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 daclatasvir. Furthermore, it has been observed, that the treatment of HCV infected cells with daclatasvir 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 daclatasvir 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 Daclatasvir 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%. Daclatasvir 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 daclatasvir independent of dose strength. Daclatasvir has a volume of distribution of 47L following an oral dose of 60 mg and an IV dose of 100 μg. History Daklinza was discovered by scientists at Bristol-Myers Squibb (BMS); the precursor was identified using phenotypic screening in which the GT-1b replicon system was implemented in Huh7 cells and bovine viral diarrhea virus also in Huh7 cells was used as a counterscreen for specificity. Bristol Myers Squibb also developed the drug, with the first Phase I trial publishing in 2010. It was approved for use in Europe in August 2014, in the US in July 2015, and in India in December 2015; it was first in the class of NS5A inhibitors to reach the market. Synthesis The daclatasvir molecule contains the two amino acids proline and valine in their natural L-form which makes it easier to synthesize in the right stereo configuration. The following describes one exemplary mechanism of synthesizing daclatasvir as its free base. The first step is a Friedel-Crafts acylation of biphenyl with chloroacetyl chloride and aluminium