the bicyclic proline derivative 18 (K, = 0.042 |xM, IC50 = 0.251 |iM) (38-46). These inhibitors are constructed around either an a-keto amide or a boronic acid moiety, well-precedented as serine protease inhibitor motifs that engage the catalytic serine residue in a covalent but reversible interaction.
However, chemical reactivity is not a prerequisite for potent inhibition in a peptidic background since phenethylamide 19 and the azapeptide 20 inhibit HCV NS3 with Ki values of 0.6 and 0.2 |iM, respectively (47,48). The more active diastereomer of the a-hydroxy amide P3 element explored in the context of 21 was found to possess the (/?)-configuration, unanticipated and explained by the presence of an intramolecular hydrogen bond that orients the lipophilic moiety of this isomer into S3, as depicted (49). Non-peptidic inhibitors of NS3/NS4A protease are much less common but some progress has been made in this direction. The bicyclic lactam 22 is a mechanism-based inhibitor of HCV NS3 whilst additional examples of bis-benzimidazole derivatives that rely upon Zn2+ to consolidate the enzyme-inhibitor complex have been described (50,51).
The NS5B RNA polymerase is another structurally-characterized viral protein that is an attractive target for therapeutic intervention (52-55). Inhibitors of HCV polymerase can be broadly divided into nucleoside and non-nucleoside derivatives. The nucleoside analog ribavirin (H) has been suggested to interfere with both the initiation and elongation steps of HCV RNA replication (52). Several HCV NS5b inhibitors incorporate modified D-ribose elements and include the 2'-Me derivative 23, EC50 = 0.25 |iM, the 4'-azido analog 24, EC50 = 1.2 |iM, and the 2'-deoxy-2'-fluoro cytidine derivative 25, EC50 = 0.74 ^M (56-59).
Several non-nucleoside inhibitors of HCV NS5B have been reported, including a series of phenylalanine derivatives of which compound 26, Ki = 2.2 ^M, is representative (60,61). This compound has been co-crystallized with NS5B and appears to bind to the inactive, open conformation of the polymerase almost 35 A from the active site (62). Other scaffolds with which HCV polymerase inhibitors have been discovered include an amino thiophene, represented by 27, the enolic rhodanine 28 (IC50 - 1.0 |iM), and structural variations of previously disclosed benzimidazole derivatives 29 (IC50 < 0.5 |iM) claimed to be active in replicons (61,63-67). Mechanistic studies with the benzo[1,2,4]thiadiazine polymerase inhibitor 30 suggest interference with the initiation step of viral RNA synthesis, allowing for a potential synergy with existing elongation inhibitors (68).
Novel approaches to HCV therapy include blocking the viral RNA internal ribosomal entry site (IRES), binding of the viral E2 envelope glycoprotein or attachement (69-72). The highly conserved IRES has been targeted by oligonucleotides and artificial ribozymes, but little progress has been made in the development of small molecule inhibitors (70,71). P2 clinical evaluation of the antisense 20-mer oligonucleotide ISIS-14803 revealed a 1-2 log™ reduction in plasma HCV RNA levels in approximately 30% of the patients after 4 weeks of treatment (73). Another approach, which may prove complementary to virus-specific HCV therapy, is the induction of interferon production in host cells. Small molecules that act via toll-like receptor activation have been identified as activators of an immune response (74). RNA interference (RNAi) is a rapidly emerging technology that has proven to be a powerful means of selectively controlling protein production in cell culture. Inhibition of HCV replication in replicons has been accomplished using this procedure whilst the demonstration of selective targeting of the liver protein Fas in vivo using RNAi holds promise for the treatment of HCV (75-78).
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