Current
Topics in Medicinal Chemistry
ISSN: 1568-0266
Current Topics
in Medicinal Chemistry
Volume 7, Number 7, 2007
Contents
Modified Nucleic Acid Substructures in
Medicinal Chemistry and Drug Development
Guest Editor: Dr. Vaijayanti A. Kumar
Editorial Pp. 639
2’-Modified Oligonucleotides for
Antisense Therapeutics Pp. 641-649
Thazha P. Prakash and Balkrishen Bhat
[Abstract]
Morpholino, siRNA, and S-DNA Compared: Impact of Structure
and Mechanism of Action on Off-Target Effects and Sequence
Specificity Pp. 651-660
James E. Summerton
[Abstract]
True Antisense Oligonucleotides with Modified Nucleotides
Restricted in the N-Conformation Pp. 661-665
Makoto Koizumi
[Abstract]
Nucleobase Modifications in Peptide Nucleic Acids
Pp. 667-679
Filip Wojciechowski and Robert H.E. Hudson
[Abstract]
Peptide Nucleic Acids with a Structurally Biased Backbone:
Effects of Conformational Constraints and Stereochemistry
Pp. 681-694
Roberto Corradini, Stefano Sforza, Tullia Tedeschi, Filbert
Totsingan and Rosangela Marchelli
[Abstract]
P-Chiral Oligonucleotides in Biological Recognition
Processes Pp. 695-713
Piotr Guga
[Abstract]
Structure-Editing of Nucleic Acids for Selective Targeting
of RNA Pp. 715-726
Vaijayanti A. Kumar and K. N. Ganesh
[Abstract]
Chemical Modifications to Improve the Cellular Uptake
of Oligonucleotides Pp. 727-737
Françoise Debart, Saïd Abes, Gaelle Deglane,
Hong M. Moulton, Philippe Clair, Michael J. Gait, Jean-Jacques
Vasseur and Bernard Lebleu
[Abstract]
Molecule
of Month
Abstracts
[Back to top]
Editorial
It gives me great pleasure to forward this special theme
issue devoted to the topic, ‘Modified Nucleic Acids
as Substructures in Medicinal Chemistry and Drug Development’.
With the discovery of siRNA being recognized by Nobel Prize
in medicine in 2006, the topic warrants such attention. The
collection of papers, contributed by several leading researchers,
illustrates the generation of therapeutically driven chemical
entities as mimics of nucleic acids. The common objective
in each case is the evolution of nucleic acids into therapeutically
viable oligonucleotide (ON) structures and to find some straightforward
answers to several technical challenges. Each contributor
has surpassed milestones on diverse roadmaps that lead to
a common destination i.e application of antisnese
(AS) principle for the therapeutic solution to several untreatable
diseases. The authoritative articles by the contributors summarize
their own research work and also aptly take note of the current
relevant literature.
Prakash and Bhat focus on the progress made in RNase H mediated
antisense approach, Summerton comprises the morpholinos, their
application and comparison with S-oligos, Koizumi details
the ONs with restricted geometry in N-sugar conformation.
Peptide nucleic acid, PNA, the highly competent DNA mimic
is further exploited in application perspective. We have two
distinct articles dedicated to the nucleobase and backbone
PNA modifications by Hudson and Corradini, respectively. The
P-chirality is a major issue in the phosphodiester modifications
in ONs and Guga has extensively reviewed this topic for both
chemistry and biology. Ganesh and myself divulge upon the
literature that highlights nucleic acid structure-editing
for RNA selectivity and the logic behind such selection. In
addition to the knockdown/down-regulating AS therapeutics,
the corrective AS principle needs such selection where small
molecular therapies are not common. The important bottleneck
for application of modified ONs as medicines is their cellular
delivery. Lebleu and co-authors have given sufficient attention
to the various ways of realizing this in their article. Considering
the nature of the special issue, there are certain quite understandable
overlaps, but authors have treated the topics in different
perspectives.
The issue thus covers concise efforts towards a common goal.
It is interesting to see that we have articles from Canada,
France, India, Italy, Japan, Russia, U.K. and U.S.A. that
also incidentally represent all the continents. I hope this
gives a comprehensive picture of today’s modified oligonucleotide
substructures and to some extent their biological evaluation.
I trust that the readers will enjoy the special issue and
it will be beneficial for the future research initiatives.
I owe all the contributors special thanks for their efforts.
I am personally grateful to Bentham Science Publications for
giving me this opportunity to bring out this issue.
Vaijayanti A. Kumar
National Chemical Laboratory,
Pune, 411008,
INDIA
[Back to top]
2’-Modified Oligonucleotides for Antisense
Therapeutics
Thazha P. Prakash and Balkrishen Bhat
Chemically modified antisense oligonucleotides are currently
progressing in multiple clinical trials. Among several chemical
modifications made, modification of the 2’-position
has proved most successful. Second generation antisense oligonucleotides
incorporating these 2’-modifications exhibit high binding
affinity to target RNA, enhanced metabolic stability, and
improved pharmacokinetic and toxicity profiles. This is, in
part, due to the enhanced biophysical properties of second
generation antisense oligonucleotides. 2'-Modifications that
influence the sugar to adopt a 3'-endo sugar pucker can improve
properties such as affinity. 2'-Modifications that provide
a gauche effect and/or a charge effect can play a significant
role in the level of nuclease resistance. The heterocyclic
base modifications such as 2-thiothymine provides additive
effect on the affinity of 2’-F and 2’-O-MOE
modifications. This review summarizes the structural and biophysical
properties of selected 2’-modified nucleosides which
are candidates for use in oligonucleotide theraputics.
[Back to top]
Morpholino, siRNA, and S-DNA Compared: Impact of Structure
and Mechanism of Action on Off-Target Effects and Sequence
Specificity
James E. Summerton
Generally a gene knockdown agent should achieve high sequence
specificity and should lack off-target effects (non-antisense
effects due to interactions with structures other than gene
transcripts). Three major gene knockdown types are compared
with respect to off-target effects and sequence specificities:
1) phosphorothioate-linked DNA (S-DNA); 2) short interfering
RNA (siRNA); and, 3) Morpholino.
S-DNAs cause multiple off-target effects, largely because
their backbone sulfurs bind to many different proteins. S-DNAs
also achieve poor sequence specificity because S-DNA/RNA duplexes
as short as 7 base-pairs are cleaved by RNase H.
siRNAs cause several off-target effects, but improved designs
may soon avoid such effects. siRNAs also provide only limited
sequence specificity because their short guide sequences largely
determine which gene transcripts will be blocked or cleaved,
and those guide sequences appear to recognize insufficient
sequence information to uniquely target a selected gene transcript.
This specificity limitation is inherent in their mechanism
of action and so probably cannot be greatly improved.
Morpholinos are virtually free of off-target effects - probably
because they cannot interact electrostatically with proteins.
Morpholinos also achieve exquisite sequence specificity -
in large part because they must bind at least about 14 to
15 contiguous bases to block a gene transcript, and this constitutes
sufficient sequence information to uniquely target a selected
gene transcript. Because of their freedom from off-target
effects, exquisite sequence specificity, complete stability
in biological systems, and highly predictable targeting, Morpholinos
dominate the most demanding of all gene knockdown applications,
studies in developing embryos.
[Back to top]
True Antisense Oligonucleotides with Modified Nucleotides
Restricted in the N-Conformation
Makoto Koizumi
As first-generation antisense oligonucleotides, more than
a dozen phosphorothioate oligodeoxynucleotides (PS ODNs) have
been clinically developed, but only one has reached the market.
To improve the drawbacks of PS ODNs, such as low affinity
to target mRNA and non-specific binding to proteins, modified
oligonucleotides with 2′-modified
sugars such as 2′
-O-(2-methoxy)ethyl and 2′-F
modification or with bridged sugars such as oxyalkylene linkages
between 2′-oxygen
and 4′-carbon,
have been synthesized as 2′-MOE,
2′-F
RNA, 2′,4′-BNA/LNA
and ENA oligonucleotides. They have shown properties of higher
affinity to complementary single-stranded RNA and DNA than
those of PS ODNs due to their preorganized N-conformation.
On the basis of the properties of these newly designed oligonucleotides,
their in vitro and in vivo applications
for gene silencing as true antisense oligonucleotides have
been reported. In this review, antisense applications with
these modified oligonucleotides are focused on.
[Back to top]
Nucleobase Modifications in Peptide Nucleic Acids
Filip Wojciechowski and Robert H.E. Hudson
Peptide nucleic acid (PNA) is an oligonucleotide mimic originally
designed upon a repeating N-(2-aminoethyl)glycine
polyamide backbone to which nucleobase heterocycles are attached
through a methylene carbonyl linkage to the α-amino
group. These molecules possess remarkable hybridization properties
with DNA or RNA forming complexes with high stability and
with excellent sequence discrimination despite the substantial
structural divergence from natural nucleic acids. Since the
disclosure of PNA, a vibrant research community with interest
in the chemistry and applications of polyamide-based nucleic
acid analogs has developed. This has led to the synthesis
and evaluation of a wide variety of modified polyamide nucleic
acids. The focus of this report is a comprehensive review
of nucleobase modifications in aminoethylglycine (aeg) PNA
with reference, where appropriate, to the same modification
in DNA or RNA.
[Back to top]
Peptide Nucleic Acids with a Structurally Biased Backbone:
Effects of Conformational Constraints and Stereochemistry
Roberto Corradini, Stefano Sforza, Tullia Tedeschi, Filbert
Totsingan and Rosangela Marchelli
Peptide nucleic acids (PNAs) are polyamidic oligonucleotide
analogs which have been described for the first time fifteen
years ago and were immediately found to be excellent tools
in binding DNA and RNA for diagnostics and gene regulation.
Their use as therapeutic agents have been proposed since early
studies and recent advancements in cellular delivery systems,
and in the so called anti-gene strategy, makes them good candidates
for drug development. The search for new chemical modification
of PNAs is a very active field of research and new structures
are continuously proposed. This review focuses on the recent
advancements obtained by the modification of the PNA backbone,
and their possible use in medicinal chemistry. In particular
two classes of structurally biased PNAs are described in details:
i) PNAs with acyclic structures and their helical preference,
which is regulated by stereochemistry and ii) cyclic PNAs
with preorganized structures, whose performances depend both
on stereochemistry and on conformational constraints. The
properties of these compounds are discussed in terms of affinity
for nucleic acids, and several recent examples of their use
in cellular or animal systems are presented.
[Back to top]
P-Chiral Oligonucleotides in Biological Recognition
Processes
Piotr Guga
Internucleotide phosphodiester linkages in non-modified oligonucleotides
are quickly degraded by nucleolytic enzymes present in the
cells and this feature practically eliminates natural DNA
and RNA molecules from medical applications and from many
structural and mechanistic studies. P-chiral oligonucleotide
analogs, in which one of the non-bridging phosphate oxygen
atoms is substituted with another heteroatom (e.g. S, Se)
or a chemical group (e.g. CH3, BH3-),
have significantly greater nuclease resistance and also offer
important possibilities for detailed studies of interactions
with other biomolecules at the molecular level. Notably, these
substitutions do not disrupt hydrogen bonding between nucleobases
and affect the overall geometry of the oligomers to only low
or moderate extent, although important changes of hydration
patterns and changes of interactions with metal ions are observed.
Such the probes, including isotopomeric species labeled with
a heavy oxygen isotope, possessing phosphorus atoms of selected
absolute configurations, have been used for elucidation of
the mode of action of many enzymes (nucleases, transferases,
kinases), ribozymes and DNA-zymes, as well as for investigations
on thermodynamic stability of nucleic acids complexes (duplexes,
triplexes, i-motif) and for studies on a mechanism of conformational
changes of B-Z type. They are also useful tools for analysis
of interactions of the phosphoryl oxygen atoms in natural
precursors with functional groups of proteins. The synthetic
routes to stereodefined forms of selected types of P-chiral
oligonucleotides are presented, as well as recently developed
methods for their configurational analysis at micromolar concentration.
Selected examples of application of diastereomerically pure
P-chiral oligonucleotides for structural, biochemical and
biological experiments are discussed.
[Back to top]
Structure-Editing of Nucleic Acids for Selective Targeting
of RNA
Vaijayanti A. Kumar and K. N. Ganesh
The synthesis of backbone-modified nucleic acids has been
an area of very intense research over the last two decades.
The main reason for this research activity is the instability
of nucleic acid based drugs in the intracellular conditions.
Changes in the sugar-phosphate backbone invariably bring about
the changes in the complementation properties of the nucleic
acids. The naturally occurring deoxyribose- (DNA) and ribose
(RNA) sugar-phosphate backbones are endowed with considerable
differences in their binding affinities towards themselves.
This occurs because of the different sugar conformations prevalent
in DNA and RNA and the subtle structural changes accruing
from these in hydrogen bonding, base-stacking interactions
and hydration of major/minor grooves. The six-atom phosphodiester
linkages and pentose-sugars give immense opportunities for
chemical modifications that lead to several backbone-modified
nucleic acid structures. This article is focused on such modifications
that impart RNA-selective binding properties to the modified
nucleic acid mimics and the rationale behind the said selectivity.
It is found that the six-atom sugar-phosphate backbone could
be replaced by either one-atom extended or one-atom edited
repeating units, leading to the folded or extended geometries
to maintain the internucleoside distance-complementarity.
Other important contributions come from electronegativity
of the substituent groups, hydration in the major/minor groove,
base stacking etc.
[Back to top]
Chemical Modifications to Improve the Cellular Uptake
of Oligonucleotides
Françoise Debart, Saïd Abes, Gaelle Deglane,
Hong M. Moulton, Philippe Clair, Michael J. Gait, Jean-Jacques
Vasseur and Bernard Lebleu
Specific control of gene expression by synthetic oligonucleotides
(ON) is now widely used for target validation but clinical
applications are limited by ON bioavailability. Moreover,
most currently used strategies for physical and chemical delivery
cannot be easily implemented in vivo. This article
reviews new strategies which appear promising for ON delivery.
The first part deals with ON chemical modifications aiming
at improving cellular uptake as for instance the grafting
of cationic groups on the ON backbone. The second part concerns
ON conjugation to cell penetrating peptides.
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