Bifunctional molecules – A possible strategy for treating Hepatitis C viral infection

Tavis Chen


Hepatitis C virus (HCV) treatment has improved over the past few years due to the development of antiviral drugs. Despite these advances, treatment remains generally expensive. One newly explored strategy to treat HCV infection is to neutralize a microRNA (miRNA) known as miRNA-122. miRNA-122 is involved in HCV development and neutralization of this miRNA has been shown to decrease HCV production in the liver. This review summarizes the current treatments for HCV and proposes a novel strategy to treat HCV by preventing miRNA-122 from being formed. To do this, a bifunctional molecule can be used to specifically inhibit the miRNA-forming enzyme Dicer from producing miRNA-122. Although popular for other applications, bifunctional molecules have not been widely applied to miRNAs. This bifunctional molecule can be synthesized to observe its effects in vitro using previously reported strategies to validate its usefulness as a new way of treating HCV.


MicroRNAs (miRNAs) are short non-coding RNAs that fulfill a crucial role in regulating gene expression.[1,2] Generally, protein synthesis in cells begins when DNA is transcribed by an RNA polymerase into RNA. From then, the RNA is translated into amino acids and eventually proteins. miRNAs mainly function in RNA silencing, a process by which they limit an RNA from being translated into a protein. miRNAs carry out gene silencing through a relatively simple mechanism. miRNAs have a complementary base pairing with the messenger RNA (mRNA) molecules that code for proteins.[3] The mRNA is silenced after base pairing with the miRNA. The RNA-induced silencing complex (RISC) is a group of proteins that mediates RNA silencing by cleaving the strand of mRNA into two pieces. This means that the protein that is made from that mRNA is not going to be made. Argonaute is a protein in RISC that degrades mRNA by cutting up the backbone of RNA. As a result, the mRNA is lost and no protein is made.[4, 5]

miRNAs are formed from precursor miRNAs (pre-miRNA). Pre-miRNAs are longer than miRNAs and contain an RNA hairpin, which is a pattern formed by complementary base pairs forming a double helix that ends with a loop.[1] One side of this hairpin is the mature miRNA. The pre-miRNA hairpin is cleaved by an enzyme called Dicer to form a mature miRNA. Dicer is a ribonuclease (RNase) III endonuclease, which functions to degrade RNA into smaller pieces. Without the activity of Dicer protein, miRNAs are not formed and the RISC is not activated.

miRNAs themselves are essential for human health, and regulate over 60% of protein-coding genes.[6] There are over 2000 discovered miRNAs in the human body. For example, one miRNA important for human health is miR-206.[7] miR-206 is prevalent in vertebrae skeletal muscle and fast-twitch muscles. Studies show that miR-206 is first detected in a nine-day-old embryo which began to increase three days after.[8] This pattern indicates that miR-206 also plays a role in the formation of muscle tissue (myogenesis). The study of miR-206 is essential for researching the treatment of skeletal muscle-related disorders.

While miRNA are crucial for regulating biological processes, they can also prove detrimental as some miRNA are linked to disease.[9] For example, one miRNA highly expressed in the liver, miRNA-122, has key involvement in the progression of Hepatitis C virus (HCV) in liver disease. miR-122 binds within HCV genomic RNA which increases the translation of HCV proteins and therefore increases viral replication.[10] This is in contrast to the normal role that miRNAs play in turning off gene expression. miR-122 can recognize two sites at the 5’ prime end of the HCV genomic RNA. miR-122 stimulates HCV replication by increasing protein expression.

The study of miRNA and different miRNA targeting technologies has yielded opportunities for treating these diseases.[11-13] One method of targeting miRNAs is through antagomirs. An antagomir can be designed to have a sequence complementary to a target miRNA so that it can bind to it and block it from binding to mRNA at the same site, preventing the degradation of the mRNA.[14] One study successfully showed that an antagomir of miR-122, which is involved in HCV infection, significantly reduced levels of HCV RNA in CNS3 and Huh 9-13 cells, which are hepatocytes that host HCV replication.[15-16] In these studies, the miRNA antagomir reduced the abundance of HCV core protein by 63% in CNS3 cells and by 84% in 9-13 replicon cells. This is expected to slow down HCV replication and alleviate its gastrointestinal effects.

Another possible method to stop a miRNA from being formed is inhibition of the Dicer enzyme. As established, Dicer is essential for the formation of miRNAs. In order to inhibit the formation of these miRNAs, a Dicer inhibitor could be used to inhibit the Dicer enzyme. This is challenging to implement, though, because Dicer inhibitors will inhibit the production of all miRNAs, whether they are beneficial or involved in the disease. Drugs that selectively inhibit the formation of only disease-associated miRNAs could be very powerful.


Hepatitis C viral infection results in long-term health issues including liver damage, liver cancer, cirrhosis, and even death.[21] According to the World Health Organization, an estimated 71 million people have chronic hepatitis C virus infection worldwide. In 2016, almost 400,000 people died from hepatitis C.[22] Previous treatment of chronic HCV was a combination of antiviral drugs known as pegylated interferon (PEG-INF) and ribavirin (RBV); however, this treatment only worked for less than half of individuals with genome 1 (75% of Americans with HCV have genome 1).[23] The introduction of direct-acting antivirals (DAAs) introduces new treatment options. DAAs are molecules that target specific steps within HCV’s life cycle, inhibiting viral replication.[24] DAAs have led to the development of protease inhibitors which are effective at treating all genomes of HCV. The price of antiviral drugs, such as Harvoni, for a full 12-week treatment, can cost up to $94,000. 

miRNA targeting drugs have a significant clinical application and can lead the way in new therapeutics as well as improving vaccines. The most relevant miRNA targeting drugs are antagomirs, such as antisense blockers known as blockmirs developed by Mirrx Therapeutics. Miravirsen is an antisense oligonucleotide to human miR-122 that has been in Phase II clinical trials since 2017.[25]

Bifunctional molecules have already revolutionized some types of disease treatment. PROTACS such as ARV-110 and ARV-471 developed by Arvinas have been proven to be successful. ARV-110, which is currently in Phase 2 clinical trials, has shown efficacy in protein degradation in patients with metastatic castration-resistant prostate cancer, and ARV-471 is a Phase 1 estrogen receptor targeting protein degrader for breast cancer.[26]

Research Aim 1: Demonstrate that the bifunctional molecule selectively inhibits Dicer-mediated miRNA production in vitro 

The first aim is to demonstrate that a bifunctional molecule can specifically inhibit the formation of miR-122. The molecules proposed can be designed to have two ends. The first is a chemical that inhibits Dicer, which is the enzyme that generates miRNA. For the Dicer inhibitor, a previously reported endonuclease inhibitor will be used.

The second part of the molecule is a sequence that is complementary to the target pre-miRNA. Complementarity is a concept that is particularly important for the assembly of DNA into a double helix. In double-stranded DNA and RNA, adenine is always present across from thymine; guanine is always present across from cytosine. This programmed assembly can be leveraged to synthesize a strand that will bind to the RNA in interest. The sequence can be identified by first identifying the sequence of the pre-miRNA target. In addition, the targeting motif could be synthesized using either phosphorothioate (PS) or phosphodiester (PO) linkers. PS linkers have one sulfur atom replacing one of the oxygen atoms which increases the targeting motif’s stability in cells.[27]

In addition to the two ends of the molecule, the chemical structure of the linker bringing them together will be varied. A simple CH2 linker was found to be more efficient than longer linkers across multiple concentrations for a similar molecule.[9]

This molecule can be expected to effectively reduce levels of miRNA-122 within liver cells and increase the pre-miRNA-122 levels. Two experiments can be conducted to confirm that the bifunctional molecule inhibits the formation of miR-122 in vitro. The first experiment will determine if the domains of the bifunctional molecule function as expected. Radioactively labeled pre-miRNA, Dicer, and the bifunctional molecule can be mixed together. The mixture will be incubated for one and a half hours. Over the course of the experiment, Electrophoresis analysis can be used to determine if the bifunctional molecule inhibited the Dicer-mediated formation of miRNA-122. To do this, a technique called electrophoresis can be used. In electrophoresis analysis, an electric charge is applied to a sample of particles. Because nucleic acid particles already have an electric charge, an electric force will act upon them. Molecules with greater charge, will enact a strong force that moves the molecule further in a gel. This is a method to separate nucleic acids by size. A successful readout would show that there is only a pre-miRNA present in the electrophoresis analysis. An unsuccessful readout would be only miRNA-122 in the analysis which indicates that Dicer was not inhibited. Running the first experiment without the Dicer enzyme is expected to demonstrate no change to the pre-miRNA. Adding Dicer and pre-miRNA should yield the only miRNA. The bifunctional molecules with shorter linkers at concentrations higher than 1 micromolar are predicted to yield the best results. These experiments will allow us to gauge how effective our molecule is.

The next experiment the specificity of my bifunctional molecule will be determined. Previous studies only confirmed that the molecule worked, but did not test for specificity. This would be an advancement to demonstrate utility. The same experiment can be run but with different pre-miRNA. This can be achieved by either increasingly altering the sequence pairing, randomizing the sequence, or using ten other predominant pre-miRNA in the liver. The molecule will specifically target pre-miRNA-122 because of the complementary base pairing on our Dicer inhibitor domain that should match pre-miRNA-122.

In order to show that this result is dependent on the presence of the bifunctional molecule, one trial can be held with just the pre-miRNA and the next can be run with both pre-miRNA and Dicer while subsequent trials will include pre-miRNA, Dicer, and the bifunctional molecule. This could also be done using an electrophoresis assay. All the pre-miRNA and miRNA could be mixed or tested separately to determine which are affected by our molecule and which are not. If the result is indeed dependent on the presence of the bifunctional molecule, the pre-miRNA would be expected to be able to produce miRNA with Dicer. There should be no miRNA when there is pre-miRNA, Dicer, and our molecule.

Figure 2: Expected results of experiment outlined in Aim 1.

Research Aim 2: Demonstrating that the bifunctional molecule reduces HCV production in vitro

HCV reproduces by entering cells and hijacking cellular machinery to make its own proteins. Therefore, the first step in the production of more HCV is the production of these proteins in cells. In order to demonstrate that our molecule will reduce the production of HCV, the molecule can be injected into cells that are engineered to make HCV core proteins. This will allow us to see the effects of our molecule on the synthesis of HCV core proteins.

In previous experiments, researchers injected an antagomir of miR-122 into liver cells (Huh-7) which were engineered to produce the HCV subgenomic protein core. The cells contained a portion of the HCV genome in a replicon, which is a nucleic acid that can replicate itself. The researchers used qRT-PCR (quantitative Reverse Transcriptase-Polymerase Chain Reaction) analysis to demonstrate the effectiveness of the miR-122 antagomir at varying concentrations for 72 hours. qRT-PCR is a method used to measure the amount of a specific RNA in cells. High levels of RNA are correlated with high levels of protein production. The method works by first using reverse transcriptase to convert the RNA into DNA. The time it takes to complete the PCR amplification can be used to calculate the initial amount of RNA in the cells. PCRs, which stands for a polymerase chain reaction, is a method of replicating millions of copies of DNA by heating it in cycles to produce more. The fold change, the degree of quantity change between an initial and final value, in the PCR were calculated to compare the two RNA samples. The fold change values were for GAPDH mRNA, which is used to normalize qPCR data. Researchers have used qRT-PCR to measure the levels of RNA corresponding to two different HCV proteins: HCV core and NS5A. NS5A is a protein with a dimeric structure that is crucial in HCV replication.[28] Experiments’ results showed that the abundance of HCV RNA decreased by 64% in CNS3 and 84% in 9-13 cells due to the antagomir.

In addition to measuring HCV mRNA levels, western blot analysis was used to measure how much the antagomir reduced HCV protein levels.[29] Western blots were used to measure levels of both HCV and GAPDH proteins. Proteins were separated based on size using a polyacrylamide gradient gel. The probes used for this blot were anti-HCV core protein and GAPDH specific antibodies.

Both of these experiments can be used with our bifunctional molecule instead of an antagomir. Both analyses would consist of the bifunctional molecule with time and increasing concentrations as a control. Furthermore, just the inhibitor and complementary base pairing nucleotide sequence would be analyzed. The treatment with the bifunctional molecule is expected to be more effective than treatment with the antagomir. Because previous experiments demonstrate that increasing concentrations and time of antagomir substantially decreased HCV core miRNA and NS5A, increased dosage and time of the bifunctional molecule is expected to yield the same result.


Although antagomirs are a workable option to treat HCV, it only silences the miRNAs that have already been produced. An alternative approach is using bifunctional small molecules, which will prevent the cleaving of pre-miRNA to miRNA.[17-19] The bifunctional molecule contains a pre-miRNA binding unit and a Dicer inhibiting unit. The binding unit recognizes the target pre-miRNA and brings the Dicer inhibiting unit into the proximity of the pre-miRNA cleavage site, thus preventing the biogenesis of the target miRNA.[9] This causes an accumulation of pre-miRNA. These molecules can also act catalytically. While it may take multiple treatments to inhibit multiple miRNAs, bifunctional molecules can inhibit multiple miRNAs at once.

Bifunctional small molecule technology can be used to inhibit miRNA-122 biosynthesis as a treatment for HCV. For treatment, this molecule could be directed to hepatocytes in the liver by utilizing trivalent N-acetylgalactosamine (GalNAc) to be recognized by the asialoglycoprotein receptor (ASGPR) for uptake.[20] ASGPR, which is known to have a high expression on hepatocytes, is a carbohydrate-binding protein that is highly specific to sugar molecules, that has a high affinity to GalNAc.

Figure 1: Schematic of the proposed bifunctional molecule which inhibits miR-122 synthesis.


This bifunctional molecule would be impactful for the treatment of HCV as it introduces a novel and robust strategy. This molecule may revolutionize biotechnology and medicine as the applications of it are vast in proteins, mRNA illnesses, and much more. The prospect of using bifunctional molecules to target miRNAs is even more new and has yet to be explored. To add on, bifunctional molecule technology itself is still relatively new and has yet to be applied to more viruses and disease. Thus, some drawbacks would include the limited scope of direction in terms of synthesizing these molecules as well as determining their effects. To improve on these drawbacks, future research could explore the effects of bifunctional molecules on a wide range of mRNA related illnesses by experimenting with different cells and mRNA. Moreover, the extent of the molecule’s applications with proteins should be further explored as well. This development will open up new possibilities in bifunctional molecule treatment for a variety of miRNA related illnesses.


About 71 million people worldwide are infected with chronic HCV. Although HCV treatment has advanced significantly over the past decade, new strategies to treat infection may help advance human health. A bifunctional molecule that utilizes an ASO and Dicer enzyme inhibitor to inhibit the production of miRNA-122 can be synthesized. Shorter linkers and higher concentrations of the molecule would demonstrate higher efficacy than other variations. The molecule should be specific to miRNA-122 and should result in a decrease in HCV core proteins. Bifunctional molecules that can bring together two biological agents to cause an effect have revolutionized several areas of disease treatment. The application of bifunctional molecules to HCV treatment could be used to inhibit the production of miRNA-122 and the replication of HCV.



I would like to express my deepest gratitude to David Caianiello for his valuable support and guidance in this project.


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About the Author

Tavis is a current high school senior at San Marino High School and will be attending the College of Chemistry at UC Berkeley in the fall. He was born and raised in the San Gabriel Valley, a suburb east of Los Angeles city. Tavis has attended multiple chemistry related programs hosted by UCLA and Caltech. His latest venture in chemistry is with a mentorship program run by Polygence, where he researched biochemistry and hepatitis C virus.

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