The changes such as DNA damage, disruption

Therelentless growth of tumors is triggered by a complex array of molecularchanges such as DNA damage, disruption of cell-cycle progression, uncontrolledproliferation and escaping cell death. Various therapies have been developed totreat cancer, many of which kill cancer cells by damaging their DNA. DNA damagein cells, including DNA strand breaks, are caused by endogenous agents, mainlyreactive oxygen species (ROS), and exogenous sources such as ionizing radiation(IR) and topoisomerase poisons, such as irinotecan.

Clinical evidence indicatesthat DNA repair is a major cause of cancer resistance. Therefore, attack on DNA repair processes renders cancercells more sensitive to radiotherapy and DNA damage chemotherapy.Targeting DNArepair enzymes is one approach to overcome resistance in cancer.

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DNA strandbreaks, major lesions generated by ROS, IR and irinotecan, are lethal to cellsif not repaired. The 3?- and 5?- termini of the DNA strand breaks are oftenmodified and do not present the correct termini for completion of DNA repair.Among the frequently generated modifications are 3?-phosphate and 5?-hydroxyltermini. Human polynucleotide kinase/phosphatase(PNKP), a bifunctional DNA repair enzyme which phosphorylates DNA5?-termini and dephosphorylates DNA 3?-termini, canprocess the unligatable DNA termini.

Moreover, cancer cells depleted of PNKPshow significant sensitivity to ionizing radiation and chemotherapeutic drugssuch as irinotecan.  Initial screening for the first generation ofsmall molecule inhibitors of PNKP phosphatase activity identified A12B4C3, animidopiperidine compound, which enhanced the radio- and chemosensitivity oflung and breast cancer cells. Based on these findings, we intended to identify more potent PNKP phosphatase inhibitors than A12B4C3 anddesign suitable nanoparticles to target inhibitors to cancer cells. First, Ideveloped a novel fluorescence-based assay inorder to screen a second generation of imidopiperidine compounds. Thisresulted in the identification of A12B4C50 and A83B4C63, which are more potentinhibitors than A12B4C3. In addition, I screened newcompounds from a natural derivative library, which resulted in the identification of two new promising3?-phosphatase inhibitors, N12 and O7.

The novel assay was used to determine theIC50 values of the newly identified inhibitors. Kinetic analysisrevealed that A83B4C63 acts as a non-competitive inhibitor, whereas N12 acts asan uncompetitive inhibitor. To test the hypothesis that nano-encapsulation would enhancethe effectiveness of the newly identified imidopiperidine-based 3?-phosphataseinhibitors in a cellular context, a series of experiments was carried out withA12B4C50 and A83B4C63. First I examined the retention of the inhibitors bypolymeric micelles of different poly(ethylene oxide)-b-poly(ester) based structures to determine suitable encapsulationmedia for each inhibitor.  Cellularstudies revealed that encapsulated A12B4C50 and A83B4C63 sensitized HCT116cells to ?-radiation and irinotecan.

Furthermore, the encapsulated inhibitorswere capable of inducing synthetic lethalilty in phosphatase and tensin homolog (PTEN)-deficient HCT116cells. In addition, actively targeteddelivery of nano-encapsulated inhibitors to colorectal cancer cellsoverexpressing epidermal growth factor receptor (EGFR) was achieved byattachment of the peptide GE11 on the surface of polymeric micelles.Preliminary studies with a human xenograft model in nude mice indicated thatencapsulated A83B4C63 has the capacity to treat PTEN deficient tumors as amonotherapeutic agent.

  Finally, investigation of the potential site of binding of3?-phosphatase inhibitors to PNKP  wasdetermined by photoaffinity crosslinking method coupled with liquidchromatography-mass spectrometry technique (LC/MS). The photoactivatable PNKPinhibitors A95B4C50, A95B4C3 and A12B4C67 revealed three distinct binding siteslocated in both the kinase and phosphatase domain of PNKP.


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