213
medium, the cellular influx transport mechanism of the essential metabolite should
also be disturbed. This needs to be done in cases where we want to inhibit bacteria
by targeting the synthesis of an essential metabolite. In these cases, two different
genes should be inhibited by two different antisense oligonucleotides. Ιt should be
mentioned that sometimes one antisense oligonucleotide is capable of inhibiting
both genes – the one that encodes an enzyme and the other responsible for the transporter protein, for instance, the FMN riboswitch. We can also target genes, which
are responsible for bacterial virulence with this technology. These genes are very
suitable targets for antibacterial drug development because their inhibition does not
exert selection to develop antimicrobial resistance (Penchovsky and
Traykovska 2015).
There are two mechanisms that antisense oligonucleotides apply to inhibit a specific RNA. As mentioned above, antisense oligonucleotides are chemically modified deoxyribonucleotide oligomers, which are designed to be complementary to the
targeted RNA. When they are transferred inside the cell with the help of a cell
penetrating peptide, they apply Watson-Crick base pairing to hybridize with the
complementary mRNA and an antisense oligonucleotide mRNA heteroduplex is
formed. In the first case, RNase H recognizes the double stranded molecule, which
is formed between the antisense oligonucleotide and mRNA and binds to it. The
binding of RNase H leads to cleavage of the targeted mRNA and these results in the
inhibition of protein expression via a multi turnover action (Penchovsky and
Traykovska 2015).
Another mechanism of antisense oligonucleotide uses peptide nucleic acids
(PNAs) or locked nucleic acids (LNA). The mode of action of PNA is a single turnover, which means that if it hybridizes it cannot be used again. The binding of PNA
to mRNA impedes the ribosomal subunits of binding to the mRNA. Thus, the procedure of translation is inhibited. Normally the ribosomal subunits bind to mRNA
and then mRNA is translated into a functional protein. Therefore, in the presence of
the antisense oligonucleotides, the binding of ribosomal subunits to mRNA is prevented which finally results in the inhibition of protein function (Chan et al. 2006;
Penchovsky and Traykovska 2015; Seth et al. 2019).
Antisense oligonucleotides are classified based on their chemical modifications
into three generations. Those, which belong to the first generation, contain a phosphorothioate backbone, in which a sulfur atom replaces one of the non bridging
oxygen in phosphodiester bonds. Antisense oligonucleotides of the first generation
are capable of inducing an RNase-H mediated cleavage of target mRNAs. The
PS-antisense oligonucleotides have higher bioavailability in comparison to unmodified nucleotides due to phosphorothioate modification, which is related to the higher
resistance to nuclease degradation. Although, it should be mentioned that
PS-antisense oligonucleotides seem to produce some non specific side effects due to
their interaction with the cell surface and intracellular proteins. The second generation antisense oligonucleotides were developed to enhance nuclease resistance and
increase the binding activity of the target mRNA. They differ because of ribose
assumed 2′-alkyl modifications (Penchovsky and Traykovska 2015).
9 Drug Discovery for Targeting Drug Resistant Bacteria
medium, the cellular influx transport mechanism of the essential metabolite should
also be disturbed. This needs to be done in cases where we want to inhibit bacteria
by targeting the synthesis of an essential metabolite. In these cases, two different
genes should be inhibited by two different antisense oligonucleotides. Ιt should be
mentioned that sometimes one antisense oligonucleotide is capable of inhibiting
both genes – the one that encodes an enzyme and the other responsible for the transporter protein, for instance, the FMN riboswitch. We can also target genes, which
are responsible for bacterial virulence with this technology. These genes are very
suitable targets for antibacterial drug development because their inhibition does not
exert selection to develop antimicrobial resistance (Penchovsky and
Traykovska 2015).
There are two mechanisms that antisense oligonucleotides apply to inhibit a specific RNA. As mentioned above, antisense oligonucleotides are chemically modified deoxyribonucleotide oligomers, which are designed to be complementary to the
targeted RNA. When they are transferred inside the cell with the help of a cell
penetrating peptide, they apply Watson-Crick base pairing to hybridize with the
complementary mRNA and an antisense oligonucleotide mRNA heteroduplex is
formed. In the first case, RNase H recognizes the double stranded molecule, which
is formed between the antisense oligonucleotide and mRNA and binds to it. The
binding of RNase H leads to cleavage of the targeted mRNA and these results in the
inhibition of protein expression via a multi turnover action (Penchovsky and
Traykovska 2015).
Another mechanism of antisense oligonucleotide uses peptide nucleic acids
(PNAs) or locked nucleic acids (LNA). The mode of action of PNA is a single turnover, which means that if it hybridizes it cannot be used again. The binding of PNA
to mRNA impedes the ribosomal subunits of binding to the mRNA. Thus, the procedure of translation is inhibited. Normally the ribosomal subunits bind to mRNA
and then mRNA is translated into a functional protein. Therefore, in the presence of
the antisense oligonucleotides, the binding of ribosomal subunits to mRNA is prevented which finally results in the inhibition of protein function (Chan et al. 2006;
Penchovsky and Traykovska 2015; Seth et al. 2019).
Antisense oligonucleotides are classified based on their chemical modifications
into three generations. Those, which belong to the first generation, contain a phosphorothioate backbone, in which a sulfur atom replaces one of the non bridging
oxygen in phosphodiester bonds. Antisense oligonucleotides of the first generation
are capable of inducing an RNase-H mediated cleavage of target mRNAs. The
PS-antisense oligonucleotides have higher bioavailability in comparison to unmodified nucleotides due to phosphorothioate modification, which is related to the higher
resistance to nuclease degradation. Although, it should be mentioned that
PS-antisense oligonucleotides seem to produce some non specific side effects due to
their interaction with the cell surface and intracellular proteins. The second generation antisense oligonucleotides were developed to enhance nuclease resistance and
increase the binding activity of the target mRNA. They differ because of ribose
assumed 2′-alkyl modifications (Penchovsky and Traykovska 2015).
9 Drug Discovery for Targeting Drug Resistant Bacteria