Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.     

Inhibitors of hepatitis C virus NS3.4A protease 2. Warhead SAR and optimization

The alpha-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3.4A protease inhibitors. Structure-activity relationships and prime side optimization are discussed.     

Inhibitors of hepatitis C virus NS3.4A protease. Part 3: P2 proline variants

We recently described the identification of an optimized alpha-ketoamide warhead for our series of HCV NS3.4A inhibitors. We report herein a series of HCV protease inhibitors incorporating 3-alkyl-substituted prolines in P(2). These compounds show exceptional enzymatic and cellular potency given their relatively small size. The marked enhancement of activity of these 3-substituted proline derivatives relative to previously reported 4-hydroxyproline derivatives constitutes additional evidence for the importance of the S(2) binding pocket as the defining pharmacophore for inhibition of the NS3.4A enzyme.     

Inhibitors of hepatitis C virus NS3.4A protease. Effect of P4 capping groups on inhibitory potency and pharmacokinetics

Reversible tetrapeptide-based compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease. Inhibition of viral replicon RNA production in Huh-7 cells has also been demonstrated. We show herein that the inclusion of hydrogen bond donors on the P4 capping group of tetrapeptide-based inhibitors result in increased binding potency to the NS3.4A protease. The capping groups also impart significant effects on the pharmacokinetic profile of these inhibitors.     

Conformational stability of hepatitis C virus NS3 protease

The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states.     

The hepatitis C virus NS2/3 protease

The hepatitis C virus NS2/3 protein is a highly hydrophobic protease responsible for the cleavage of the viral polypeptide between non-structural proteins NS2 and NS3. However, many aspects of the NS2/3 protease's role in the viral life cycle and mechanism of action remain unknown. Based on the recently elucidated crystal structure of NS2, NS2/3 has been proposed to function as a cysteine protease despite its lack of sequence homology to proteases of known function. In addition, although shown to be required for HCV genome replication and persistent infection in a chimpanzee, the role of NS2/3 cleavage in the viral life cycle has not yet been fully investigated. However, several recent studies are beginning to clarify possible roles of the cleaved NS2 protein in modulation of host cell gene expression and apoptosis.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.     

Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex

The backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex have been determined by NMR spectroscopy. Residues I34 to S181 of NS3 and the central three residues of the NS4A cofactor were assigned and the secondary structure was verified for these residues. In several X-ray structures of NS4A-bound NS3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the A0 strand and alpha0 helix. In solution, these N-terminal residues are largely unassigned and no evidence of a well-structured A0 strand or alpha0 helix was detected. NOEs between residues in the E1-F1 loop (containing D81) and the alpha1 helix (containing H57) together with the detection of a D81-H57 hydrogen bond indicate that in solution the catalytic triad (D81, H57, S139) of the protease is better ordered in the presence of the NS4A cofactor. This is consistent with the earlier crystallographic results and may explain the observed increase in catalytic activity of the enzyme due to NS4A binding. A model-free analysis of our relaxation data indicates substantial exchange rates for residues V51-D81, which comprise the upper part of the N-terminal beta-barrel. A comparison of chemical-shift differences between NS3 protease and the NS3 protease-NS4A complex shows extensive chemical-shift changes for residues V51-D81 indicating that non-local structural changes occur upon NS4A binding to the NS3 protease that are propagated well beyond the protease-cofactor interaction site. This is consistent with crystallographic data that reveal large structural rearrangements of the strand and loop regions formed by residues V51-D81 as a result of NS4A binding. The coincidence of large exchange rates for the NS3 protease-NS4A complex with chemical-shift differences due to NS4A binding suggests that residues V51-D81 of the NS3 protease NS4A complex are in slow exchange with a NS4A-free conformation of NS3 protease.     

Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease

RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.     

A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus

Translating proteases as inactive precursors, or zymogens, protects cells from the potentially lethal action of unregulated proteolytic activity. Here, we impose this strategy on bovine pancreatic ribonuclease (RNase A) by creating a zymogen in which quiescent ribonucleolytic activity is activated by the NS3 protease of the hepatitis C virus. Connecting the N-terminus and C-terminus of RNase A with a 14-residue linker was found to diminish its ribonucleolytic activity by both occluding an RNA substrate and dislocating active-site residues, which are devices used by natural zymogens. After cleavage of the linker by the NS3 protease, the ribonucleolytic activity of the RNase A zymogen increased 105-fold. Both before and after activation, the RNase A zymogen displayed high conformational stability and evasion of the endogenous ribonuclease inhibitor protein of the mammalian cytosol. Thus, the creation of ribonuclease zymogens provides a means to control ribonucleolytic activity and has the potential to provide a new class of antiviral chemotherapeutic agents.     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C^pro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G₅T) as a 3C^pro protease inhibitor. Using ¹H NMR spectroscopy, G₅T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of ¹H, ¹⁵N-HSQC experiments the binding site for G₅T was located to the C-terminal β-barrel of HAV 3C^pro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G₅T-binding site, nor does G₅T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.     

Azapeptides as inhibitors of the hepatitis C virus NS3 serine protease

Truncation and substitution SAR studies of azapeptide-based inhibitors of the Hepatitis C virus (HCV) NS3 serine protease have been performed. These azapeptides were designed from the HCV polyprotein's NS5A-NS5B trans cleavage junction and contained an azaamino acid residue at the P1 position. These azapeptides exhibited predominantly non-acylating, competitive inhibition, contrary to classical azapeptides.     

An RNA ligand inhibits hepatitis C virus NS3 protease and helicase activities

The hepatitis C virus non-structural protein 3 (HCV NS3) possesses both protease and helicase activities that are essential for viral replication. In a previous study, we obtained RNA aptamers that specifically and efficiently inhibited NS3 protease activity (G9 aptamers). In order to add helicase-inhibition capability, we attached (U)14 to the 3'-terminal end of a minimized G9 aptamer, DeltaNEO-III. NEO-III-14U was shown to inhibit the NS3 protease activity more efficiently than the original aptamer and, furthermore, to efficiently inhibit the unwinding reaction by NS3 helicase. In addition, NEO-III-14U has the potential to diminish specific interactions between NS3 and the 3'-UTR of HCV-positive and -negative strands. NEO-III-14U showed effective inhibition against NS3 protease in living cells.     

Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic alpha-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.     

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.     

SAR and pharmacokinetic studies on phenethylamide inhibitors of the hepatitis C virus NS3/NS4A serine protease

SAR on the phenethylamide 1 (Ki 1.2 microM) in the P2- and the P'-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.