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

Abstract

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...

Combination Therapy with Ombitasvir/Paritaprevir/Ritonavir for Dialysis Patients Infected with Hepatitis C Virus: A Prospective Multi-Institutional Study

“…4 [http://www.abbvie.co.jp/content/dam/ abbviecorp/japan/docs/if_Viekirax_j.pdf]). V170I is not known to be associated with resistance to NS3/4A protease inhibitors (Romano et al 2010;Halfon and Locarnini 2011). His comorbidities were diabetes mellitus and hypertension.…”

“…There were no mutations in the NS3 or NS5A regions of the infected HCV/1b, which are known to be associated with resistance to PTV and OBV, respectively (Table 3). Although Q80K, which is known to be an RAV against the NS3/4A protease inhibitor TMC435 (simeprevir) (Romano et al 2010;Halfon and Locarnini 2011), was found, this mutation is unlikely to be associated with resistance to PTV (Pilot-Matias et al 2015; Interview form of VIEKIRAX ® ver. 4 [http://www.abbvie.co.jp/content/dam/ abbviecorp/japan/docs/if_Viekirax_j.pdf]).…”

“…pdf]). V170I is not known to be associated with resistance to NS3/4A protease inhibitors (Romano et al 2010;Halfon and Locarnini 2011). In addition, there were no mutations at aa 155, 156, or 168 in the NS3 region or at aa 28, 31, or 93 in the NS5A region, which are known to be associated with resistance to PTV and OBV, respectively (Krishnan et al 2015;; Interview form of VIEKIRAX ® ver.…”

“…He had not received interferon-based therapy. He had no RAVs known to be associated with resistance to NS3/4A protease inhibitors, including linear ketoamids and macrocyclic compounds (Romano et al 2010;Halfon and Locarnini 2011), or NS5A inhibitors, including DCV (Fridell et al 2011) and OBV (Krishnan et al 2015) (Table 3). He had several comorbidities, such as atrial septal defect, atrial fibrillation, hypertension, upper gastrointestinal disease, and liver cysts with calcification.…”

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

“…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).…”