways in mammalian cells. It plays a central role in NER because it removes CPD and chemically induced helix-distorting lesions by incising the damaged DNA strand 5´ and 3´, respectively. Therefore, ERCC1–XPF has become an interesting therapeutic target to manipulate the DNA repair pathways, and its inhibition has the potential of sensitizing cancer cells towards cytotoxic chemotherapeutic agents and ionizing radiation (IR). One of the potential sites on ERCC–XPF to target for inhibiting its endonuclease activity is the interaction site between ERCC and XPF which is essential for the protein stability and activity. A few groups have developed hits that interfere with the interaction site and inhibit its endonuclease activity successfully. However, the activities of the reported hits to date are suboptimal in terms of clinical properties, including potency and further optimization is required. In Chapter 2, we used the reported F06 (compound 1) as a reference hit that was subjected in docking-based virtual screening. The in-silico screening results yielded compounds with a better binding energy and ligand efficiency values using the XPF interaction domain. Synthesis of seven novel analogues of F06 (1) is discussed. Two of the new F06-based derivatives were shown to have a potent inhibitory effect on ERCC1–XPF activity relative to F06 in vitro. The cell-based assays showed that compound 4 significantly inhibited NER by inhibiting the removal of CPDs in UV-irradiated cells. Also, it successfully showed a significant sensitization of colorectal cancer cells to cyclophosphamide and UV radiation. Chapter 3 describes the use of a multi-step CADD strategy to identify better inhibitors than F06 (1) based on the modification of one site of F06 (1), methoxy-acridine functional group. The in silico screening study identified two compounds that showed improved inhibitory effect on the ERCC1–XPF nuclease activity compared to the parent compound F06 (1). The in vitro endonuclease assay revealed that B9 has a better ERCC1–XPF inhibition with an IC50 of 0.49 µM, showing 3-fold improvement in inhibition activity compared to 1. Detailed analysis of the predicted binding mode of B9 to XPF derived from molecular dynamics simulations is discussed. This analysis showed that the hydroxyl group substituting the methoxy in F06 (1) was involved in a new hydrogen bond interaction with side chain of V859, not observed for F06 (1) or our previous inhibitor compound 4. Therefore, having a polar hydrogen bond donor group in this site is responsible for the better in vitro activity of these two compounds in comparison to F06 (1).Chapter 4 discusses an extensive structure activity relationship analysis that has been conducted based on the previously identified hit compound, F06 (1), as a reference compound. Two different series of generations have been developed by structure-based design and were synthesized through various modifications on two different sites of F06 (1), according to the corresponding pharmacophore model. Unfortunately, lack of piperazine moiety in Gen C compounds was accompanied by a lost activity. Also, replacing the acridine moiety with smaller aromatic group such as quinoline (Gen D compounds) was responsible for losing the inhibitory effect on ERCC1-XPF. Therefore, B9 has been selected for carrying out further cell-based/ cell-free assays. B9 elicit high binding affinity to the ERCC1–XPF complex with a Kd of 85 nM relative to 140 nM for 1. B9 also showed a significant sensitization of lung and colorectal cells towards cisplatin, cyclophosphamide, and Mitomycin C. The proximity ligation assay suggested that the mechanism of inhibition of B9 is mediated by heterodimerization disruption.

• Subjects / Keywords

  • Structure activity relationship
  • DNA targeted therapy
  • Synthesis of different generations
  • Computer aided drug design
  • Medicinal chemistry
  • ERCC1-XPF inhibitors
  • In vitro biological evaluations
  • Organic chemistry
  • Nucleotide excision repair
  • Synergism

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-6fj5-kb12

• License

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