mRNA as Targets for developing anticancer agents.

1. microRNA AS TARGETS FOR DEVELOPING
ANTICANCER COMPOUNDS
1
Dushyant Kumar Dewangan
M.S. (Pharm.) Medicinal Chemistry
ID- 427/MS-MC /2017
Department of Medicinal Chemistry
NIPER, Raebareli
5/3/2018
5th Dec 2017

2. • What is miRNA ?
• miRNA in cancer and possible treatment approaches
• Small molecules that target miRNA
• Designing of Bi-functional miR-21 inhibitors
• Synthesis of Bi-functional miR-21 inhibitors
• Analysis of synthesized compound
• Study of activity of synthesized compound
Outline
25/3/2018

3. What is miRNA
• Non-protein coding RNA of 19-25 (approx) nucleotides.
• Regulates gene expression by translational process.
• Their abnormal levels in tumors have an important
pathogenetic consequences.
• miRNAs overexpressed in tumors contribute to oncogenesis
by downregulating tumor suppressor miRNAs.
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4. miRNA in cancer and possible treatment approaches
45/3/2018 https://www.frontiersin.org/articles/10.3389/fonc.2013.00019/full

5. miRNA with experimental data supporting a tumor supressor or
oncogene function in cancer
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MicroRNA Expression in patients Confirmed targets Function
miR-15a
miR-16-1
Down regulated in CLLa Bcl-2, Wt-1 Tumor supressent
miR-29 (a,b,c)
Down regulated in CLL, AMLa
(11q23),lung and breast cancers
TCL-1, MCL1, DNMT3s Tumor supressent
miR-34 (a,b,c)
Down regulated in pancreatic,
colon, and breast cancers
CDK4, CDK6,
cyclinE2, E2F3
Tumor supressent
miR-155
Up regulated in CLL, DLBCL,a
FLT3-ITDa AML, BL,a and lung
and breast cancers
c-maf Oncogene
miR-21
upregulated in breast, colon,
pancreas, lung, prostate,liver, and
stomach cancer;
and glioblastoma
PTEN,
PDCD4,
TPM1
Oncogene
Garzon, R., Calin, G. A., & Croce, C. M. (2009). MicroRNAs in cancer. Annual review of medicine, 60, 167-179.5/3/2018

6. Small molecules that target miRNA
 MRX34 : a liposome based miRNA-34a mimic in combination
with Erlotinib
 Anti-miR Oligonucleotides (AMOs)
 MIRAVIRSEN : a locked nucleic acid (LNA) based miRNA inhibitor
 Enhancers of DICER mediated miRNA processing
 Inhibitors of DICER mediated miRNA processing
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7. 75/3/2018

8. MIRAVIRSEN: A locked nucleic acid (LNA) -based miRNA inhibitor:
• It is LNA-modified anti- sense oligonucleotide.
• It has an additional methylene bridge connecting the 2’ oxygen and 4’
carbon. Thereby, the ribose is ‘locked’ in the 3’-endo conformation, which
enhances base stacking and backbone pre-organization in cancer cells.
• Recently tested successfully in Phase II clinical trials for the treatment of
hepatitis C virus (HCV) infection.
• It targets miRNA122-Argonaute 2 complex responsible for promoting viral
RNA stability and propagation, and blocks the interaction with HCV RNA
for viral replication.
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9. Small molecule enhancers of miRNA processing
• Exonaxin, tend to induce a general upregulation of miRNA.
• Enoxacin binds to the HIV transactivating response RNA-binding protein-2
(TRBP), inducing DICER-mediated miRNA maturation.
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Small molecule inhibitors of miRNA processing
• Velagapudi et.al. identified a small molecule that binds selectively to
pre-miRNA-96, thus inhibiting maturation of miRNA-96.
5/3/2018 Schmidt, M. F. (2014). Drug target miRNAs: chances and challenges. Trends in biotechnology, 32(11), 578-585.

10. Designing of new Bi-functional miR-21 inhibitors
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• Contains an intercalating unit to enhance binding of neomycin, thus serve as
inhibitors for oncogenic miR-21a
• This bi-functional molecules is designed to deliver a specific dicer inhibitor
to the dicer-catalyzed cleavage site in the target pre-miRNA/Dicer complex.
• Recognition of a target pre-miRNA by the RNA binding unit in the conjugate
brings the dicer inhibitor unit into close proximity with the premiRNA
cleavage site of bound dicer, thus causing blockage of the activity of dicer.
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11. Synthesis of Bi-functional miR-21 inhibitors
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Consists of three Parts: (1) Synthesis of inhibitor building blocks
(2) Synthesis of bi-functional small molecules.
(3)Linking of 1 and 2 to give final Product
Yan, H., Bhattarai, U., Guo, Z. F., & Liang, F. S. (2017). Regulating miRNA-21 Biogenesis By Bifunctional Small Molecules.
Journal of the American Chemical Society, 139(14), 4987-4990.
Bi-functional miR-21 inhibitor
5/3/2018
00
(1)
(2)

12. 125/3/2018
Yan, H., Bhattarai, U., Guo, Z. F., & Liang, F. S. (2017). Regulating miRNA-21 Biogenesis By Bifunctional Small
Molecules. Journal of the American Chemical Society, 139(14), 4987-4990.
Reagents and conditions:
(a) H2SO4, MeOH, reflux; (b) Propargyl bromide, K2CO3, DMF; (c) DIAD, Ph3P, THF; (d) i) LiOH, MeOH/H2O; ii) HCl;
(e) O-(4-Methoxybenzyl)-hydroxylamine, toluene, reflux (f) trifloroacetic acid, DCM
Synthesis of Inhibitor Building blocks (Part-1)

13. Synthesis of bi-functional small molecules (Part-2)
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14. Final Part (Linking 1 & 2 )
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15. ACTIVITY STUDY OF SYNTHESISED COMPOUNDS
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0
0.2
0.4
0.6
0.8
1
1.2
(A)
RelativemiRNA-21level
Yan, H., Bhattarai, U., Guo, Z. F., & Liang, F. S. (2017). Regulating miRNA-21 Biogenesis By Bifunctional
Small Molecules. Journal of the American Chemical Society, 139(14), 4987-4990.
0
0.5
1
1.5
2
2.5
Control Compound 12
RelativePDCD4Levels
(B)
• (A) RT-qPCR analysis of mature miR-21 expression levels in HEK293T cells with pCMV-
miR21 plasmid expressing pre-miR-21.
• (B)RT-qPCR analysis of PDCD4 levels in pre-miR-21 expressing HEK293T cells with or
without 7A treatment.
Compound 12

16. CONCLUSION
• A new approach to regulate miRNA maturation was developed as a Dicer-
inhibiting bi-functional small molecule which contains neomycin as a pre-
miR-21 binding unit and the N-hydroxyimide 1 as the Dicer inhibitory unit,
inhibits miR-21 maturation in vitro and in cells.
• Together, these observations demonstrate new bi-functional strategy for
the design of active miR-21 inhibitors based on non-active pre-miR-21
binder.
• The therapeutic potential of miRNAs is unexplored and miRNA-based
cancer therapy offers targeting of multiple gene networks that are
controlled by a single miRNA.
• Compound 12 has shown better miR-21 inhibition than Neomycin alone
and commercially available phosphorothioate-based anti-miR-21.
• It also shows to have better selectivity towards miR-21 inhibition than the
inhibition of anothers like miRNA-34a.
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17. ACKNOWLEDGEMENT
 Dr. S.J.S Flora , Director
 Dr. Atul Kumar , Dean & Course co-ordinator
 Dr. Nihar Ranjan , Asst Professor
 Dr. Abha Sharma , Asst Professor
 Dr. K.N Tiwari , Asst Professor
 Department of Pharmaceuticals, Ministry of chemicals and fertilizers
Govt. of India
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18. 18
ANY QUESTIONS…
5/3/2018