The role of NS5A in HCV replication NS5A is a zinc-binding protein dimer serving as a regulator in the replication cycle of HCV. It contains an amino-terminal amphipathic helix and three different domains with different binding affinities. The N-terminus binds to cellular membranes and is essential for the formation of replication complexes. Domain 1 (amino acid 401-60012) is an RNA binding region of NS5A and domain 2 serves in the RNA replication. Virus assembly is associated with domain 3. NS5A not only binds to cellular membranes, other non-structural proteins of the HCV replication-complex and viral RNA, but also to co-factors like PI4KA. PI4KA is a kinase, which phosphorylates a hydroxyl group of phosphatidylinositol in its inositol-ring at position four. This phosphorylation generates phosphatidylinositol 4-phosphate (PI4P), a docking site normally located at the Golgi membrane. PI4P is necessary for the recruitment of effector proteins participating in transport vesicles of lipid transfer. Resulting from that PI4P is crucial for maintaining the Golgi membrane structure and Golgi-secretion. By binding PI4KA on its domain 1 and accumulating PI4P away from the Golgi apparatus, NS5A induces the formation of a membranous web, a membrane structure arising from the endoplasmic reticulum via recruitment of lipid vesicles. The replication complex of HCV is located on this web-like structure. Out of these membranes and lipid drops, which had been recruited via PI4P, the virions can be assembled after viral-RNA replication. The RNA replication and assembly of virions depend on the ratio between two different forms of NS5A. Depending on the phosphorylation rate of NS5A, it is differentiated between NS5A (p56) and the hyperphosphorylated NS5A (p58). A higher rate of p58 seems to induce HCV assembly, whereas a lack of p58 leads to RNA replication. But the actual role of phosphorylation of NS5A is not clear. However, NS5A does not only regulate HCV replication and assembly, it also inhibits apoptosis, so the infected cell cannot disintegrate by itself and contain the infection, as cells normally do. Because of the lack of apoptosis, mutations within the cellular genome also do not trigger cells death. This causes the promotion of tumours. Mechanism of action NS5A contains a binding pocket in domain 1, where<unk>  can insert. Because of the symmetric and amino acid related structure of<unk> , it comes to H-bond interactions between<unk>  and the amino acids of the binding pocket. That causes a structural modification of NS5A, leading to a loss of action and resulting in an inhibition of virion formation. By altering the structure of NS5A, the recruitment of PI4KA and therefore the accumulation of PI4P get disabled. New lipid-drops and membranous components can no longer get recruited by PI4P in the membranous web and the web can no longer maintain. But it is important to mention that<unk>  does not inhibit PI4KA directly, because it was observed that in cells, which were treated with<unk> , PI4P is still present in the plasma membrane and Golgi apparatus.<unk>  also does not inhibit the initial PI4KA delocalization induced by NS5A because it was witnessed that<unk>  had no impact on the function of NS5A within cells expressing only NS5A. It is likely that<unk>  merely inhibits functions of NS5A associated with other proteins within the HCV replication complex just like the RNA-replicase NS4B. Concluding,<unk>  presumably only inhibits PI4P hyper-accumulation in the context of full HCV polypeptide expression. This phenomenon is associated with the structure of domain 1 of NS5A. It was observed that domain 1 has two different conformations.<unk>  binds to one of these and as a result, locks domain 1 in this conformation. Though, NS5A is still able to bind PI4KA, what can explain the remaining function of NS5A in uninfected cells mentioned before. The second conformation of domain 1, which appears in non-treated cells, is then most likely to be associated with PI4P hyper-accumulation that is necessary for the formation and maintainment of the replication-complex and membranous web. So, PI4KA can still interact with NS5A, but due to the structural blockade, NS5A is not able to activate PI4KA as efficiently as normal. The inhibition of hyper-accumulation of PI4P results in the collapse of the membranous web and the formation of large aggregated structures formed out of the proteins of the replication complex. Moreover, NS5A seems to delocalize from the membranous web to lipid drops within the cytosol. Resulting from this observation, it is likely that the HCV replication-complex and virion hulls still get formed but are highly impaired by the treatment with<unk> . Furthermore, it has been observed, that the treatment of HCV infected cells with<unk>  leads to a lack of the hyper-phosphorylated form of NS5A (p58). It is not clear if the formation of p58 is directly inhibited by<unk>  or if it is just a side effect. But it is likely that the lack of p58 is crucial for the inhibition of virion assembly. Pharmacokinetics<unk>  reaches steady state in human subjects after about 4 days of once-daily 60 mg oral administration, with a peak dose in concentration occurring about 2 hours after administration. It comes in the form of an oral tablet, with a bioavailability of 67%.<unk>  is predominantly metabolized by the liver enzyme CYP3A4, and is also a P-gp substrate. It is highly protein bound. Protein binding was measured to be around 99% in people dosed multiple times with<unk>  independent of dose strength.<unk>  has a volume of distribution of 47L following an oral dose of 60 mg and an IV dose of 100 μg.
Daclatasvir
Balapiravir
Odalasvir
Telaprevir
Remdesivir
Favipiravir
Ledipasvir
Entecavir