Nucleic acids are central molecules in the transmission, expression and conservation of genetic information. Recognition of duplex DNA by oligonucleotides, forming DNA triple helices, provides a promising approach to a chemical solution for DNA recognition. These triplex forming oligonucleotides (TFOs) have aroused significant interest as potential inhibitors of the expression of particular genes. The ets2 gene encodes a transcription factor that plays a critical role in controlling cell proliferation and differentiation and also has a key role in the pathogenesis and progression of breast and prostate cancer. In the Catapano (co- PI) lab, triplex formation has been shown to sequence-specifically inhibit transcription at specific sites in the ets2 promoter sequences and also directly inhibit transcription by blocking RNA polymerase. Triplex technology however remains to be improved before health applications to these specific areas can be made. The need for an improved target affinity in the therapeutic regulation of specific gene expression in cancer cells remains, as does the delivery of these TFOs and all oligonucleotides in cells. Our preliminary results show the ability of neomycin to stabilize key triplex forming targets in Ets2 gene and aid in delivery of oligonucleotides. The objective of this project is to develop neomycin-TFOs that directly target the ets2 gene, found amplified in breast and prostate cancer. The proposed work will overcome important challenges such as TFO affinity to the duplex and delivery to the cells, that traditional TFOs face. The hypothesis :} Neomycin-mediated delivery of TFO-neomycin conjugates can be used to develop sequence-specific anticancer agents with improved delivery properties and enhanced duplex affinity.} We propose to develop neomycin conjugated TFOs that (1) enhance the binding affinity of the TFO to its target duplex, (2) improve the delivery and uptake of the TFO to the cancer cells. Using the recent findings from our labs, we will synthesize neomycin-TFO conjugates for triplex formation with sites of therapeutics interest in Ets-2 gene. We will then use neomycin- TFO conjugates to determine their efficacy in stabilizing the triplexes on Ets-2. Appropriate neomycin-TFO conjugates will be synthesized on a DNA synthesizer, and evaluated for anti- transcriptional and anticancer activity in binding to the Ets-2 targets of interest. The cellular and nuclear uptake of neomycin-conjugated TFOs is being done in collaboration with Carlo Catapano, M.D., Director, Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland (IOSI) Bellinzona, Switzerland. This testing will involve studies focused on the stability of the conjugate within cancer cells, examination of the sequence and target selectivity of the conjugate, systematic determination of the number of neomycin molecules per oligonucleotide needed for optimal delivery and anti-gene activity, the ability of the neomycin-TFO to discriminate for target sequences and test the effects of these high binding TFOs on endogeneous gene expression and growth of human breast cancer cells. The development of neomycin-TFO conjugates has the potential for bringing the much needed improvements in cancer therapy by vastly improving the permeability of TFOs and deregulating expression of ets2 gene in proliferation of breast and prostate cancer cells. The success of this approach will open up new avenues of TFO development and delivery not only as they relate to cancer, but in other oligonucleotide based therapies as well. Health Relevance Statement One of the challenges of research in oncology is to find ways to use the increasing knowledge of the mechanisms underlying neoplastic transformation and tumor progression to develop novel therapeutic strategies for cancer. Targeting specific genes, such as Ets2 or c-myc, which are involved in proliferation and survival of cancer cells is a promising approach. Our preliminary results show the ability of neomycin to stabilize key triplex forming targets in Ets2 gene and aid in delivery of oligonucleotides. This property of neomycin will now be extended to develop aminoglycoside-TFO conjugates to target Ets2 promoter sequences that bind with high affinity and can be delivered without external transfection agents. These conjugates can then be used to down regulate gene expression and inhibit proliferation of cancer cells. Proposed studies in our labs, using undergraduate and graduate students, and collaborative efforts with the Catapano lab will further help establish the efficacy of this approach. The work proposed here, a multidisciplinary effort encompassing organic synthesis, biophysical chemistry, and cancer research describes the development and neomycin mediated delivery/anticancer activity of novel positively charged TFOs (neomycin-TFOs). In particular, this work proposes to initiate a multidisciplinary team that leads to the creation and use of TFO conjugates that have novel properties and functions because of their conjugation with neomycin. The success of the proposed work would be a significant addition to currently available approaches in breast and prostate cancer drug development and TFO (oligonucleotide) delivery. We propose using 25 base Ets2 target sequences to design neomycin-TFO conjugates that can be employed to inhibit cancer growth, opening possibilities for the development of small sequence- designed anti-cancer agents. Using neomycin's ability to stabilize triplexes and deliver TFOs, neomycin-TFO (PS) conjugates will be developed as triplex-forming anticancer agents. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15CA125724-01A2
Application #
7458423
Study Section
Special Emphasis Panel (ZRG1-GGG-F (90))
Program Officer
Lees, Robert G
Project Start
2008-04-10
Project End
2012-01-31
Budget Start
2008-04-10
Budget End
2012-01-31
Support Year
1
Fiscal Year
2008
Total Cost
$222,376
Indirect Cost
Name
Clemson University
Department
Chemistry
Type
Schools of Engineering
DUNS #
042629816
City
Clemson
State
SC
Country
United States
Zip Code
29634
Ranjan, Nihar; Arya, Dev P (2016) Linker dependent intercalation of bisbenzimidazole-aminosugars in an RNA duplex; selectivity in RNA vs. DNA binding. Bioorg Med Chem Lett 26:5989-5994
Kumar, Sunil; Ranjan, Nihar; Kellish, Patrick et al. (2016) Multivalency in the recognition and antagonism of a HIV TAR RNA-TAT assembly using an aminoglycoside benzimidazole scaffold. Org Biomol Chem 14:2052-6
Kumar, Sunil; Spano, Meredith Newby; Arya, Dev P (2015) Influence of linker length in shape recognition of B* DNA by dimeric aminoglycosides. Bioorg Med Chem 23:3105-9
Ranjan, Nihar; Fulcrand, Geraldine; King, Ada et al. (2014) Selective Inhibition of Bacterial Topoisomerase I by alkynyl-bisbenzimidazoles. Medchemcomm 5:816-825
Kellish, Patrick C; Kumar, Sunil; Mack, Todd S et al. (2014) Multivalent Amino Sugars to Recognize Different TAR RNA Conformations. Medchemcomm 5:1235-1246
Kumar, Sunil; Newby Spano, Meredith; Arya, Dev P (2014) Shape readout of AT-rich DNA by carbohydrates. Biopolymers 101:720-32
Watkins, Derrick; Norris, F A; Kumar, Sunil et al. (2013) A fluorescence-based screen for ribosome binding antibiotics. Anal Biochem 434:300-7
Ranjan, Nihar; Kumar, Sunil; Watkins, Derrick et al. (2013) Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates. Bioorg Med Chem Lett 23:5689-93
Ranjan, Nihar; Arya, Dev P (2013) Targeting C-myc G-quadruplex: dual recognition by aminosugar-bisbenzimidazoles with varying linker lengths. Molecules 18:14228-40
Ranjan, Nihar; Davis, Erik; Xue, Liang et al. (2013) Dual recognition of the human telomeric G-quadruplex by a neomycin-anthraquinone conjugate. Chem Commun (Camb) 49:5796-8

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