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. 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. 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. 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 𝛾-phosphate of nucleotide triphosphates which are added to the RNA chain in the ongoing RNA replication. The pyrophosphates are a necessary component of the nucleotide incorporation in the RNA replication. Pyrophosphate inhibitors bind to the active site of the polymerase in competition both with the nucleotides and pyrophosphate, which results in a nucleophilic attack on the terminal phosphodiester bond of the newly released pyrophosphate and thereby stops the RNA replication. Drug discovery and development Discovery The NS5B crystal structure was first reported in literature by three different research groups in 1999. After that, the NS5B RNA dependent RNA polymerase was pursued as a target for the development of treatments for hepatitis C. With crystallized X-ray structures, high-throughput screening, enzyme and cell based replicon assays, and animal models for screening, many potential drug candidates were discovered but few have been approved. Development Many structurally different compounds inhibit NS5B polymerase at different binding sites, but relatively few candidates meet the requirements for selectivity, acceptable pK and toxicity profiles. Direct-acting antivirals were an important advancement in the treatment of chronic hepatitis C. In December 2013, sofosbuvir was approved by the FDA and in October 2014, ledipasvir and sofosbuvir were approved in a combination tablet. The progress in the development of direct-acting antivirals has been very rapid and there