Combatting Drug Resistance: Lessons from the viral proteases of HIV and HCV

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target's function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. "Resilient" targets are less susceptible to drug resistance due to their key location in a particular pathway. This rationale was derived through crystallographic studies elucidating substrate recognition and drug resistance in HIV-1 protease and Hepatitis C (HCV) NS3/4A protease. Both are key therapeutic targets and are potentially "resilient" targets where resistant mutations occur outside of the substrate binding site. To reduce the probability of drug resistance inhibitors should be designed to fit within what we define as the "substrate envelope". These principals are likely more generally applicable to other quickly evolving diseases where drug resistance is quickly evolving. http://www.umassmed.edu/schifferlab/index.aspx

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Substrate Envelope-Designed Potent HIV-1 Protease Inhibitors to Avoid Drug Resistance

Chemistry & Biology ◽

10.1016/j.chembiol.2013.07.014 ◽

2013 ◽

Vol 20 (9) ◽

pp. 1116-1124 ◽

Cited By ~ 36

Author(s):

Madhavi N.L. Nalam ◽

Akbar Ali ◽

G.S. Kiran Kumar Reddy ◽

Hong Cao ◽

Saima G. Anjum ◽

...

Keyword(s):

Drug Resistance ◽

Protease Inhibitors ◽

Hiv 1 ◽

Substrate Envelope

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In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile

Biological Chemistry ◽

10.1515/bc.2011.076 ◽

2011 ◽

Vol 392 (10) ◽

pp. 927-935 ◽

Cited By ~ 3

Author(s):

Morgan M. Martin ◽

Stephanie A. Condotta ◽

Jeremy Fenn ◽

Andrea D. Olmstead ◽

François Jean

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Biochemical Study ◽

Substrate Recognition ◽

Host Cells ◽

Protein Substrates ◽

Viral Proteases ◽

Endoproteolytic Cleavage ◽

Infected Cells ◽

Membrane Anchoring

AbstractThe need to identify anti-Flaviviridaeagents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1006370107 ◽

2010 ◽

Vol 107 (49) ◽

pp. 20986-20991 ◽

Cited By ~ 129

Author(s):

K. P. Romano ◽

A. Ali ◽

W. E. Royer ◽

C. A. Schiffer

Keyword(s):

Drug Resistance ◽

Substrate Recognition ◽

Inhibitor Binding

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A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation, and Subsite−Pocket Interactions Analyzed at Atomic Resolution

Journal of Medicinal Chemistry ◽

10.1021/jm031105q ◽

2004 ◽

Vol 47 (8) ◽

pp. 2030-2036 ◽

Cited By ~ 18

Author(s):

Jiri Brynda ◽

Pavlina Rezacova ◽

Milan Fabry ◽

Magdalena Horejsi ◽

Renata Stouracova ◽

...

Keyword(s):

Atomic Resolution ◽

Inhibitor Binding ◽

Hiv 1

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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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Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 A resolution crystal structures of HIV-1 protease mutants with substrate analogs

FEBS Journal ◽

10.1111/j.1742-4658.2005.04923.x ◽

2005 ◽

Vol 272 (20) ◽

pp. 5265-5277 ◽

Cited By ~ 51

Author(s):

Yunfeng Tie ◽

Peter I. Boross ◽

Yuan-Fang Wang ◽

Laquasha Gaddis ◽

Fengling Liu ◽

...

Keyword(s):

Drug Resistance ◽

Crystal Structures ◽

Molecular Basis ◽

Substrate Recognition ◽

Substrate Analogs ◽

Hiv 1

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

mBio ◽

10.1128/mbio.00172-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 2

Author(s):

Ashley N. Matthew ◽

Jacqueto Zephyr ◽

Desaboini Nageswara Rao ◽

Mina Henes ◽

Wasih Kamran ◽

...

Keyword(s):

Drug Resistance ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Treatment Failure ◽

Protease Inhibitors ◽

Active Site ◽

Rational Design ◽

Parent Compound ◽

Major Health Problem ◽

Substrate Envelope

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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