NS5B inhibitor
Non-structural protein 5B (NS5B) inhibitors are a class of direct-acting antivirals widely used in the treatment of chronic hepatitis C.[1] Depending on site of action and chemical composition, NS5B inhibitors may be categorized into three classes—nucleoside active site inhibitors (NIs), non-nucleoside allosteric inhibitors, and pyrophosphate analogues.[2] Subsequently, all three classes are then subclassified.[3] All inhibit RNA synthesis by NS5B but at different stages/sites resulting in inability of viral RNA replication.[2] Expression of direct-acting NS5B inhibitors does not takes place cells that are not infected by hepatitis C virus, which seems to be beneficial for this class of drugs.[3]
Low efficacy, serious side effects, development of resistance of previously available hepatitis C treatments were the greatest concerns prior to the development of direct-acting antivirals, and remained a problem at the beginning of their development. Therefore, combinational direct-acting antiviral therapies were preferable.[4][5] Research has demonstrated that specific anti-hepatitis C virus agents such as NS5B inhibitors lead to improved efficacy and tolerability.[5]
Medical uses[]
Hepatitis C is one of the most significant diseases to affect humans but despite its global impact, there are no vaccines or effective therapies without major side effects. The hepatitis C virus non-structural protein 5B (NS5B) has become a target of choice for the development of anti-hepatitis C virus drugs, as it is not expressed in cells that are not infected by hepatitis C virus.[6] The treatment for patients with chronic hepatitis C that was used prior to the development of direct-acting antivirals was ribavirin plus pegylated interferon alfa-2a. This treatment was dissatisfactory in many cases, both for the lack of efficacy and because of side effects.[3]
Sofosbuvir, a highly potent inhibitor of the NS5B polymerase, is active across all genotype of hepatitis C virus. It has high efficacy in combination with several other drugs, like ledipasvir, either with or without peginterferon-alfa. It has many advantages due to its tolerability, high potency, high resistance barrier, low side effects and oral administration.[7] Another advantage of sofosbuvir is the low rates of interactions with other drugs, as its metabolism does not go through the CYP3A4 pathway.[8] However, its cost can be a challenge, as all the new direct-acting antiviral drugs can be very expensive.
Adverse effects[]
Comparing the virological response of NS5B inhibitors to previous therapy of chronic hepatitis C with the combination of interferon and ribavirin, it was possible to observe a significant difference in efficacy and safety. The efficacy rate of interferon plus ribavirin varied within 44–46% while it increases up to 90% by direct-acting antivirals such as NS5B inhibitors/[7][9] Data on the frequency of side effects may vary between researches for both drug categories. Nevertheless, there is a significant difference between the safety profiles of NS5B inhibitors, particularly sofosbuvir and the combination of interferon and ribavirin.[7][8] Comparing interferon containing therapies, sofosbuvir does not cause neutropenia, thrombocytopenia, or any other serious adverse effects. Common side effects associated with sofosbuvir are headache, tiredness, insomnia, dizziness, itching, infections of upper respiratory tract, rashes, back pain, anemia, and lymphopenia.[7] With the advent of direct-acting antivirals, the duration of the hepatitis treatment has also been impressively reduced. Severity of adverse effects is strongly associated with duration of therapy and the dose used for hepatitis C treatment. Therefore, direct-acting antivirals have become the initial treatment used for most patients with chronic hepatitis C.
Hepatitis C virus[]
Hepatitis C virus belongs to the family Flaviviridae and genus hepavirus. It is a small enveloped, single stranded RNA virus with positive polarity. A lipid bilayer which contains two viral glycoproteins, E1 and E2, forms the envelope and virion.[10]
Hepatitis C virus is transmitted through exposure to infected blood or body fluids, and the most common route is through injection by infected needles.[11] In the beginning of the hepatitis C virus life cycle, virions bind to specific receptors on hepatocytes. The virus is internalized and the nucleocapsid is released into the cytoplasm of the hepatocyte after binding with the receptor. The NS5B protein, which is an RNA-dependent RNA polymerase, catalyzes hepatitis C virus replication.[10]
The hepatitis C virus can cause acute infection but most patients are asymptomatic upon exposure. About one-third of patients develop symptoms such as fatigue, arthralgia and jaundice. About 15–45% of patients diagnosed with acute infection may clear the virus without treatment.[11] After acute infection, most infected patients will develop chronic infection which increases the risk for later developing life-threatening conditions, like liver cirrhosis and hepatocellular carcinoma.[10]
In 2015, about 71 million individuals throughout the world were living with chronic hepatitis C and there were 1.75 million new hepatitis C virus infections that same year. With highly effective direct-acting antivirals, hepatitis C can be cured within 2 to 3 months.[12]
NS5B receptor[]
NS5B is a viral protein that can be found in the hepatitis C virus and is an important enzyme in the replication process.[13] The protein is an RNA-dependent RNA polymerase, which means that it uses single-stranded RNA (ssRNA) as a template to form double-stranded RNA (dsRNA).[14] It contains 591 amino acids with the last 21 amino acids at the C-terminal end functioning as a cell membrane anchor that is hydrophobic in nature. NS5B is therefore categorized as a membrane protein, termed as a "tail-anchored protein".[6]
The structure of NS5B is similar to other viral polynucleotide polymerases and is made up of three subdomains. The structure resembles a right hand with a palm domain that acts as the foundation for the active site, a finger domain, and a thumb domain. The thumb and finger domains encircle the active site of the enzyme.[6]
The finger domain has two loops, named Λ1 and Λ2, that reach over and interact with the top of the thumb domain to enclose the active site. These two loops promote the NS5B to maintain its closed conformation that is necessary for nucleic acid binding and for cramping movement of the enzyme across the RNA template elongation process. NS5B also has allosteric sites in addition to the active site. Palm I, domain close to the active site, palm II, partly overlapping palm I and towards the active site, thumb I, thumb domain near the fingertips and thumb II, the outer surface of the thumb domain.[6]
Mechanism of action[]
There are three known classes of NS5B inhibitors: non-nucleoside analogue inhibitors, nucleoside/nucleotide analogue inhibitors, and pyrophosphate analogues.[2] The classes differ in their structure and where they bind to the NS5B protein: at allosteric binding sites, the enzyme active site, or the pyrophosphate binding site, respectively.[13]
Nucleoside analogue inhibitors[]
Nucleoside analogue inhibitors terminate the RNA synthesis essential for RNA replication. They do so by being incorporated by the RNA-dependent RNA polymerase, which prevents incoming nucleotides from being added to the RNA chain. It has been proposed that steric hindrance by the nucleoside inhibitors, which contain a 3'-hydroxyl group, is the reason for the chain termination. Because of this mechanism, nucleoside inhibitors are sometimes called chain terminating inhibitors. Nucleoside inhibitors of hepatitis C virus are developed as prodrugs and eventually get cleaved at their site of action in the liver by hepatic enzymes, and go through phosphorylation into a triphosphate form to become active to target the polymerase at its highly conserved active site.[15]
Non-nucleoside analogue inhibitors[]
Non-nucleoside analogue inhibitors are a diverse class of NS5B inhibitors. They can be arranged into three categories based on their binding sites: active site inhibitors, allosteric site inhibitors and miscellaneous site inhibitors. The miscellaneous site inhibitors are those inhibitors do not yet have a defined binding site.[3] As of 2015, five different allosteric binding sites have been discovered for the non-nucleoside analogue inhibitors of hepatitis C virus. Two of them are located at the thumb domain of the polymerase and the other three in the palm domain. The non-nucleoside analogue inhibitors inhibit the polymerase activity in different ways, which depend on the allosteric site to which the inhibitor binds.[15][16] In contrast to the nucleoside inhibitors, non-nucleoside analogue inhibitors do not compete with nucleotides nor the RNA template. Instead, by binding to RNA-dependent RNA polymerase, they inhibit polymerase activity by inhibiting conformational changes which are necessary for NS5B activity.[8][15][17]
Pyrophosphate analogue inhibitors[]
Pyrophosphate analogue inhibitors mimic the inorganic pyrophosphate. During the nucleotidyl-transfer reaction, an enzymatic reaction fundamental for DNA synthesis, pyrophosphate is released, which binds to certain binding sites at the NS5B polymerase. This binding site is also the binding site for β- and