T007E

Drug discovery (Gene Therapy by DNA specific recognition) & Biodetection
Recently, gene therapy with genome editing is expected as the novel medical treatment.
HiPep Laboratory has been performed Peptide Nucleic Acid (PNA) and Pyrrole-Imidazole Polyamide (PIPA).
Both can be performed by the contract syntheses. Moreover the facility of GMP-production is under construction.
PNA is a kind of peptide consisting of base units with amide bonds. PNA can bind complementary with DNA, such as A-T; C-G, andrecognize to specific nucleotide sequence. DNA/PNA double-stranded structure is more stable than dsDNA. DNA expression can beblocked by hybridization with PNA and DNA. However, the limitation of PNA is poor membrane permeability. So devised to delivery
to pass through the cell membrane and nuclear membrane is required. Especially efficient introduction technology into the nucleusof the target cell is indispensable. In addition, molecules recognition can be applied for biodetection (e.g. FISH etc.). In order todeliver PNA into the cell, we propose a conjugate of module type molecule (designated “Peptide-vehicle” ; “Bio-shuttle”).

Peptide Vehicle; Bio-shuttle module type molecule (Middle Molecular )Nuclear localization
signal peptide

Fluorescent
DyeNLS

Cell-penetrating
peptideCleavage(Enzyme)

PNA

Cleavage(Chemical)

wire: PIPA

balls: DNA molecule
red, blue: recognition part

CPP

Advantages of bioconjugate
1.
Improvement of drug delivery: Introduced only in target (cancer) cells
2.
Cell specific; reduction of side effects: Target cells rather than molecular targets
3.
New patent: Drugs out of patent secured new intellectual property right4.
Improve selectivity for cell lines
5.
Activity even in cancer-resistant cancer cells (peptide vehicle with anticancer drug)
PIPA is a peptide containing N-methylpyrrole (Py) and N-methylimidazole (Im) as major building units. PIPA can bind to specificnucleotide sequences in the minor groove of double-helical DNA with high affinity and specificity, suggesting that PIPA blocks
binding of transcription factors inhibiting gene expression, thus PIPA can be used for gene-control. PIPA is stable in cells or bodies
because of nuclease resistance and enters into cell nucleus without any DDS.

Comparison of PNA and PIPA
Form of recognition
DNAPNA

PIPA

single-stranded DNA

double-stranded DNA

Bonding mechanism

Bind complementary such as A-T；C-G

Stability of binding
state with DNA

more stable than dsDNA

Bind in the minor groove of dsDNA: Py/Py with A-T& T-A; Py/Im with C-G; Im/Py with G-C
Binding to the DNA double helix structure containing
the target DNA sequence shows high affinity as nano
M order (Kd value)

Average molecularweight / unite

300/base

Optional modification・Conjugation with modules to pass into
membranes (e.g. CPP, NLS)
・N-terminus acetylation; C-terminus amidation

Optional modification
Common・Fluorescent label for imaging (selectable fluorescent dye suitable for wavelength to be observed)
・Biotinylation ・PEG modification ・Introduction of non-natural amino acid120/unite (smaller molecules than PNA)
・N-terminus acetylation; C-terminus amidation or
diamine derivative (Improve recognition)[Literatures in our activities] upon requests reprints are available
Pipkorn, R. et al., Int. J. Med. Sci., 9, 1-10, 2012. Improved Synthesis Strategy for
Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging
Braun, K. et al. Int. J. Med. Sci. 9, 339-352, 2012. BioShuttle Mobility in Living Cells
Studied with High-Resolution FCS & CLSM Methodologies
Nokihara, K. et al. Peptide Science 2012: K. Sugimura (Ed) The Japanese Peptide
Society, pp 167-170, 2013. Improved Coupling Methods for the Difficult Amide-Bond
Formation in Solid-Phase Assembly
Hirata, A. et al. Peptide Science 2011: K. Sakaguchi (Ed), The Japanese Peptide
Society , pp 377-380, 2012. Synthesis and Mass-Spectrometric Characterization ofthe Peptide-Vehicle, Peptide that Carry Molecules into Cells

Nokihara, K. et al. Peptide Science 2011: K. Sakaguchi (Ed), The JapanesePeptide Society pp 73-76, 2012. Studies of Bioconjugates Focusing on Drug
Delivery; Peptide-Vehicles Consisting of Recognition and Cell Penetrating
Peptide-Modules with Anticancer Drugs
Hirata, A. and Nokihara, K., Tetrahedron Lett. 55, 4091-4094, 2014.
Construction of peptide-vehicles, bioconjugates having modules of cancer cellsurface capture and cell-penetrating peptide with anticancer agents, focusing
on efficient drug delivery
Hirata,A., Nokihara, K et al. J. Am. Chem. Soc., 136, 11546–11554, 2014.
Structural Evaluation of Tandem Hairpin Pyrrole–Imidazole Polyamides
Recognizing Human Telomeres

HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan
Tel +81-75-813-2101 E-mail: PepTenChip@hipep.jp URL: http://www.hipep.jp/