1

Molecular Cancer Therapeutics

Antisense OligonucleotidesRNA interference (RNAi)

Vũ Mạnh Huỳnh

Tiến Sĩ Hóa Học

Concepts Mechanisms Progresses Developments in the sequencing of human genome led to

the use of short fragments of nucleic acid, antisense. Antisense technology is the use of a complementary seq.

of Watson-Crick bp hybridization, to a specific mRNA can inhibit its expression and then induce a blockade in the transfer of genetic information from DNA to protein.

The first antisense, Vitravene, for cytomegalovirus (CMV) retinitis, is approved by FDA in 1998.

Many others are in clinical trials for disease treatments. Antisense companies merged to major Pharmaceutical

companies: ISIS/Ely Lilly, Coley, Idera, Genentech,… Although antisense are commonly in use in laboratory

and clinic, Scientists believed there are many questions concerning the molecular mechanism of action of these compounds.

Purines & Pyrimidines Hybridization

DNA Structure:

HO

N

NN

N

NH2

O

HOH

HH

HH

2'-Deoxy Adenosine : dA

HO

NH

N

N

O

NH2N

O

HOH

HH

HH

2'-Deoxy Guanosine : dG

HO

O

HOH

HH

HH

N

N

NH2

O

2'-Deoxy Cytidine : dC

HO

O

HOH

HH

HH

N

NH

O

O

2'-Deoxy Thymidine : T

Blockade of Translation by Antisense: Antisense complementary to a mRNA bind mRNA, preventing translation by steric effect or by inducing degradation of mRNA by RNase

Antisense Directed against HIV

Antisense Designs for HIV

Oligonucleotides have been designed to bind at various sites along the genome of the human immunodeficiency virus (HIV).

Antisense nucleotides can bind to the proviral long terminal repeats, which are regulatory regions that have an important role in integrating the proviral DNA into the human genome (a necessary step in the life cycle of HIV).

Several groups are studying antisense oligonucleotides against HIV mRNA, especially the mRNAs for proteins that regulate viral expression, such as tat and rev.

It has been possible to inhibit viral replication in vitro with such antisense oligonucleotides.

Inhibition of Angiotensinogen by Antisense Oligonucleotides

DNA transcription is followed by translation of mRNA to form angiotensinogen, which in turn is converted To angiotensin I by renin and To angiotensin II by angiotensin-converting enzyme

(ACE). This cascade results in increased blood pressure. ACE inhibitors are one form of pharmaceutical therapy

that interrupts this cascade and lowers blood pressure. Antisense oligonucleotides have been used in animal

models to prevent the translation of mRNA into angiotensinogen, with decreases in blood pressure.

Inhibition of Angiotensinogen by Antisense Oligonucleotides

Antisense therapy could be used in Cardiovascular disease.

Another possibility for the use of oligonucleotides is in the treatment of leukemia.

DNA/RNA to block protein function to prevent the translation of messenger RNA (mRNA) into protein.

DNA or RNA that contains the information for the amino acid sequence of the protein is called the “sense” strand. The other sequence is complementary to the sense strand and is called the “antisense” strand.

Antisense applications of DNA/RNA

Technical issues of Antisense

In an attempt to overcome the various nonspecific

problems, new chemical modifications have been developed.

These "second-generation" oligonucleotides are resistant to degradation by cellular nucleases

Hybridize specifically to their target mRNA Higher affinity than phosphodiester or

phosphorothioate.

Antisense with new backbone

O BO

OPOO

S-

BO

O

5'

3'

O BO

OPOO

O-

BO

O

5'

3'

O BO

OPOO

Me

BO

O

5'

3'

O BO

NHPOO

O-

BO

HN

5'

3'

OPOO

N(CH3)2

O

N

OB

ON

OB

5'O B

O

OO

PO

O

O-

BO

OO3'

H2N

N

NH

O

N

CONH2

BaseO

BaseO

PNAMorpholino

Phosphoramidate

Methylphosphonate

Phosphorothioate

LNA : 2'-O, 4'-C Methylene bicyclo nucleoside

TETD (Tetraethylthiuram disulfide)Sulfurization1

• TETD in ACN is available for synthesizing Phosphorothioate Oligo.

• Enzymatic Digestion shows no detectable base modification.

TETD converts cyanoethyl phosphite to the phosphorothioate triester.

1 Vu, H. and Hirschbein, L. B. "Internucleotide Phosphite Sulfurization WithTetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.", Tetrahedron Lett.,1991, 32, 3005-3008.

Synthesis of (N3’P5’) Phosphoramidate

DMTOO

NH

N

N

OPh

O

PN O

CN

DMTO

N

N

N

OCONPh2

N=CH-NMe2N

O

NH

PN O

CN

DMTOO

NH

N

N

N=CHN(iBu)2

O

PN O

CN

DMTO

N

NN

N

N=CHN(iBu)2

O

NH

PN O

CN

Pyrimidines 3'amino-2',3'-dideoxynucleoside phosphoramidites

Purine 3'amino-2',3'-dideoxynucleoside phosphoramidites

Vu, H., Rao, T. S., Akiyama, T., Hogan, M. E., Ojwang, J. O., Rando, R. F., Revankar, G. R. “Automated synthesis of oligonucleotide (N3P5) phosphoramidates using 3-amino-2,3-dideoxynucleoside phosphoramidites” presented at 213th ACS National Meeting, April 13-17, 1997, San Francisco, CA.

Phosphoramidate Antisense

Another example of a "second-generation" oligonucleotide is the N3'P5' PN.

Oxygen of 3’-position of ribose is replaced by an Amine.

Can form very stable complexes with RNA, and single or double stranded DNA.

Can exhibit highly selective and specific antisense activity in vitro and in vivo.

Inhibited efficiently the growth of treated BV173 cells. Inhibited selectively the c-myc protein expression and

the proliferation of HL-60 cells.

HOO

N3

N

NH

O

O DMTOO

N3

N

NH

O

O

DMTOO

N3

N

N

N

O

N

NNO2

DMTOO

R

N

N

OPh

O

DMTOO

N3

N

N

NH2

ODMTO

O

R

N

N

N=CHN(iBu)2

O

PN

O CN

NH

PN

O CN

NH

Synthesis of Pyrimidine 3-Amino-2',3'-dideoxynucleoside Phosphoramidite

1i 2 34, R = N3

5, R = NH2

6, R =

7

8, R = N3

9, R = NH2

10, R =

ii iii iv

v

vi

vii

viiiix

vi

i. Rao, T.S.; Reese, C.B., J. Chem. Soc., Chem. Commun., 1989, 997; ii. DMTCl, pyr., 61%; 3-Nitrotriazole, Ph2POCl, pyr.; iv. Phenol, CH3CN, NEt3, 61.5%; v. 10% Pd/C in MeOH, 40 psi, 6 h, 87%; vi. DIPEA, CEDICP, CH2Cl2, 80%; vii. NH4OH, dioxane, RT, 1h, 57%; viii. (iBu)2NCH(OCH3)2, DMF, RT, 6h, 96%; ix. Bu3SnH, AIBN, toluene, reflux, 15 min, 83%.

TolOO

N3

OMe

N

NN

N

Cl

R

TolOO

N3

N

NN

N

NH2

HOO

N3

HN

NN

N

O

H2N

HOO

N3

N

NNH

N

Cl

R

Synthesis of 3'-Azido-2',3'-dideoxyadenosine/guanosine

11, R = H12, R = NH2

+

13i

+OtherIsomers

ii

14, R = H15, R = NH2

16

iiiiv

17

i. Dyatkina, N.B.; Azhayev, A.B. Sinthesis 1984, 961; ii. HMDS, (NH4)2 SO4, Cl(CH2)2Cl, TMStriflate;iii. MeOH/NH3, 75 C, 8 h; iv. HSCH2CH2OH, MeOH/MeONa, Reflux, 5 h or !N NaOH, 80 C, 4 h.

N

NN

N

NH2

HOO

N3

N

NN

N

N=CHN(iBu)2

HOO

N3

N

NN

N

N=CHN(iBu)2

DMTOO

N3

N

NN

N

N=CHN(iBu)2

DMTOO

NH2

N

NN

N

N=CHN(iBu)2

DMTOO

NH

PN O

CN

PCl

O CN

N

16

Synthesis: 3'-Amino-2',3'-dideoxyadenosine Phosphoramidite

i

85%

ii

70%

i. (iBu)2NCH(OCH3)2, DMF; ii. DMTCl, pyr.; iii. AIBN, Bu3SnH, toluene; iv. CH2Cl2, DIPEA

49%

iii

63%

18 19

2021

HN

NN

N

O

H2N

HOO

N3

HN

NN

N

O

Me2HNC=N

HOO

N3

HN

NN

N

O

Me2HNC=N

DMTOO

N3

N

NN

N

OCONPh2

Me2HNC=N

DMTOO

N3

N

NN

N

OCONPh2

Me2HNC=N

DMTOO

NH2

N

NN

N

OCONPh2

Me2HNC=N

DMTOO

NH

PN O

CN

PCl

O CN

N

17

Synthesis: 3'-Amino-2',3'-dideoxyadenosine Phosphoramidite

22 23

2425

26

i

89%

ii

70%

iii69%

iv

63%

v

50%

i. (Me)2NCH(OC2H5)2, DMF; ii. DMTCl, pyr.;iii.Ph2NCOCl, pyr., DIPEA;iv. AIBN, Bu3SnH, toluene;v.CH2Cl2,DIPEA

Phosphoramidate Antisense

Another example of a "second-generation" oligonucleotide is the N3'P5' PN.

Oxygen of 3’-position of ribose is replaced by an Amine. Can form very stable complexes with RNA, and single or

double stranded DNA. Melting Temperature shows significant increase (10.9 °C). Can exhibit highly selective and specific antisense activity

in vitro and in vivo. Inhibited TNF production in phorbol myristate acetate

andinterferon gamma (PMA/IFN) stimulated THP-1 cells. Inhibited efficiently the growth of treated BV173 cells. Inhibited selectively the c-myc protein expression and the

proliferation of HL-60 cells.

Locked Nucleic Acids - LNAs

• LNAs a class of restricted nucleotide analogs.

• LNA increases the affinity of RNA or DNA.

• LNA increases the melting temperature (Tm) of duplex.

• Differentiate effectively between a perfect matched target and a mismatched target

• Highly sensitive, and discriminatory in miRNAs

HOB

O

OOH

HH

H

B: A, C, G, T

5'O B

O

OO

PO

O

O-

B

O

O3'

2'-O, 4'-C Methylene bicyclo nucleoside

Oligo contains LNA

Advantages of Antisense

Antisense oligonucleotides have potential as a unique way to treat a variety of diseases.

There is concern about the mechanism of action of the oligonucleotides, drug-delivery systems, cellular-uptake systems, and long-term effects.

Oligonucleotide therapy does not have the safety and efficacy issues associated with expressed-vector gene therapy,

Its use in some applications is advancing on the road to approval by the Food and Drug Administration.