Induced‐Fit Binding of the Macrocyclic Noncovalent Inhibitor TMC435 to its HCV NS3/NS4A Protease Target

Cummings, Maxwell D.; Lindberg, Jimmy; Lin, Tse‐I; Kock, Herman de; Lenz, Oliver; Lilja, Elisabet; Felländer, Sara; Baraznenok, Vera; Nyström, Staffan; Nilsson, Magnus; Vrang, Lotta; Edlund, Michael; Rosenquist, Åsa; Samuelsson, Bertil; Raboisson, Pierre; Simmen, Kenneth

doi:10.1002/anie.200906696

Cited by 85 publications

(47 citation statements)

References 30 publications

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“…Overall, many mutations in NS3 previously associated with exposure to NS3/4A protease inhibitors (reviewed in reference 17) were observed during the selection experiments. However, as expected for a macrocyclic NS3 protease inhibitor (7,24) with a large S2 group and consistent with the results from population sequencing, the most pronounced amino acid changes in selection experiments with TMC380765 were detected at NS3 positions 156 (3 different mutations) and 168 (5 different mutations). As determined by 454 deep sequencing in the experiments with stepwise-increasing TMC380765 concentrations, initial low concentrations selected for a mixed pool of replicons carrying the low-level resistance mutations Q41R, F43S, Q80R, and A156G at frequencies between 10 and 25%, which were suppressed with increasing TMC380765 concentrations and gradually replaced by replicons carrying the high-level resistance mutations A156V, D168A, and D168V.…”

Section: Discussionsupporting

confidence: 88%

Tracking the Evolution of MultipleIn VitroHepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Verbinnen¹,

Marck²,

Vandenbroucke³

et al. 2010

J Virol

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Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitroChronic hepatitis C virus (HCV) infection can lead to liver fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Approximately 170 million people worldwide are infected with HCV (54a). The current standard of care consists of pegylated alpha interferon (Peg-IFN) plus ribavirin (RBV), providing limited efficacy for genotype 1-infected patients, i.e., a sustained virological response (SVR) in 40 to 50% of the patients. Moreover, Peg-IFN/RBV therapy is associated with significant adverse events (9). Therefore, direct antiviral agents (DAA) (previously also known as "specifically targeted antiviral therapies for hepatitis C" or STAT-C) have been a major focus of drug discovery efforts over the last 2 decades. Several NS3/4A (protease), NS5A, and NS5B (polymerase) inhibitors either alone or in combination with Peg-IFN/RBV have recently shown potent antiviral effects in HCV-infected patients (22,36). However, viral resistance to these novel agents can occur rapidly when they are dosed as monotherapy (43,49).Because of the high mutation rate of the HCV polymerase (10 Ϫ3 to 10 Ϫ5 misincorporations per nucleotide copied [11]) and the high viral production rates in vivo (approximately 10 12 viruses per patient per day [37]), it can be assumed that HCV exists as a diverse population of nonidentical but closely related viral genomes, referred to as a quasispecies (10). A viral quasispecies is characterized by a dominant nucleotide sequence, called a master sequence, and a surrounding mutant spectrum, which can harbor minority subpopulations (42). Although in theory all single and double mutants are produced daily in an infected person (6, 40), it is important to note that mutation rates are not equally distributed over the entire genome and that additional factors, such as viral fitness and the replication environment, determine whether a mutation becomes fixed in a viral quasispecies population (12). The diversity of the viral variants present in an infected individual facilitates the adaptation of the quasispecies to external pressure, such as antiviral treatment, improving the survival chances of the population (53). The speed of such adaptation depends mainly on the turnover of the viral nucleic acid acting as a source of new viral genomes. Whereas in HIV the rapid turnover of infected CD4 ϩ T lymphocytes is responsible for the rapid turnover of nucleic acids, in HCV rapid turnover is explained by the short half-life (ϳ10 h) of HCV RNA strands in the hepatocyte (47). However, if mutation rates exceed a certain limit, called the error threshold, deleterious mutations will accumulate and the viral population will become extinct (4).Recent reports have demonstrated that mutations known to affect the activities of DAA compounds in vitro are present in some treatment-naive patients as either dominant or minority species

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Section: Discussionsupporting

confidence: 88%

Tracking the Evolution of MultipleIn VitroHepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Verbinnen¹,

Marck²,

Vandenbroucke³

et al. 2010

J Virol

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“…Finally, the summation of these four consensus maps was generated to construct the substrate envelope, depicting the van der Waals volume shared by any three of the four products. The previously determined boceprevir complex (PDB code 2OC8) (24) and TMC435 complex (PDB code 3KEE) (23) were used in this study (66 …”

Section: Methodsmentioning

confidence: 99%

“…Four crystal structures of the NS3/4A protease domain in complex with the N-terminal products of viral substrates reveal a conserved mode of substrate binding, with the consensus volume defining the substrate envelope. The protease inhibitors ITMN-191 (3M5L), TMC435 (3KEE) (23), and boceprevir (2OC8) (24) protrude extensively from the substrate envelope in regions that correlate with known sites of resistance mutations. Most notably, the P2 moieties of all three drugs protrude to contact A156 and R155, which mutate to confer high-level resistance against nearly all drugs reported in the literature (25)(26)(27)(28)(29)(30).…”

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Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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“…Currently approximately 50 HCV NS3/4A protease structures are available in the Protein Data Bank (PDB). In most cases the protease has been complexed with inhibitors, including two macrocyclic acylsulfonamides (11,12). The NS3/4A full-length protease-helicase apo crystal structure was reported in 1999 (13) and recently several complexes with helicase ligands have been published (14).…”

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confidence: 99%

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Schiering

D’Arcy

Villard

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed proteasehelicase domain assembly in solution.structure-based drug design | X-ray structure | solution scattering | medicinal chemistry C hronic hepatitis C virus (HCV) infection affects more than 3% of the world's population and is a leading cause of chronic liver diseases (1). Therapeutic options have been suboptimal, especially for HCV genotype 1, the most prevalent genotype in developed countries. Recently, the addition of direct-acting antiviral agents (DAAs) to the previous standard of care (combination therapy with pegylated interferon and ribavirin) have demonstrated considerable improvement in sustained virological response rates in patients infected with HCV genotype 1 (2). With the first DAAs now having been introduced into clinical practice, it is to be expected that in the near future standard therapy will change to a triple therapy including an HCV NS3/4A protease inhibitor in combination with pegylated interferon and ribavirin.The positive-strand RNA genome of HCV encodes a polyprotein precursor, which is proteolytically processed by host and viral proteases into 10 individual structural and nonstructural (NS) proteins. The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3, 4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairin...

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Induced‐Fit Binding of the Macrocyclic Noncovalent Inhibitor TMC435 to its HCV NS3/NS4A Protease Target

Cited by 85 publications

References 30 publications

Tracking the Evolution of MultipleIn VitroHepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Tracking the Evolution of MultipleIn VitroHepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

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