1. Introduction
Antisense technology is one of the therapeutic platforms to regulate
gene expression in vivo1. The synthetic antisense
oligonucleotides would influence gene expression and inhibit protein
synthesis. Proteins have a crucial role in all the cellular processes of
human metabolism. The majority of human diseases are the result of
inappropriate protein production or disordered protein
performance2. To inhibit the production of
disease-causing proteins, antisense technology based drugs could be
designed to bind sequence specifically to concerned mRNA. A wide
spectrum of diseases including infectious, inflammatory, cancer, and
cardiovascular diseases can be treated by specially designed and
synthesized antisense drugs. Antisense oligonucleotides (ASOs) are short
chemically modified oligonucleotides that bind to their complementary
mRNA by Watson-Crick base-pairing and modulate its
function3. The ASOs can be more effective and
less toxic in targeting any disease because they can have the capacity
to bind sequence specifically to the target mRNA. Single-stranded DNA
based oligonucleotides activate the RNase H antisense
mechanism4. The RNase H1 enzyme is ubiquitously
expressed which cleaves the RNA strand selectively from the RNA/DNA
hetero duplex5. The investigation of therapeutic
applications of antisense oligonucleotides against various diseases is
moving at a fast pace6. There are six antisense
technology based drugs namely Fomivirsen7,8,
Pegaptanib6, Mipomersen9,
Eteplirsen9, Defibrotide9 and
Nusinersen9 that have been approved by the FDA from
1998 to 2016. It has been observed in recent years that there is a rapid
increase in the number of antisense molecules entering into phase III
clinical trials8. In 1970, first time Zamecnik and
Stephenson proposed ASOs as therapeutic agents10. The
standard unmodified nucleic acids have confined stability in biological
media and undergo rapid degradation by nucleases11,12.
Chemical modifications would be required to protect the oligonucleotides
from the cellular nucleases, enhance the stability, improve binding
affinity for the target RNA, and improve pharmacokinetic properties in
animals to elicit a functional antisense response. The protective
modifications could be introduced at three different sites on the
nucleotide13. The nitrogen base can be altered or
changes in phosphate backbone can be made for DNA and RNA nucleotides.
Apart from these two, in RNA nucleotides, the 2’ hydroxyl group also can
be modified. These modifications are categorized into three generations.
First generation antisense modifications are majorly backbone based
modifications like phosphorothioates, methyl phosphonates, and
phosphoramidates modifications. One of the non-bridged oxygen attached
to the phosphate is replaced by sulfur in the phosphodiester backbone of
the nucleotide (Phosphorothioates), a methyl group (methylphophonates)
and amines (phosphoramidates). In all these backbone modifications,
phosphorothioates (PS) are successful and are widely used for
gene-silencing because of their resistance against nucleases and the
ability to induce the RNase H functions14. However,
the binding affinity to the target sequences, specificity and cellular
uptake profiles of phosphorothioates are less
satisfactory15. The issues raised with first
generation antisense modifications are solved up to some extent with
second generation modifications that are majorly based on sugar based
modifications. The 2’-O-methyl (OMe) and 2’-O-methoxyethyl (MOE) are
well explored and important members of second generation modifications.
These sugar based modifications OMe and MOE can be further combined with
the phosphorothioate backbone linkage16. It was
reported that the antisense oligonucleotides having the
2’-O-methoxyethyl modification are less toxic than phosphorothioate
antisense oligonucleotides and also shows enhanced affinity towards
their complementary RNAs and improved pharmacokinetic
properties17,18. To improve the thermal stabilities of
the antisense oligomer bound to either complementary DNA or RNA, several
nucleic acid analogs have been studied and developed. The third
generation modifications are peptide nucleic acids (PNAs), locked
nucleic acids (LNAs) or bridged nucleic acids, hexitol nucleic acids,
and morpholino oligonucleotides to name a few19-22.
PNAs are oligonucleotide analogues in which the sugar-phosphate backbone
has been completely replaced by pseudo peptide linkages. This
modification offers increased stability and favourable hybridization
kinetics. However, these constructs have problems of solubility and
delivery difficulties and they can’t activate the RNase H cleavage
mechanism23. LNAs are the most promising third
generation modifications. LNA nucleotides are a class of nucleic acid
analogues in which the ribose ring is locked by a methylene bridge
connecting the 2’-O atom and the 4’-C atom. The LNAs show increased
thermodynamic stability and improved nucleic acid
recognition24,25. Apart from these generations of
modifications, several nucleobase based modifications, backbone based
modifications, furanose sugar based modifications, six-membered ring
analogues, bicyclo and tricyclo modifications, constrained nucleic
acids, etc. have been designed by medicinal
chemists26. The novel “chimera” modifications in
which more than one modification has been made to improve the nuclease
resistance and target binding affinity as well. For example, single
nucleotide modification can have phosphorothioate backbone modification
and MOE sugar based modification. Another advanced strategy being
explored recently is the concept of gapmer design. Gapmer is a designed
antisense oligomer strand in which both the ends (2-5nt) have particular
modification for example MOE or LNA to increase binding affinity,
improve pharmacokinetic properties and in middle region phosphorothioate
modification to increase nuclease resistance and activate RNase H
activity27-31.
The importance of antisense technology and other oligonucleotide based
therapeutics is continuously increasing. Several studies are being
carried out by various labs to design novel drugs against some critical
diseases. There is a strong need to identify or design novel
modifications to improve the performance of existing antisense
oligonucleotides32. Various modified antisense
oligonucleotides are used in targeting different regions of the mRNA of
disease-causing genes. However, the structural information about these
antisense modifications is limited. The structural parameters calculated
through computational methods at the quantum mechanical level or
molecular dynamics level of the modifications are very useful for
understanding the mechanism of action of these modifications and also
help in designing novel modifications. The quantum chemical and
molecular dynamics simulations studies of some of the antisense
modifications have been reported in the
literature33-36. Many antisense molecules are
available in the market as FDA approved drugs, where PS, MOE, LNA
modified molecules are being used. There is a tremendous scope to
develop new modifications that can have strong binding affinity with
DNA/RNA and less toxicity. In the present study, five new antisense
modifications have been proposed. All the modifications have been
introduced in the cytidine nucleotide for the monomer level study. This
has been done to understand the effect of particular modification
considering any one nucleobase (here cytosine) at monomer level by
comparing it with its standard nucleotide with the same nitrogen base.
The efforts of quantum chemical studies at the monomer and base-pair
level would be helpful to understand the various properties of the
modifications in order to identify better modifications. Modification of
nucleic acids can affect chemical stability, pairing, and conformation.
The structures of these proposed modifications are designed based on the
LNA structure. LNA has strong binding properties but higher toxicity
compared to MOE37-40. The proposed modifications are
designed by changing various atoms or by adding different oxy or nitro
groups to the LNA. The backbone and sugar based chemical modifications
are known to improve the pairing affinity, metabolic stability, and
cellular uptake of RNA. These parameters are absolutely essential in the
discovery and development of highly active oligonucleotide / RNA based
therapeutic candidates. To enhance the potency and reduce the potential
toxicity of antisense oligomers, numerous chemical modification
geometries have been established and tested. The basic idea behind
proposing these modifications is to increase the binding affinity as
well as to reduce the toxic effects and improve the pharmacokinetic
properties. The proposed modifications are designed in such a way that
they share the structural components from LNA, MOE and other
electronegative groups / bulky groups attached to methylene bridge
carbon of LNA. In the proposed modifications, the methylene bridge
carbon is replaced with nitrogen in A1 and A2 modifications. In A2, an
extra methoxy group is added to the bridged nitrogen. For A3, A4 and A5
modifications, dimethyl amine group, amine group and dihydroxy amine
group are added to the bridged carbon respectively. For proposed
modifications, the methoxy group and other amine groups are added
because they are already well established as 2’-O sugar modifications
and incorporation of these proved to show good pharmacokinetic
properties17,18,41. In all proposed modifications, the
electronegative groups and other small chemical groups were added to the
basic LNA structure to improve the pharmacokinetics and reduce the
toxicity without disturbing the strong binding nature of LNA. All these
proposed modifications are studied thoroughly through quantum
calculations. The 2D structural representation of all the modifications
used in the current study has been given in Figure 1 and the
modification details have been given in Table 1. The study also aims to
identify the stable conformations of the antisense molecules selected
from the literature and the proposed novel modifications. Comparison has
been made in terms of quantum chemical descriptors which helps to
evaluate the different standard antisense modifications and the proposed
new modifications.
The conformational search and optimization of all modifications at the
monomer level was carried out and identified the most stable
conformation for each modification by using in-house developed
conformation generation and optimization tool TANGO42.
The quantum chemical calculations have been carried out for the most
stable conformations of all the modifications and derived various
quantum chemical descriptors. This study may help in understanding the
structural and functional significance of these novel antisense
modifications in exhibiting lower toxicity with higher binding affinity
and increased potency. Structural insight and information regarding
various quantum chemical properties could be useful in their functional
understanding, which may guide in the design of better modifications.