New Details of HCV NS3/4A Proteinase Functionality Revealed by a High-Throughput Cleavage Assay

Background: The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a noncovalent heterodimer of the N-terminal,,180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/ 4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target. Methodology/Principal Findings: Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1–P19) and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the viru

Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals

Improved clinical investigation and evaluation of high-risk medical devices: the rationale and objectives of CORE-MD (Coordinating Research and Evidence for Medical Devices)

: In the European Union (EU) the delivery of health services is a national responsibility but there are concerted actions between member states to protect public health. Approval of pharmaceutical products is the responsibility of the European Medicines Agency, whereas authorizing the placing on the market of medical devices is decentralized to independent 'conformity assessment' organizations called notified bodies. The first legal basis for an EU system of evaluating medical devices and approving their market access was the medical device directives, from the 1990s. Uncertainties about clinical evidence requirements, among other reasons, led to the EU Medical Device Regulation (2017/745) that has applied since May 2021. It provides general principles for clinical investigations but few methodological details-which challenges responsible authorities to set appropriate balances between regulation and innovation, pre- and post-market studies, and clinical trials and real-world evidence. Scientific experts should advise on methods and standards for assessing and approving new high-risk devices, and safety, efficacy, and transparency of evidence should be paramount. The European Commission recently awarded a Horizon 2020 grant to a consortium led by the European Society of Cardiology and the European Federation of National Associations of Orthopaedics and Traumatology, that will review methodologies of clinical investigations, advise on study designs, and develop recommendations for aggregating clinical data from registries and other real-world sources. The CORE-MD project (Coordinating Research and Evidence for Medical Devices) will run until March 2024; here we describe how it may contribute to the development of regulatory science in Europe

Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 μM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro

Effects of resistance mutations on the inhibitory activity of compounds 1–8.

<p>The IC₅₀ values of the compounds were determined in the cleavage assays <i>in vitro</i> using the recombinant wild-type (WT) and mutant (V36M, R155K, A156T, D168A and V170A) NS3/4A constructs and Ac-DE-D(Edans)-EE-Abu-ψ-[COO]AS-K(Dabcyl)-NH₂ as a substrate.</p>¥<p>The IC₅₀ was directly measured <i>in vitro</i> using the purified recombinant WT and mutant NS3/4A constructs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040029#pone.0040029-Zhou1" target="_blank">[75]</a>.</p>*<p>The IC₅₀ was measured using the mutant HCV replicon with luciferase readout <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040029#pone.0040029-Romano1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040029#pone.0040029-Lenz1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040029#pone.0040029-He1" target="_blank">[15]</a>. FC, the fold change in the IC₅₀ for the mutant compared to that the WT proteinase. ND, not determined, n/a, not applicable.</p

Three docking sites in NS3/4A and VLS of the NCI/DTP compound library.

<p>Left panels, positions of docking sites 1, 2 and 3 in the PDB 3EYD X-ray structure of NS3/4A, surface model. The catalytic triad (His-57, Asp-81, and Ser-139) is green. Docking sites 1, 2 and 3 are red. Right panels, VLS of the 275,000-compound NCI library against docking sites 1, 2 and 3. VLS led to identification of the top 84, 87 and 88 hits, from which 7, 15 and 18 available compounds for sites 1, 2 and 3, respectively, were tested in the NS3/4A inhibitory assays. Compounds were ranked according to their relative binding energy. Black, grey and open circles correspond to the tested compounds with the IC₅₀ values below 1 µM, below 10 µM and above 10 µM, respectively. Predicted (but untested) hits are shown as small back dots. E, relative binding energy. Inset, relations between the molecular weight (MW) and ranking of the ligands.</p

Structural similarity of the flaviviral NS3 proteinases.

<p>(A) Sequence alignment of NS3 proteinases. Asterisks mark the catalytic triad. Identical and homologous residue positions are shaded gray. (B) Sequence alignment of the flaviviral WNV and DV2 NS2B and HCV NS4A (PDB 3EYD) co-factors. Dots indicate identical residues. Secondary structure elements above and below the sequences are for WNV NS2B-NS3 proteinase (PDB 2IJO) and HCV NS3/4A (PDB 3EYD), respectively. The secondary structure of the minimal, 14-residue, NS4A co-factor required for activation of the NS3 proteinase <i>in vitro</i> is shown. WNV, West Nile virus. DV2, Dengue virus type 2.</p

Selected compounds efficiently inhibit the catalytic activity of NS3/4A: representative dose-response curves.

<p>Before the addition of the Ac-DE-D(Edans)-EE-Abu-ψ-[COO]-AS-K(Dabsyl)-NH₂ substrate (10 µM), NS3/4A (10 nM) was co-incubated for 30 min at ambient temperature with increasing concentrations of compounds <b>1, 4</b> and <b>7</b>. The residual activity was then monitored continuously at λ_ex = 355 nm and λ_em = 500 nm to determine the initial velocity of the reactions. The initial velocity was calculated as a percentage of residual activity versus the untreated proteinase (control). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040029#pone.0040029.s001" target="_blank">Table S1</a> for the compound structures.</p

VLS and SAR optimization of the focused compound sub-library.

<p>The 665-compound sub-library was docked into docking site 3 of NS3/4A. Compounds were ranked according to their relative binding energy (inset, a complete ranking curve, each dot represents a single compound). The screening led to the identification of the top 100 compounds with the lowest binding energy. Compounds were visually inspected and the 20 available compounds were ordered from the NCI/DTP for the follow-up <i>in vitro</i> activity tests. Black, grey and open circles correspond to the tested compounds with the IC₅₀ values below 1 µM, below 10 µM and above 10 µM, respectively. Predicted (but untested) hits are shown as small back dots. E, relative binding energy.</p