The
relentless growth of tumors is triggered by a complex of molecular changes such
as DNA damage, disruption of cell-cycle progression, uncontrolled proliferation
and escaping cell death. Various therapies have been developed to treat cancer,
many of which kill cancer cells by damaging their DNA. DNA damage in cells,
including DNA strand breaks, are caused by endogenous agents, mainly reactive
oxygen species (ROS), and exogenous sources such as ionizing radiation (IR) and
topoisomerase poisons, such as irinotecan. Clinical evidence indicates that DNA
repair is a major cause of cancer resistance. Therefore, attack on DNA repair processes renders cancer
cells more sensitive to radiotherapy and DNA damage chemotherapy.

Targeting DNA
repair enzymes is one approach to overcome resistance in cancer. DNA strand
breaks, major lesions generated by IR and irinotecan, are lethal to cells if
not repaired. The 3?- and 5?- termini of the DNA strand breaks are often
modified and do not present the correct termini for completion of DNA repair.
Among the frequently generated modifications are 3?-phosphate and 5?-hydroxyl
termini. Human polynucleotide kinase/phosphatase
(PNKP), a bifunctional DNA repair enzyme which phosphorylates DNA
5?-termini and dephosphorylates DNA 3?-termini, can
process the unligatable DNA termini. Moreover, cancer cells depleted of PNKP
show significant sensitivity to ionizing radiation and chemotherapeutic drugs
such as irinotecan.  Initial screening for the first generation of
small molecule inhibitors of PNKP phosphatase activity identified A12B4C3, an
imidopiperidine compound, which enhanced the radio- and chemosensitivity of
lung and breast cancer cells. Based on these findings, we intended to identify more potent PNKP phosphatase inhibitors than A12B4C3 and
design suitable nanoparticles to target inhibitors to cancer cells.

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First, I
developed a novel mix and read fluorescence-based
assay in order to screen a second generation of imidopiperidine compounds. This
resulted in the identification of A12B4C50 and A83B4C63, more potent inhibitors
than A12B4C3. In addition, I screened new compounds from a natural
derivative library, which resulted in the
identification of two new promising 3?-phosphatase inhibitors, N12 and O7. The novel
assay was used to determine the IC50 values of the newly identified
inhibitors. Kinetic analysis revealed that A83B4C63 acts as a non-competitive
inhibitor, whereas N12 acts as an uncompetitive inhibitor.

To test the hypothesis that nano-encapsulation would enhance
the effectiveness of the newly identified imidopiperidine-based 3?-phosphatase
inhibitors in a cellular context, a series of experiments was carried out with
A12B4C50 and A83B4C63. First I examined the retention of the inhibitors by
polymeric micelles of different poly(ethylene oxide)-b-poly(ester) based structures to determine suitable encapsulation
media for each inhibitor.  Cellular
studies revealed that encapsulated A12B4C50 and A83B4C63 sensitized HCT116
cells to ?-radiation and irinotecan. Furthermore, the encapsulated inhibitors
were capable of inducing synthetic lethalilty in phosphatase and tensin homolog (PTEN)-deficient HCT116
cells. In addition, actively targeted
delivery of nano-encapsulated inhibitors to colorectal cancer cells
overexpressing epidermal growth factor receptor (EGFR) was achieved by
attachment of the peptide GE11 on the surface of polymeric micelles.
Preliminary studies with a human xenograft model in nude mice indicated that
encapsulated A83B4C63 has the capacity to treat PTEN deficient tumors as a
monotherapeutic agent. 

Finally, investigation of the potential site of binding of
3?-phosphatase inhibitors to PNKP  was
determined by photoaffinity crosslinking method coupled with liquid
chromatography-mass spectrometry technique (LC/MS). The photoactivatable PNKP
inhibitors A95B4C50, A95B4C3 and A12B4C67 revealed three distinct binding sites
located in both the kinase and phosphatase domain of PNKP.

 

 

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