Amity Institute of Pharmacy
Amity Institute of Pharmacy
Pharm D., 3rd Year
Medicinal Chemistry (BP319)
Antisense Molecule
1
Amity Institute of Pharmacy
Basic Science:
Genes contain the information necessary to produce
proteins.
Protein production occurs in two
phases called transcription and
translation.
In the transcription phase, the DNA strand is
used as a template for manufacturing an
mRNA molecule.
Amity Institute of Pharmacy
mRNA is responsible for communicating the genetic
message in the DNA to the cell so that protein production
can take place.
In the translation phase, the mRNA
travels to the ribosome, and carry out
protein synthesis.
Amity Institute of Pharmacy
Overview:
Currently, a total of ~4,000 genetic disorders are
known.
The mutated genes produce proteins that
cannot function properly, leading to the
occurance of the diseases.
Examples: Sickle-cell anemia, Cystic
fibrosis, Color blindness
Amity Institute of Pharmacy
Amity Institute of Pharmacy
How to stop genetic disorder using
DNA drugs?
Design a short DNA sequence that
matches the sequence of mRNA that is
transcribed from the mutated gene
(which causes diseases).
The DNA drug binds to the mRNA
The mRNA cannot be translated to
protein
Amity Institute of Pharmacy
What are the ways?
The available possible ways to achieve this
include the use of :
Oligonucleoti
des Ribozyme
RNAi
Newer
techniques
like LNA and
CeNA
Locked nucleic acid
(LNA) ,Cyclohexene nucleic acids
(CeNA)
The Collective
use of these
Amity Institute of Pharmacy
https://youtu.be/mcEV3m9SG9M
Amity Institute of Pharmacy
• Oligonucleotides, or oligos, are short single strands of
synthetic DNA or RNA that serve as the starting point for
many molecular biology and synthetic biology applications.
• A ribozyme is a ribonucleic acid (RNA) enzyme that
catalyzes a chemical reaction. The ribozyme catalyses
specific reactions in a similar way to that of protein enzymes.
Also called catalytic RNA, ribozymes are found in the
ribosome where they join amino acids together to form
protein chains.
• The term RNA interference (RNAi) was coined to describe a
cellular mechanism that use the gene's own DNA sequence of
gene to turn it off, a process that researchers call silencing. In
a wide variety of organisms, including animals, plants, and
fungi, RNAi is triggered by double-stranded RNA (dsRNA).
RNA interference (RNAi)
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Antisense Technology
Antisense technologies are a suite of
techniques that, together, form a very
powerful weapon for studying
Gene function (functional genomics)
For discovering new and more specific
treatments of diseases in
Humans,
Animal
s,
Amity Institute of Pharmacy
Antisense Technology
Antisense technology interrupts the translation
phase of the protein production process by
Preventing the mRNA instructions from
reaching the ribosome.
Inhibiting the protein systhesis.
Antisense drugs are short, chemically
modified complementary nucleotide chains
that hybridize to a specific complementary
area of mRNA.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Pharm D., 3rd Year
Medicinal Chemistry (BP319)
Antisense Molecule
14
Amity Institute of Pharmacy
What are antisense oligonucleotides?
• Antisense Oligonucleotides are unmodified or
chemically modified ssDNA, RNA or their analogs.
•They are 13-25 nucleotides long and are specifically
designed to hybridize to the corresponding mRNA by
Watson-Crick binding .
Amity Institute of Pharmacy
In this technique Short segments of single stranded DNA
called Oligodeoxynucleotides are
introduced in to the cell.
TheseOligonucleotides are
complementary to the mRNA, and
physically bind to it.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
The antisense effect of a oligonucleotide
sequence was first demonstrated in 1970s
by Zamecnik and Stephenson, in Rous
sarcoma virus.
When these oligonucleotides combined
with target mRNA, a DNA/RNA hybrid is
formed,which is degraded by the enzyme
RNase H.
Amity Institute of Pharmacy
Despite the simplicity of the idea behind the
Antisense oligonucleotides, several problems
have to be overcome for successful
application:
Accessible sites of the target
RNA for oligonucleotide binding
have to be identified.
Antisense agents have to be protected against
nuclease enzyme attack.
Cellular uptake and correct intracellular
Amity Institute of Pharmacy
It is therefore necessary to chemically
modify antisense oligonucleotides to
make them stable in cells.
Modification of the phosphodiester backbone
is likely to inhibit nuclease action and
several phosphodiester backbone analogues
have been developed with this goal in mind.
Amity Institute of Pharmacy
On the basis of mechanism of action, two
classes of antisense oligonucleotide can
be discerned:
The RNase H-dependent
oligonucleotides,
which induce the degradation of mRNA
and
The steric-blocker oligonucleotides,
which physically prevent or inhibit the
progression of splicing or the
Amity Institute of Pharmacy
First generation Antisense
oligonucleotides:
First synthesized by Eckstein and colleagues
in 1960s .
Phosphoro-thioate -deoxy-nucleotides are the
first generation oligonucleotides and have a
sulfur atom replacing the non-bridging oxygen
of the sugar phosphate backbone. It preserves
the overall charge and can also activate
RNaseH for the degradation of mRNA.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Characterstics of First generation
Antisense oligonucleotides:
Better stability to nucleases but still
degrades.
Can activate RNase H.
Are highly soluble and have excellent
antisense activity.
They were first used as Antisense
oligonucleotides
Amity Institute of Pharmacy
Cannot cross the lipid bilayer because of their charge and
polarity.
Complement activation due to their polyanoinic
nature.
Once in the circulation they can be taken up by
many cell types and not just the cell targeted
leading to potential side-effects.
Amity Institute of Pharmacy
Observed side effects seen in clinical
studies performed on humans
include
Thrombocytope
nia Fatigue
Fever
Rashes
Leukopen
ia
There is also a transient inhibition of the
clotting times shown by an increased
Amity Institute of Pharmacy
Second generation Antisense
oligonucleotides :
Second generation Antisense
oligonucleotides containing nucleotides with
alkyl modifications at the 2’ position of the
ribose.
2’-O-methyl and 2’-O-methoxy-ethyl RNA
are the most important member of this
class.
Amity Institute of Pharmacy
2’-O- 2’-O-methoxy-
methyl ethyl
Amity Institute of Pharmacy
• These “second-generation” oligonucleotides are resistant to
degradation by cellular nucleases and hybridize specifically to
their target mRNA with higher affinity than the phosphodiester
or phosphorothioate.
However, such antisense effects result from Rnase H
independent mechanisms.
Amity Institute of Pharmacy
Characterstics of second generation
Antisense oligonucleotides :
Mechanisms of action for the 2’ modified
oligonucleotides do not rely on RNase H
activation but on translation arrest by
blocking 80S ribosome complex formation as
well as with splicing interference.
They were developed to try and avoid the
Amity Institute of Pharmacy
Show high binding affinity to target mRNA.
Best stability to nucleases.
Less toxic than first generation AS-ON.
Higher lipophilicity compared to first
generation AS- Ons.
What is Oligonucleotide Synthesis?
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Third generation Antisense oligonucleotides
Newest and most promising.
Enhance binding affinity and
biostability.
Peptide nucleic acids
(PNAs) Locked nucleic acid
(LNA) Tricyclo-DNA
(tcDNA) Cyclohexene
Amity Institute of Pharmacy
Peptide nucleic acids
(PNA)
In PNAs the :
deoxyribose phosphate
backbone is replaced by polyamide
linkages.
The property of high-affinity nucleic acid
binding can be explained by the lack of
electrostatic repulsion because of the absence
of negative charges on the PNA oligomers.
The antisense mechanism of PNAs depends
on steric hindrance.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Locked nucleic acid (LNA) :
The ribose ring is connected
by a methylene bridge
(orange) between the 2’-O
and 4’-C atoms thus
“locking” the ribose ring in
the ideal conformation for
Watson- Crick binding.
Thus the Pairing with a
complementary nucleotide
strand is more rapid and
increases the stability of
the resulting duplex.
Amity Institute of Pharmacy
LNA oligonucleotides exhibit
unprecedented thermal stability when
hybridized to a complementary DNA or
RNA strand.
LNA based hepatitis C drug called Miravirsen,
targeting miR-122, is in Phase II clinical
testing as of late 2010.
Amity Institute of Pharmacy
Tricyclo-DNA (tcDNA) :
Chemically, tc-DNA deviates from natural DNA by three
additional C-atoms between C(5’) and C(3’).
Tricyclo-DNA belongs to the family of conformationally
restricted oligodeoxynucleotide analogues. It differs
structurally from DNA by an additional ethylene bridge
between the centers C(3') and C(5') of the nucleosides, to
which a cyclopropane unit is fused for further enhancement
of structural rigidity.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Cyclohexene nucleic acids (CeNA)
:
The replacement of the furanose moiety
of DNA by a cyclohexene ring gives
Cyclohexene nucleic acids or CeNA.
CeNA is stable against degradation in serum
and a CeNA/RNA hybrid is able to activate
RNase H, resulting in cleavage of the RNA
strand.
Amity Institute of Pharmacy
These chemical modifications change the
properties of natural oligodeoxynucleotides
in the following way:
Increased RNA
affinity. Increased
hydrophobicity.
Increased stability towards nucleolytic
Amity Institute of Pharmacy
Delivery vectors:
Delivery vectors can take care of both toxicity
and drug delivery problems .The vector can
also protect the drug from degradation and
also from rapid clearance from the body. The
vector must:
Be of small size to allow intercalation
between tissues.
To allow intracellular transport, they must
be non- toxic and stable in the blood
stream
Amity Institute of Pharmacy
They must retain the drug when in the
circulation, and
Must release it at its target before
elimination.
These are quite challenging tasks but
many ideas have been developed such as
liposomes, protein or peptide constructs
and polymers.
Amity Institute of Pharmacy
Liposomes are small
microscopic spheres of
one or more
concentric, closed
phospholipid bilayer.
Polar
2 n drugs
generati such as 1st
andon
oligonucleotides
d can
be entrapped in the
internal space.
Amity Institute of Pharmacy
Advantage to liposomes is that they tend to
accumulate at sites of infection, inflammation
and tumors.
Liposomes protect the oligonucleotides from
degradation and clearance and promise a
long half-life in the body.
Another potential delivery vector is composed of
polymerized nanoparticles . One example is the
commercially available NanoGel and can be
used for oral delivery of antisense drugs.
Amity Institute of Pharmacy
Application of Antisense Oligonucleotides:
Antisense drugs are being researched to treat a
variety of diseases such as :
Lung cancer,
Colorectal
carcinoma
Pancreatic
carcinoma
Malignant
melanoma
Diabetes
Amyotrophic lateral sclerosis
Amity Institute of Pharmacy
Most potential therapies have not yet
produced significant clinical results
though two antisense drugs have been
approved by the U.S. FDA :
Fomivirsen -marketed as
Vitravene as a treatment for
cytomegalovirus retinitis.
Mipomersen- marketed as
Kynamro for homozygous familial
hypercholesterolemia.
Fomivirsen Amity Institute of Pharmacy
Mipomersen Amity Institute of Pharmacy
Amity Institute of Pharmacy
Ribozymes:
Ribozymes are RNA molecules that have
catalytic activity.
Ribozyme Bind to the target RNA moiety and
inactivate it by cleaving the phosphodiester
backbone at a specific cutting site.
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Ribozymes in clinical trials:
Angiozyme : VEGF
receptor and
angiogenesis inhibitors -
treatment of kidney
cancer.
Herzyme : Anti-
HumanEpidermal growth
factor Receptor type 2
(HER2)- treatment of
breast and ovarian
cancer
Heptazyme : Reduces
Amity Institute of Pharmacy
RNA interference (RNAi) :
RNAi is an antisense mechanism that
involves using small interfering RNA, or
siRNA, to target a mRNA sequence. With
siRNA, the cell utilizes a protein complex
called RNA-induced silencing complex
(RISC) to destroy the mRNA, thereby
preventing the production of a disease-
causing protein.
Applications of RNAi :
Canc
er
HIV
Amity Institute of Pharmacy
Amity Institute of Pharmacy
Conclusion:
Antisense oligonucleotides show a great
potential as a molecular biology
investigative tool as well as highly selective
therapeutic agents.
They can produce non-specific effects
such as hematologic disturbances or
activation of immune system components.
They have limited uptake due to their
polarity and specific delivery to target
Amity Institute of Pharmacy
2n
generation antisense
d oligonucleotides
promise show to
as an alternative 1st as they
can function at other levels than RNase H.
Drug delivery systems may be the key in
making antisense oligonucleotides better
therapeutic agents, as they can produce
enhanced uptake, they protect from
degradation or prevent non-specific effects
Amity Institute of Pharmacy
Tutorial Class
Qus 1. Define the antisense molecule and give its
classification with specific characteristics.
Amity Institute of Pharmacy
Thank
you
