TIBTEC-1195; No. of Pages 8
Review
Drug target miRNAs: chances and challenges Marco F. Schmidt University Chemical Laboratory, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK
miRNAs, short ribonucleic acid gene regulators, are increasingly popular drug targets. Traditionally ‘undruggable’ proteins can be targeted via their miRNA gene regulators, enabling the treatment of diseases that, at present, seem impossible to cure. However, addressing miRNAs requires innovation at the level of drug discovery. This review article outlines the potential of miRNAs as drug targets, focuses on the challenges of developing miRNA-targeting drugs, and surveys new advances. The aim is to provide an orientation guide for scientists, as well business analysts, to help them navigate the jungle of different approaches in miRNA drug discovery. The need for new drug targets Developing new medicines is becoming an increasingly expensive business. For example, the average cost per approved new drug molecule over 5 years from 2002 to 2006 was US $2.8 billion, whereas from 2007 to 2011, the average cost was already US $4.2 billion (www.pwc.com/ pharma2020). This recent development has been termed the ‘compound crisis’ and it has been postulated to result from inefficient methods and management mistakes in the pharmaceutical industry [1]. One major aspect of the pharmaceutical industry’s poor R&D productivity is drug target selection. Less than 1% of all approved drugs do not bind to proteins, whereas more than 80% of all drugs target only two protein classes: enzymes or receptors [2,3]. Reported protocols in drug discovery, for example, compound selection (Lipinski’s Rule of Five [3]), have been developed only for protein drug targets. Nevertheless, the number of protein drug targets is limited. Although the human genome encodes 25 000 genes, it is estimated that only 600 disease-modifying protein drug targets exist [3]. The focus in target selection has now shifted to other macromolecules, such as RNAs. Due to their involvement in gene regulation, RNAi [4] miRNAs have been identified as high-value targets for therapy. Only 20 years after first reports about their existence, several miRNA-targeting drugs are now in clinical trials or even close to market launch. This review article discusses the fate of miRNAs as drug targets, briefly describing their biological functions, the challenge of developing miRNA-targeting drugs, and Corresponding author: Schmidt, M.F. ([email protected]). Keywords: miRNA; drug discovery; RNAi; miRNA-induced silencing complex; antisense agents; small molecule miRNA modulators. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.09.002
developments that extend beyond established drug discovery protocols. miRNAs: post-transcriptional gene regulators and drug targets miRNAs, non-coding, single-stranded RNA molecules that are, on average, only 22– 23 nucleotides long, are posttranscriptional gene regulators. Although only approximately 2000 miRNAs exist in humans (www.mirbase.org), they regulate 30% of all genes. Therefore, they are crucial in almost all biological processes [4,5]. miRNAs silence genes through a well-studied pathway (Box 1). miRNAs have been identified to play a crucial role in various human diseases, and therefore, they are interesting for drug discovery. However, this is not the full story. As mentioned before, not every protein can be targeted or modulated by a drug molecule. These proteins are also referred to as being ‘undruggable’. By contrast, intervention at the miRNA level may allow specific manipulation of every protein population: miRNA inhibitors induce selective upregulation of one protein population; by contrast, miRNA mimics induce gene silencing, thus resulting in downregulation of the target protein. Consequently, also former ‘undruggable’ proteins can be modulated via their miRNA gene regulators, enabling the cure of diseases, which, at present, seems impossible. As a result, miRNAs are regarded as high-value drug targets. Anti-sense agents: miRNA mimics and inhibitors A reasonable strategy in miRNA modulation is the use of anti-sense agents or small interfering RNAs (siRNAs) [6]. As mentioned before, oligonucleotides can either mimic miRNA, thus inducing gene silencing in a similar manner to RNAi, or bind to a target miRNA and block the translational arrest. Nevertheless, it has been shown that, in practice, single as well double-stranded oligonucleotides are poorly cell-permeable, and consequently, cannot enter the cell by diffusion [6]. Therefore, their general application in therapy is limited. Only under certain circumstances have anti-sense agents been successful in clinical trials. MRX34: a liposome-based miRNA-34a mimic in combination with Erlotinib An example for gene silencing intervention by anti-sense agents is MRX34, developed by Mirnarx Therapeutics, Inc. (www.mirnarx.com) [7,8]. As human miRNA-34a is a downregulated tumor suppressor in numerous cancers that regulates more than 20 oncogenes, including the epidermal growth factor receptor (EGFR) pathway, it has been Trends in Biotechnology xx (2014) 1–8
1
TIBTEC-1195; No. of Pages 8
Review
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Box 1. The miRNA-induced gene silencing pathway miRNAs encoding genes are transcribed by RNA polymerase II/III, producing the so-called primary miRNAs (pri-miRNAs). In the nucleus, these transcripts are processed by the nuclease Drosha, forming the precursor miRNAs (pre-miRNAs), which are up to 70–80 nucleotides long and are organized in a double-stranded hairpin conformation. The pre-miRNAs are transported out of the nucleus. In the cytosol, the ribonuclease DICER, in association with two proteins, protein activator of the interferon induced protein kinase (PACT) and HIV transactivating response RNA-binding protein-2 (TRBP), cleaves the double-stranded pre-miRNA. Finally, miRNA guides the Argonaute 2 (AGO2) protein to its target mRNA by sequence-specific complementarity [4]. The AGO2 protein itself recruits several other proteins: glycine(g)-tryptophan(w) repeat-containing protein of
182 kDa (GW182), poly(A)binding protein (PABP), the cellular mRNA deadenylase complex CCR4-NOT, poly(A) nucleases 2 and 3 (PAN2– PAN3), and likely some unknown proteins, all forming together the so-called miRNA-induced silencing complex (miRISC) [5]. miRISC-mediated gene silencing is executed via three different molecular actions (Figure I). (i) The Argonaute protein is an RNase and catalyzes the cleavage of target mRNA, sequence-specifically guided by bound miRNA. (ii) GW182 inhibits translation initiation by preventing ribosomal complex formation. miRISC also might block translation at post-initiation steps by interfering with ribosome elongation. (iii) CCR4-NOT and PAN2–PAN3 facilitate deadenylation of the poly(A) tail (AAA). mRNA decay is terminated by exonucleases followed by removal of the 5-terminal cap (m7G) [5].
Transcripon by RNA Pol II/III
RNA Pol II Processing by Drosha pre-miRNA Drosha DGCR8
pri-miRNA
Exporn 5
Nucleus
PACT
DICER
TRBP
DICER processing
Cystosol Translaon block 40S m7G
? ? ?
GW182
AGO2
mRNA
CCR4-NOT PAN2-PAN3 PABP
TRBP AGO2 PACT
AAA Deadenylaon Decay
STOP
miRNA
Translaonal arrest of target mRNA by miRNA-induced silencing complex (miRISC)
Passenger strand unwinding TRENDS in Biotechnology
Figure I. Steps of gene silencing. Abbreviations: AAA, poly(A) tail; AGO2, Argonaute 2 protein; DGCR8, a microprocessor complex subunit; GW182, glycine(g)tryptophan(w) repeat-containing protein of 182 kDa; PABP, poly(A)binding protein; PACT, protein activator of the interferon induced protein kinase; PAN2 and PAN3, poly(A) nucleases 2 and 3; RNA pol, RNA polymerase; TRBP, HIV transactivating response RNA-binding protein-2.
demonstrated that addition of artificial miRNA-34a to cancer cells provides a possible therapy. Nonetheless, anti-sense agents are poorly cell-permeable. Scientists at Mirnarx Therapeutics, Inc. developed a formulation for the anti-sense agent delivery into the cell: a liposomal nanoparticle loaded with synthetic miRNA-34a mimics (MRX34), which has recently entered Phase I clinical trials [7]. Additionally, MRX34 has been tested against cancer cells in combination with the EGFR tyrosine kinase inhibitor (EGFR-TKI) Erlotinib, giving evidence that several cancers previously not suited for Erlotinib may prove sensitive to the drug when used in combination with MRX34 [8]. MRX34 displays two trends in miRNA mimic drug discovery: firstly, using advanced formulations such as liposomal nanoparticles to increase the delivery of oligonucleotides into the cell; and secondly, searching for synergistic effects while miRNA mimics are used in combination with reported drugs. Accordingly, Dicerna 2
Pharmaceuticals, Inc. (www.dicerna.com) developed a similar approach only using a lipid nanoparticle system for the delivery of double-stranded RNA as substrate for the RNAi-initiating enzyme DICER. The double-stranded DICER-substrate small interfering RNA (DsiRNA) acts as a prodrug and is more stable than single-stranded miRNA mimics [9]. Additionally, Dicerna Pharmaceuticals, Inc. reported that silencing of the traditionally ‘undruggable’ oncogene protein MYC by their DsiRNA technology in co-treatment with the multi-kinase inhibitor Sorafenib showed significantly higher anti-tumor activity than the single use of Sorafenib (www.dicerna.com/ media-publications.php). Miravirsen: a locked nucleic acid-based miRNA inhibitor Anti-sense agents can also be used as RNAi inhibitors by binding to target miRNA. Kru¨tzfeldt et al. reported in 2004 the use of modified ribonucleic acids, so-called
TIBTEC-1195; No. of Pages 8
Review
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
antagonistic miRNAs (antagomiRs) [10]. These antagomiRs are inappropriate for therapeutic use due to their poor pharmacokinetic properties [6]. From there, efforts were made to design oligonucleotide derivatives with increased lipophilicity compared with ribonucleic acids, in the hope of overcoming inappropriate pharmacokinetic properties. One nucleotide derivative is locked nucleic acid (LNA) [11]: Here, the ribose moiety of an LNA nucleotide has an additional methylene bridge connecting the 20 oxygen and 40 carbon (Figure 1A). Thereby, the ribose is ‘locked’ in the 30 -endo conformation, which enhances base stacking and backbone pre-organization. The most prominent LNA-modified antisense oligonucleotide drug candidate is Miravirsen. The 15mer Miravirsen has been developed by Santaris Pharma A/S (www.santaris.com; recently acquired by F. Hoffmann-La Roche AG for US $ 450 Mio.), and recently tested successfully
in Phase II clinical trials for the treatment of hepatitis C virus (HCV) infection [12]. Miravirsen targets human liverexpressed miRNA-122. MiRNA-122 is an important host factor for HCV. It interferes with the 50 untranslated region (50 UTR) of the virus RNA by binding to two miRNA-122 sites in complex with the Argonaute 2 protein (AGO2) [13]. Here, by binding its 50 UTR region to the miRNA-122-Argonaute 2 complex, the HCV virus genome is protected from nucleolytic degradation (Figure 1B, left), thereby promoting viral RNA stability and propagation [14,15]. By contrast, Miravirsen binds to the miRNA-122–AGO2 complex, blocking the essential interaction with HCV RNA for viral replication (Figure 1B, right) [16]. However, Miravirsen is an exception in miRNA drug discovery. Its phosphorothioate modification results in Miravirsen accumulation in the liver after injection, where its target miRNA-122 is exclusively expressed [17].
(A)
Miravirsen: (5′-3′)(P-thio)(mC-dC-A-dT-dT-G-T-dC-dA-mC-dA-mC-dT-mC-mC) –O
A:
O
P
O
–O
S P
O O
–O
O
mC:
O
N
N
O
S P
N
O
O O
N
HO
O
O
N
HO
H N
NH
O O
O
O
HO
O
N
N
S P
G:
O
HO
–O
NH2
N O
T:
H2N
N
S
NH2
N
O
(B) AGO2
miRNA-122
+ HCV RNA
LNA-based drug Miravirsen
AGO2
AGO2
miRNA-122
+
HCV RNA Miravirsen
Protected from degradaon
HCV RNA degradaon
Viral replicaon
STOP
No viral replicaon TRENDS in Biotechnology
Figure 1. Structure and mechanism of the locked nucleic acid (LNA)-based drug Miravirsen (simplified) [12]. (A) Structure of Miravirsen. Abbreviations: P-thio, phosphorothioate; A, adenosine; dA, 20 -deoxyadenosine; dT, 20 -deoxythymidine; T, thymidine; G, guanosine; dC, 20 -deoxycytidine; mC, 5-methylcytidine. (B) Hepatitis C virus (HCV) is either protected by the Argonaute 2 protein (AGO2) in the absence of drugs (left) or allowed to degrade in the presence of Miravirsen (right).
3
TIBTEC-1195; No. of Pages 8
Review Therefore, targeting other miRNAs outside of the liver with anti-sense oligonucleotides is still a challenge. In conclusion, anti-sense miRNA modifiers only work under certain circumstances, such as liposomal nanoparticles, in combination with other drugs, or in case of Miravirsen, as an exception. However, their general application is hampered due to their poor pharmacokinetic properties, likely because of their molecular size of >6000 Da and their negatively charged backbone [6]. In addition, anti-sense oligonucleotides have to be applied by injection, thereby making the treatment less convenient. In contrast to that, the success of protein-targeting small molecule drugs relies mostly on their oral administration and their ability to reach in-cell targets by simple diffusion [3]. The question may, therefore, be asked: why can the concept of protein-based drug discovery not be adopted to target miRNAs with small molecules? Small molecule modulators: adopting lessons learned from protein-based drug discovery to miRNAs The use of small molecules in protein drug discovery has been established several years ago and offers the possibility of addressing targets within a cell by simple diffusion. Parameters for designing a molecule to fulfill this challenge have been summarized by Lipinski’s Rule of Five [molecular weight (MW)
Review
Drug target miRNAs: chances and challenges Marco F. Schmidt University Chemical Laboratory, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK
miRNAs, short ribonucleic acid gene regulators, are increasingly popular drug targets. Traditionally ‘undruggable’ proteins can be targeted via their miRNA gene regulators, enabling the treatment of diseases that, at present, seem impossible to cure. However, addressing miRNAs requires innovation at the level of drug discovery. This review article outlines the potential of miRNAs as drug targets, focuses on the challenges of developing miRNA-targeting drugs, and surveys new advances. The aim is to provide an orientation guide for scientists, as well business analysts, to help them navigate the jungle of different approaches in miRNA drug discovery. The need for new drug targets Developing new medicines is becoming an increasingly expensive business. For example, the average cost per approved new drug molecule over 5 years from 2002 to 2006 was US $2.8 billion, whereas from 2007 to 2011, the average cost was already US $4.2 billion (www.pwc.com/ pharma2020). This recent development has been termed the ‘compound crisis’ and it has been postulated to result from inefficient methods and management mistakes in the pharmaceutical industry [1]. One major aspect of the pharmaceutical industry’s poor R&D productivity is drug target selection. Less than 1% of all approved drugs do not bind to proteins, whereas more than 80% of all drugs target only two protein classes: enzymes or receptors [2,3]. Reported protocols in drug discovery, for example, compound selection (Lipinski’s Rule of Five [3]), have been developed only for protein drug targets. Nevertheless, the number of protein drug targets is limited. Although the human genome encodes 25 000 genes, it is estimated that only 600 disease-modifying protein drug targets exist [3]. The focus in target selection has now shifted to other macromolecules, such as RNAs. Due to their involvement in gene regulation, RNAi [4] miRNAs have been identified as high-value targets for therapy. Only 20 years after first reports about their existence, several miRNA-targeting drugs are now in clinical trials or even close to market launch. This review article discusses the fate of miRNAs as drug targets, briefly describing their biological functions, the challenge of developing miRNA-targeting drugs, and Corresponding author: Schmidt, M.F. ([email protected]). Keywords: miRNA; drug discovery; RNAi; miRNA-induced silencing complex; antisense agents; small molecule miRNA modulators. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.09.002
developments that extend beyond established drug discovery protocols. miRNAs: post-transcriptional gene regulators and drug targets miRNAs, non-coding, single-stranded RNA molecules that are, on average, only 22– 23 nucleotides long, are posttranscriptional gene regulators. Although only approximately 2000 miRNAs exist in humans (www.mirbase.org), they regulate 30% of all genes. Therefore, they are crucial in almost all biological processes [4,5]. miRNAs silence genes through a well-studied pathway (Box 1). miRNAs have been identified to play a crucial role in various human diseases, and therefore, they are interesting for drug discovery. However, this is not the full story. As mentioned before, not every protein can be targeted or modulated by a drug molecule. These proteins are also referred to as being ‘undruggable’. By contrast, intervention at the miRNA level may allow specific manipulation of every protein population: miRNA inhibitors induce selective upregulation of one protein population; by contrast, miRNA mimics induce gene silencing, thus resulting in downregulation of the target protein. Consequently, also former ‘undruggable’ proteins can be modulated via their miRNA gene regulators, enabling the cure of diseases, which, at present, seems impossible. As a result, miRNAs are regarded as high-value drug targets. Anti-sense agents: miRNA mimics and inhibitors A reasonable strategy in miRNA modulation is the use of anti-sense agents or small interfering RNAs (siRNAs) [6]. As mentioned before, oligonucleotides can either mimic miRNA, thus inducing gene silencing in a similar manner to RNAi, or bind to a target miRNA and block the translational arrest. Nevertheless, it has been shown that, in practice, single as well double-stranded oligonucleotides are poorly cell-permeable, and consequently, cannot enter the cell by diffusion [6]. Therefore, their general application in therapy is limited. Only under certain circumstances have anti-sense agents been successful in clinical trials. MRX34: a liposome-based miRNA-34a mimic in combination with Erlotinib An example for gene silencing intervention by anti-sense agents is MRX34, developed by Mirnarx Therapeutics, Inc. (www.mirnarx.com) [7,8]. As human miRNA-34a is a downregulated tumor suppressor in numerous cancers that regulates more than 20 oncogenes, including the epidermal growth factor receptor (EGFR) pathway, it has been Trends in Biotechnology xx (2014) 1–8
1
TIBTEC-1195; No. of Pages 8
Review
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
Box 1. The miRNA-induced gene silencing pathway miRNAs encoding genes are transcribed by RNA polymerase II/III, producing the so-called primary miRNAs (pri-miRNAs). In the nucleus, these transcripts are processed by the nuclease Drosha, forming the precursor miRNAs (pre-miRNAs), which are up to 70–80 nucleotides long and are organized in a double-stranded hairpin conformation. The pre-miRNAs are transported out of the nucleus. In the cytosol, the ribonuclease DICER, in association with two proteins, protein activator of the interferon induced protein kinase (PACT) and HIV transactivating response RNA-binding protein-2 (TRBP), cleaves the double-stranded pre-miRNA. Finally, miRNA guides the Argonaute 2 (AGO2) protein to its target mRNA by sequence-specific complementarity [4]. The AGO2 protein itself recruits several other proteins: glycine(g)-tryptophan(w) repeat-containing protein of
182 kDa (GW182), poly(A)binding protein (PABP), the cellular mRNA deadenylase complex CCR4-NOT, poly(A) nucleases 2 and 3 (PAN2– PAN3), and likely some unknown proteins, all forming together the so-called miRNA-induced silencing complex (miRISC) [5]. miRISC-mediated gene silencing is executed via three different molecular actions (Figure I). (i) The Argonaute protein is an RNase and catalyzes the cleavage of target mRNA, sequence-specifically guided by bound miRNA. (ii) GW182 inhibits translation initiation by preventing ribosomal complex formation. miRISC also might block translation at post-initiation steps by interfering with ribosome elongation. (iii) CCR4-NOT and PAN2–PAN3 facilitate deadenylation of the poly(A) tail (AAA). mRNA decay is terminated by exonucleases followed by removal of the 5-terminal cap (m7G) [5].
Transcripon by RNA Pol II/III
RNA Pol II Processing by Drosha pre-miRNA Drosha DGCR8
pri-miRNA
Exporn 5
Nucleus
PACT
DICER
TRBP
DICER processing
Cystosol Translaon block 40S m7G
? ? ?
GW182
AGO2
mRNA
CCR4-NOT PAN2-PAN3 PABP
TRBP AGO2 PACT
AAA Deadenylaon Decay
STOP
miRNA
Translaonal arrest of target mRNA by miRNA-induced silencing complex (miRISC)
Passenger strand unwinding TRENDS in Biotechnology
Figure I. Steps of gene silencing. Abbreviations: AAA, poly(A) tail; AGO2, Argonaute 2 protein; DGCR8, a microprocessor complex subunit; GW182, glycine(g)tryptophan(w) repeat-containing protein of 182 kDa; PABP, poly(A)binding protein; PACT, protein activator of the interferon induced protein kinase; PAN2 and PAN3, poly(A) nucleases 2 and 3; RNA pol, RNA polymerase; TRBP, HIV transactivating response RNA-binding protein-2.
demonstrated that addition of artificial miRNA-34a to cancer cells provides a possible therapy. Nonetheless, anti-sense agents are poorly cell-permeable. Scientists at Mirnarx Therapeutics, Inc. developed a formulation for the anti-sense agent delivery into the cell: a liposomal nanoparticle loaded with synthetic miRNA-34a mimics (MRX34), which has recently entered Phase I clinical trials [7]. Additionally, MRX34 has been tested against cancer cells in combination with the EGFR tyrosine kinase inhibitor (EGFR-TKI) Erlotinib, giving evidence that several cancers previously not suited for Erlotinib may prove sensitive to the drug when used in combination with MRX34 [8]. MRX34 displays two trends in miRNA mimic drug discovery: firstly, using advanced formulations such as liposomal nanoparticles to increase the delivery of oligonucleotides into the cell; and secondly, searching for synergistic effects while miRNA mimics are used in combination with reported drugs. Accordingly, Dicerna 2
Pharmaceuticals, Inc. (www.dicerna.com) developed a similar approach only using a lipid nanoparticle system for the delivery of double-stranded RNA as substrate for the RNAi-initiating enzyme DICER. The double-stranded DICER-substrate small interfering RNA (DsiRNA) acts as a prodrug and is more stable than single-stranded miRNA mimics [9]. Additionally, Dicerna Pharmaceuticals, Inc. reported that silencing of the traditionally ‘undruggable’ oncogene protein MYC by their DsiRNA technology in co-treatment with the multi-kinase inhibitor Sorafenib showed significantly higher anti-tumor activity than the single use of Sorafenib (www.dicerna.com/ media-publications.php). Miravirsen: a locked nucleic acid-based miRNA inhibitor Anti-sense agents can also be used as RNAi inhibitors by binding to target miRNA. Kru¨tzfeldt et al. reported in 2004 the use of modified ribonucleic acids, so-called
TIBTEC-1195; No. of Pages 8
Review
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
antagonistic miRNAs (antagomiRs) [10]. These antagomiRs are inappropriate for therapeutic use due to their poor pharmacokinetic properties [6]. From there, efforts were made to design oligonucleotide derivatives with increased lipophilicity compared with ribonucleic acids, in the hope of overcoming inappropriate pharmacokinetic properties. One nucleotide derivative is locked nucleic acid (LNA) [11]: Here, the ribose moiety of an LNA nucleotide has an additional methylene bridge connecting the 20 oxygen and 40 carbon (Figure 1A). Thereby, the ribose is ‘locked’ in the 30 -endo conformation, which enhances base stacking and backbone pre-organization. The most prominent LNA-modified antisense oligonucleotide drug candidate is Miravirsen. The 15mer Miravirsen has been developed by Santaris Pharma A/S (www.santaris.com; recently acquired by F. Hoffmann-La Roche AG for US $ 450 Mio.), and recently tested successfully
in Phase II clinical trials for the treatment of hepatitis C virus (HCV) infection [12]. Miravirsen targets human liverexpressed miRNA-122. MiRNA-122 is an important host factor for HCV. It interferes with the 50 untranslated region (50 UTR) of the virus RNA by binding to two miRNA-122 sites in complex with the Argonaute 2 protein (AGO2) [13]. Here, by binding its 50 UTR region to the miRNA-122-Argonaute 2 complex, the HCV virus genome is protected from nucleolytic degradation (Figure 1B, left), thereby promoting viral RNA stability and propagation [14,15]. By contrast, Miravirsen binds to the miRNA-122–AGO2 complex, blocking the essential interaction with HCV RNA for viral replication (Figure 1B, right) [16]. However, Miravirsen is an exception in miRNA drug discovery. Its phosphorothioate modification results in Miravirsen accumulation in the liver after injection, where its target miRNA-122 is exclusively expressed [17].
(A)
Miravirsen: (5′-3′)(P-thio)(mC-dC-A-dT-dT-G-T-dC-dA-mC-dA-mC-dT-mC-mC) –O
A:
O
P
O
–O
S P
O O
–O
O
mC:
O
N
N
O
S P
N
O
O O
N
HO
O
O
N
HO
H N
NH
O O
O
O
HO
O
N
N
S P
G:
O
HO
–O
NH2
N O
T:
H2N
N
S
NH2
N
O
(B) AGO2
miRNA-122
+ HCV RNA
LNA-based drug Miravirsen
AGO2
AGO2
miRNA-122
+
HCV RNA Miravirsen
Protected from degradaon
HCV RNA degradaon
Viral replicaon
STOP
No viral replicaon TRENDS in Biotechnology
Figure 1. Structure and mechanism of the locked nucleic acid (LNA)-based drug Miravirsen (simplified) [12]. (A) Structure of Miravirsen. Abbreviations: P-thio, phosphorothioate; A, adenosine; dA, 20 -deoxyadenosine; dT, 20 -deoxythymidine; T, thymidine; G, guanosine; dC, 20 -deoxycytidine; mC, 5-methylcytidine. (B) Hepatitis C virus (HCV) is either protected by the Argonaute 2 protein (AGO2) in the absence of drugs (left) or allowed to degrade in the presence of Miravirsen (right).
3
TIBTEC-1195; No. of Pages 8
Review Therefore, targeting other miRNAs outside of the liver with anti-sense oligonucleotides is still a challenge. In conclusion, anti-sense miRNA modifiers only work under certain circumstances, such as liposomal nanoparticles, in combination with other drugs, or in case of Miravirsen, as an exception. However, their general application is hampered due to their poor pharmacokinetic properties, likely because of their molecular size of >6000 Da and their negatively charged backbone [6]. In addition, anti-sense oligonucleotides have to be applied by injection, thereby making the treatment less convenient. In contrast to that, the success of protein-targeting small molecule drugs relies mostly on their oral administration and their ability to reach in-cell targets by simple diffusion [3]. The question may, therefore, be asked: why can the concept of protein-based drug discovery not be adopted to target miRNAs with small molecules? Small molecule modulators: adopting lessons learned from protein-based drug discovery to miRNAs The use of small molecules in protein drug discovery has been established several years ago and offers the possibility of addressing targets within a cell by simple diffusion. Parameters for designing a molecule to fulfill this challenge have been summarized by Lipinski’s Rule of Five [molecular weight (MW)
