Catching a Moving Target: Comparative Modeling of Flaviviral NS2B-NS3 Reveals Small Molecule Zika Protease Inhibitors.

pubs.acs.org/acsmedchemlett Letter Catching a Moving Target: Comparative Modeling of Flaviviral NS2B-NS3 Reveals Small Molecule Zika Protease Inhibitors Szymon Pach, Tim M. Sarter, Rafe Yousef, David Schaller, Silke Bergemann, Christoph Arkona, Jörg Rademann, Christoph Nitsche, and Gerhard Wolber* Cite This: ACS Med. Chem. Lett. 2020, 11, 514−520 ACCESS Metrics & More Read Online sı Supporting Information * Article Recommendations ABSTRACT: The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs. KEYWORDS: Flavivirus, protease, inhibitors, PyRod, 3D pharmacophores, Dynophores F NS2B-NS3 represents a promising drug target, since blocking proteases in other virus species, e.g. human immunodeficiency virus18 or hepatitis C virus,19 leads to disruption of the replication cycle, which has already yielded several antiviral drugs. Despite high scientific efforts, only a few small molecule Zika virus protease (ZIKVPro) inhibitors20−28 have been reported to date. Several reported nonpeptide compounds targeting the active site of the protease show undesirable properties for lead optimization, such as instability in aqueous solution20 or high molecular weight21,28 (>500 Da). As random findings in high-throughput screening campaigns, most active small-molecular competitive inhibitors have poorly characterized binding modes,21 rendering further development even more challenging. Allosteric inhibitors may lead to fast resistance development.29 Hence, we strive for the development of druglike NS2B-NS3 protease inhibitors targeting the substratebinding site by combining in-silico design and biochemical experiments. Our novel, rationally discovered inhibitors with laviviruses cause millions of infections and thousands of fatalities annually.1 Despite a high medicinal need, no approved antiflaviviral treatment is currently available.2 Vaccines preventing infections with frequently prevalent viruses such as yellow fever virus,3 Japanese encephalitis virus,4 tickborne encephalitis virus,5 or dengue virus6 are approved, but not against emerging species such as West Nile virus (WNV) or Zika virus (ZIKV).2,7 Due to the high conservation of all flaviviral nonstructural (NS) proteins,8 designing broad-spectrum antivirals is a viable strategy for the treatment of recently emerged species. Flaviviruses encode for seven NS proteins,9 whose functions are only understood well for the NS2B-NS3 and NS5.10 The NS2B-NS3 protease complex is essential for the flaviviral replication cycle by processing the viral polyprotein into functional units of the virion. The nonstructural protein 3 (NS3) forms the catalytically active domain of the protease complex.11 NS2B acts as a cofactor for the protease domain, supporting substrate binding.12,13 NS2B-NS3 is a serine protease showing substrate specificity and catalytic triad (S135, H51, and D75, Figure 1) similar to trypsin.14 This enzyme recognizes dibasic peptide sequences with a cleavage site between an arginine or lysine and amino acids with small side chains (alanine or serine).14−17 © 2020 American Chemical Society Received: December 19, 2019 Accepted: March 3, 2020 Published: March 3, 2020 514 https://dx.doi.org/10.1021/acsmedchemlett.9b00629 ACS Med. Chem. Lett. 2020, 11, 514−520 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter flaviviral species14 and accept lysine and arginine.14 S3 and S4 subpockets show sequence-variability and accept various residues. Both substrate binding sites are highly flexible,13 hydrophilic, and shallow,33 rendering the NS2B-NS3 protease a challenging target for drug discovery. In order to address binding pocket flexibility of WNVPro, we employed our novel application PyRod.34 In this tool, the protein environment of water molecules is analyzed over the course of an MD simulation. Pharmacophoric binding site characteristics can subsequently be visualized with dynamic molecular interaction fields (dMIFs, Figure 2A). Features outside the Figure 2. (A) Dynamic molecular interaction fields (dMIFs) and (B) focused (B1) 3D-pharmacophore model obtained from WNVPro MD simulations and PyRod analysis. Pink letters and numbers indicate protease subpockets. Color code: yellow spheres and clouds, lipophilic contacts; purple rings and blue clouds, aromatic interactions; red arrows and clouds, hydrogen bond acceptors; purple stars and clouds, cationic interactions. Figure 1. Comparison of ZIKVPro (top, PDB-ID: 5YOF20) and WNVPro (bottom, PDB-ID: 5IDK30) binding pockets. The key-residues are highlighted with black letters and numbers. Pink letters and numbers indicate protease-subpockets. Gray backbone, NS2B; green backbone, NS3. This figure was generated using UCSF Chimera 1.13.1.31 validated binding modes and low molecular weight represent promising starting points for future hit optimization. Literature research revealed a lack of high-quality bioactivity data for ZIKVPro. Reported competitive ligands show either low potency, high molecular weight, or low stability in aqueous solutions.20,21,28 The substrate binding site of WNV protease (WNVPro) and ZIKVPro shows a sequence identity of 83% (Figure 1), and several nonpeptidomimetic ligands for WNVPro were reported with activity below 50 μM8 (Supporting Information Table S1). Hence, WNVPro was used as a starting point for the identification of novel drug-like ZIKVPro inhibitors. Substrate binding sites of WNVPro and ZIKVPro only differ at three residue positions (Figure 1). The S1 and S2 subpockets (Schechter−Berger nomenclature32) are highly conserved in highly conserved S1 and S2 subpockets were removed, and dMIFs were used to prioritize features inside the binding pocket to generate a focused 3D pharmacophore model consisting of 16 independent features (B1, Figure 2B). Identified cationic interactions exploit contacts in the S1 subpocket to D129 and in the S2 subpocket to D75 and H51, while aromatic interactions are present facing Y161 and H (...truncated)