Cooperative Effects of Drug-Resistance Mutations in the Flap Region of HIV-1 Protease

Foulkes-Murzycki, Jennifer E.; Rosi, Christina; Yilmaz, Neşe Kurt; Shafer, Robert W.; Schiffer, Celia A.

doi:10.1021/cb3006193

Cited by 20 publications

(39 citation statements)

References 40 publications

(76 reference statements)

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“…Cooperative resistance is a known phenomenon exemplified, for instance, by HIV escape from antiretrovirals (48). However, interdependent escape mutations located in distal regions of the native influenza virus HA protein, as identified in our study, are unusual and highlight the possibility that mechanistically distinct avenues may be available for IAV escape from entry inhibitors.…”

Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 57%

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCEThis study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale highthroughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for smallmolecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism ...

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Section: Viral Adaptation For Resistance Profilingmentioning

confidence: 57%

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Weisshaar

Cox

Morehouse

et al. 2016

J Virol

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“…Numerous computational studies have been aimed at the understanding of flap opening dynamics through standard computational molecular dynamics (MD) simulations (Chetty et al, 2015;Foulkes-Murzycki et al, 2013;Simmerling, 2007 and, in which it is imperative to assign protonation states correctly (Chen and Tropsha, 1995;Lindahl, 2015;Sussman et al, 2013 andXie et al, 1997). However, accurate protonation assignment is difficult because there are multiple sites that can bind or release protons, and these are coupled to one another in a complex, time and conformation-dependent manner.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, experimental studies have shown that there is a relationship between conformational sampling and drug resistance in HIV-PR (Cai et al, 2014;Cai et al 2012Foulkes-Murzycki et al, 2013Gibbs, 2014 andde Vera et al, 2013). Therefore, understanding functional dynamics of the flaps of HIV-PR is of great importance to the development of new HIV-PR inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

Soares

Tôrres

Silva

et al. 2016

Journal of Structural Biology

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a b s t r a c tThe active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pHdependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25 0 and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding.

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“…In HIV-1, multi-drug resistant patient isolates contain multiple mutations with up to 25% of the amino acids in the enzyme able to mutate and contribute to resistance [65, 66]. The impact of such combinations of mutations on conferring drug resistance is not simply additive, but instead these mutations can have complex interdependent effects [67, 68]. One remaining challenge is to unravel the interdependency of multiple mutations in conferring drug resistance.…”

Section: Remaining Challenges In Overcoming Resistancementioning

confidence: 99%

Improving Viral Protease Inhibitors to Counter Drug Resistance

Yilmaz

Swanstrom

Schiffer

2016

Trends in Microbiology

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Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design.

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Cooperative Effects of Drug-Resistance Mutations in the Flap Region of HIV-1 Protease

Cited by 20 publications

References 40 publications

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

Improving Viral Protease Inhibitors to Counter Drug Resistance

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