1

2

http://www.cancer.gov/cancertopics/types/pancreatic

Pancreatic cancer is the fourth most common cause of cancer-related deaths in the United States.

Estimated new cases and deaths from pancreatic cancer in the United States in 2013:

New cases: 45,220 Deaths: 38,460

3

Genes and Disorders

Gene Silencing

4

Any technique or mechanism in which the expression of a gene is prevented.

5

•Protein Inhibition

•Inhibition of translation

•Degradation of the mRNA

•Regulation of

transcription

•siRNA •AONs

Strategies for Gene Silencing

Milhavet, O.; Gary, D.S.; Mattson, M.P. Pharmacol. Rev. 2003, 55, 629.

6

Gene Silencing

Food and agriculture

Gene therapy

Cancer

Cardiovascular and Cerebrovascular

Diseases

Neurodegenerative Disorders

(Huntington's disease)

Viral infections

Gene Silencing

Robinson, R. RNAi therapeutics: PLoS Biol. 2, E28 (2004).

7

Antisense Oligonucleotides (AONs)

2012 Pearson education, Inc 8

Short Interfering RNA (siRNA)

siRNA

Deleavey, G.F.; Damha, M.J. Chem. Biol. 2012, 19, 937.

9

si-RNA

Fire, A.; Xu, S.; Montgomery, M.K.; Kostas, S.A.; Driver, S.E. Mello, C.C. Nature 1998, 391, 806.

Elbashir, S.M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Nature 2001, 411, 494.

10

Importance of siRNA

Andrew Fire (1998) “double-stranded RNA was substantially more effective at producing interference than was either strand individually.”

Thomas Tuschl (2001) First report of using synthetic siRNA in mammalian cell line

siRNA: More resistant to nuclease degradation

Zhou, J.; Shum, K.T.; Burnett, J.C.; Rossi, J.J. Pharmaceuticals 2013, 6, 85.

Liver cancer

Hypercholesterolemia

11

siRNAs in Clinical Pipeline

Asthma

12

Outline

Nucleotides

Double stranded structures

siRNA Synthesis Techniques

13

Nucleotides Structure

14

Natural Nucleotides

Molecular Cell Biology, Sixth edition 2008 W.H. Freeman and Company 15

Double Stranded Structures

Characteristic A-DNA B-DNA Z-DNA

Helix Direction Right-handed Right-handed Left-handed

Average base pairs per turn 11 10 12

Rotation per base pair 32.7 36 -30

Distance between adjacent bases

0.26 nm 0.34 nm 0.37nm

Diameter 2.3 nm 1.9 nm 1.8 nm

Overall shape Short and wide Long and narrow Elongated and narrow

16

Synthesis of siRNA

Synthesis of siRNA

Enzymatically Generated siRNAs

Chemically Synthesized siRNAs

T7 RiboMAX™ Express RNAi System protocol

Advantages: Quick

Cost effective

Disadvantage: Limited to natural nucleotides

17

Enzymatically generated siRNA

Somoza, A. Chem. Soc. Rev. 2008, 37, 2668.

18

Chemically Synthesized siRNAs

Stability Targeting Side effect

Endonuclease Target delivery Off target effects

19

General Problems for siRNA as a Drug Candidate

Modification of siRNA

• A/U at the 5'end antisense strand (guide strand)

• Avoiding immunostimulators (GU rich sequences, blunt structure at 3´ end etc.)

• siRNA conjugation and delivery systems

20

Frequently Utilized Modifications

Chen, Y.; Wang , X.; Huang, Y.; Zhang, L. H.; Yang, Z. JCPS 2012, 21, 5

21

Frequently Utilized Modifications

Internucleotide linkage Modifications

Sugar Modifications

Nucleobase Modifications

Chemical Modification of Nucleotides

Chen, Y.; Wang , X.; Huang, Y.; Zhang, L. H.; Yang, Z. JCPS 2012, 21, 5

22

Internucleotide Linkage Modifications

Decreasing Charge Density

Increasing Lipophilicity

Increasing Stability

Hall, A.H.; Wan, J.; Shaughnessy, E.E.; Ramsay Shaw, B.; Alexander, K.A. Nucleic Acids Res. 2004, 32, 5991.

23

Sugar Modification

Deleavey, G.F.; Damha, M.J. Chem. Biol. 2012, 19, 937.

24

Sugar Conformations

0.34 nm 0.26 nm

2´-O-Me modification

• Pegaptanib(FDA approved 2004)

2´-O-MOE modification

• Mipomersen(FDA approved 2013)

25

Commercial Available Products

Peacock, H.; Kannan, A.; Beal, P.A.; Burrows, C.J. J. Org. Chem. 2011, 76, 7295.

26

Nucleobases Modification

Affecting Hydrogen bonding in order to control:

Duplex Stability OTEs

Nucleobases modification is less common than sugar and backbone modifications

Wang, Y.; Juranek, S.; Li, H.; Sheng, G.; Tuschl, T.; Patel, D. J. Nature, 2008, 456, 921. 27

Crystal Structure of Thermus thermophilus Argonaute

Puthenveetil, S.; Whitby, L.; Ren, J.; Kelnar, K.; Krebs, J. F.; Beal, P. A. Nucleic Acids Res., 2006 34, 4900.

Peacock, H.; Maydanovych, O.; Beal, P. A. Org. Lett. 2010, 12, 1044.

28

siRNA instability

•RNA-dependent Protein Kinase (PKR) •Adenosine Deaminases (ADAR-1)

Molecular Biology of the Gene, Watson et al, 5th Edition Chapter 6 Page 98 29

Minor Groove & Major Groove

30

Minor Groove Modification: a New Strategy for Increasing the Stability of siRNA

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

31

Development of a Method for Switching Steric Groups away from the Minor Groove when the Guide Strand Enters RISC

siRNA Guide Strand of siRNA +

Target mRNA

Nikolova, E. N.; Kim, E.; Wise, A. A.; O’Brien, P. J.; Andricioaei,I.; Al-Hashimi, H. M. Nature, 2011, 470, 498.

32

Hoogsteen Base Pairing

Bruner, S. D.; Norman, D. P. G.; Verdine, G. L. Nature 2000, 403, 859.

Cheng,X.;Kelso,C.;Hornak,V.; de los Santos,C.;Grollman,A. P.;Simmerling, C. J. Am. Chem. Soc. 2005, 127, 13906.

33

8-Oxopurines as a potential frameworks

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

34

35

N²-alkyl-8-oxo-7,8-dihydroguanosine

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

Kannan, A.; Burrows, C. J. J. Org. Chem. 2011, 76, 720–723 36

Preparation of N²-Alkyl-8-oxo-7,8-dihydro-2´- deoxyguanosine Phosphoramidites

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

37

Design of siRNAs

38 http://www.atdbio.com/content/53/DNA-duplex-stability

The temperature at which under specific condition double stranded structure is changed(50%) to single stranded nucleic acid.

Thermal Stability of siRNA

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

39

Thermal Stability Tm

R = H (O) > R = propyl (P) > R = benzyl

55 °C

60 °C

the presence of a single OG:A Hoogsteen pair in the RISC does not diminish the activity

The location of addition of single alkyl group (propyl or benzyl) is important for siRNA function.

40

Knockdown Studies with Single Modification

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

Propyl Benzyl

Multiple substitutions with the benzyl substituent compromise the activity of siRNA more severely!

41

Knockdown Studies with 2 or 3 Modification

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

Substitution of a U in two or three positions of the guide strand with an OG was detrimental to knockdown activity.

Propyl Benzyl

42

PKR Quantification

MW

siRNA1 siRNA2 siRNA3 siRNA4

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

43

PKR Binding Assay

Kannan, A.; Fostvedt, E.; Beal, P.A.; Burrows, C.J. J. Am. Chem. Soc. 2011, 133, 6343.

44

PKR binding assay

45

So far ….

Hoogsteen base pair formed by 8-oxoguanine in the syn conformation pairing with a target adenosine is tolerated in the seed region.

Single Site Modifications of this type led to knockdown of protein expression that was more efficient than the unmodified siRNA. (only in specific locations)

PKR binding assay supported the model.

Collins, L. V.; Hajizadeh, S.; Holme, E.; Jonsson, I.M.; Tarkowski, A. J. Leukocyte Biol. 2004, 75, 995.

Pope, B. L.; MacIntyre, J. P.; Kimball, E.; Lee, S.; Zhou, L.;Taylor, G. R.; Goodman, M. G. Cell. Immunol. 1995, 162, 333.

46

Improvement of Model

Loxoribine 8-oxo-2´-deoxyguanosine

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

47

Alternative Purine Modifications

8-alkoxyadenosine phosphoramidites

Ghanty U, Fostvedt E, Valenzuela R, Beal PA, Burrows CJ: J Am Chem Soc 2012, 134: 17643–17652. 48

Base Switch

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

49

8-AlkoxyA

Delivery as Hoogsteen pair Watson-Crick pairing in the RISC

50

Synthesis of 8-AlkoxyA

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

51

Synthesis of 8-AlkoxyA

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

52

siRNA design

Alkoxy group appears to introduce instability in the RNA

duplexes.

In Watson−Crick Model hydrogen bonding is

still preferred.

53

Thermal analysis

Ghanty U, Fostvedt E, Valenzuela R, Beal PA, Burrows CJ: J Am Chem Soc 2012, 134: 17643–17652.

Ghanty U, Fostvedt E, Valenzuela R, Beal PA, Burrows CJ: J Am Chem Soc 2012, 134: 17643–17652. 54

Thermal analysis

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

Knockdown Ability of the modified nucleotides is sensitive to position.

55

Knockdown Studies with Modified Adenosine-Containing siRNAs

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

Multiple 8-ROA substitutions at the guide strands showed significantly reduced silencing efficacy.

56

Knockdown Studies (2 sites modification)

57

Importance of Switching the Steric Blockade from the Minor to the Major Groove in the RISC

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

Ghanty, U.; Fostvedt, E.; Valenzuela, R.; Beal P.A.; Burrows, C.J. J. Am. Chem. Soc. 2012, 134, 17643.

58

PKR binding assay

59

Summary

Chemical modification is a promising strategy to increase the potency and specificity of siRNAs .

A purine ribonucleoside can exist in both syn and anti conformations around the glycosidic bond depending on the base-pairing partner.

Singly modified siRNAs were capable of inhibiting PKR−siRNA interactions.

Acknowledgement

• Dr Huang

• Group members: Hovig, Herbert, Isaac, Steven

Qian, Berm, Peng, Jicheng, Zeren, Weizhun,

Zhao-Jun, Claire, Joe

• My Friends

• And you for your attention

60

61