Avoiding Drug Resistance by Substrate Envelope-Guided Design: Toward Potent and Robust HCV NS3/4A Protease Inhibitors

ABSTRACT Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles. IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

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The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors

PLoS Pathogens ◽

10.1371/journal.ppat.1002832 ◽

2012 ◽

Vol 8 (7) ◽

pp. e1002832 ◽

Cited By ~ 142

Author(s):

Keith P. Romano ◽

Akbar Ali ◽

Cihan Aydin ◽

Djade Soumana ◽

Ayşegül Özen ◽

...

Keyword(s):

Drug Resistance ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitors ◽

Molecular Basis

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Evaluating the Role of Macrocycles in the Susceptibility of Hepatitis C Virus NS3/4A Protease Inhibitors to Drug Resistance

ACS Chemical Biology ◽

10.1021/cb400100g ◽

2013 ◽

Vol 8 (7) ◽

pp. 1469-1478 ◽

Cited By ~ 41

Author(s):

Akbar Ali ◽

Cihan Aydin ◽

Reinhold Gildemeister ◽

Keith P. Romano ◽

Hong Cao ◽

...

Keyword(s):

Drug Resistance ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitors

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Evaluating the Substrate-Envelope Hypothesis: Structural Analysis of Novel HIV-1 Protease Inhibitors Designed To Be Robust against Drug Resistance

Journal of Virology ◽

10.1128/jvi.02531-09 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5368-5378 ◽

Cited By ~ 70

Author(s):

Madhavi N. L. Nalam ◽

Akbar Ali ◽

Michael D. Altman ◽

G. S. Kiran Kumar Reddy ◽

Sripriya Chellappan ◽

...

Keyword(s):

Drug Resistance ◽

Protease Inhibitors ◽

Active Site ◽

Crystallographic Analysis ◽

Van Der Waals Interactions ◽

Drug Resistant ◽

Design Strategies ◽

Resistance Mutations ◽

Hiv 1 ◽

Substrate Envelope

ABSTRACT Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to “lock” into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors.

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Comparative Study of the Genetic Barriers and Pathways towards Resistance of Selective Inhibitors of Hepatitis C Virus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00294-11 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4103-4113 ◽

Cited By ~ 47

Author(s):

Leen Delang ◽

Inge Vliegen ◽

Mathy Froeyen ◽

Johan Neyts

Keyword(s):

Drug Resistance ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Comparative Study ◽

Protease Inhibitors ◽

Selection Process ◽

Drug Resistant ◽

Resistance Mutations ◽

Polymerase Inhibitors ◽

Genetic Barriers

ABSTRACTHepatitis C virus (HCV) inhibitors include direct-acting antivirals (DAAs) such as NS3 serine protease inhibitors, nucleoside and nonnucleoside polymerase inhibitors, and host-targeting antivirals (HTAs) such as cyclophilin inhibitors that have been developed in recent years. Drug-resistant HCV variants have been reported bothin vitroand in the clinical setting for most classes of drugs. We report a comparative study in which the genetic barrier to drug resistance of a representative selection of these inhibitors is evaluated employing a number of resistance selection protocols. The NS3 protease inhibitors VX-950 and BILN 2061, the nucleoside polymerase inhibitor 2′-C-methylcytidine, three nonnucleoside polymerase inhibitors (thiophene carboxylic acid, benzimidazole, and benzothiadiazine), and DEB025 were included. For each drug and passage in the selection process, the phenotype and genotype of the drug-resistant replicon were determined. For a number of molecules (BILN 2061 and nonnucleoside inhibitors), drug-resistant variants were readily selected when wild-type replicon-containing cells were directly cultured in the presence of high concentrations of the inhibitor. Resistance to DEB025 could be selected only following a lengthy stepwise selection procedure. For some DAAs, the signature mutations that emerged under inhibitor pressure differed depending on the selection protocol that was employed. Replication fitness of resistant mutants revealed that the C445F mutation in the RNA-dependent RNA polymerase can restore loss of fitness caused by a number of unfit resistance mutations. These data provide important insights into the various pathways leading to drug resistance and allow a direct comparison of the genetic barriers of various HCV drugs.

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Detection of drug resistance mutations of hepatitis C virus in patients with failure of the treatment with direct acting antivirals

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-47 ◽

2021 ◽

Vol 98 (1) ◽

pp. 18-27

Author(s):

D. E. Valutite ◽

A. V. Semenov ◽

Yu. V. Ostankova ◽

K. V. Kozlov ◽

A. G. Borisov ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Treatment Failure ◽

Chronic Hepatitis C ◽

Direct Acting Antivirals ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Direct Acting

Background. The development of direct acting antivirals (DAAs) has spurred a revolution in treatment of patients with chronic hepatitis C. However, there are cases showing no response to treatment. In 5% of cases, the viral breakthrough is most likely caused by DAA resistance mutations in the hepatitis C virus genome.The purpose of the study is to detect drug resistance mutations of hepatitis C virus in patients with DAA treatment failure.Materials and methods. The study was performed on plasma samples from 3 patients diagnosed with chronic hepatitis C virus infection and demonstrating DAA virological treatment failure. All isolates had genotype 1b. Drug resistance mutations were detected by using direct sequencing of NS3, NS5A, and NS5B genome regions. The detection technique was developed at the Pasteur Research Institute of Epidemiology and Microbiology.Results. Drug resistance mutations were detected in all cases. By using the Geno2pheno [hcv] 0.92 tool, nucleotide substitutions were detected in different viral genome regions and presumably caused resistance or decreased sensitivity to antivirals both present and absent in the sofosbuvir + daclatasvir combination therapy. Antiviral treatment failure in patients with chronic hepatitis C is caused by drug resistance mutations.Conclusions. The developed technique is efficient for detection of drug resistance mutations in NS3, NS5A, and NS5B regions in cases of virological failure of DAA treatment.

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