The the first generation of small molecule inhibitors of

relentless growth of tumors is triggered by a complex array 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 ROS, 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 fluorescence-based assay in
order to screen a second generation of imidopiperidine compounds. This
resulted in the identification of A12B4C50 and A83B4C63, which are 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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