Proposed communication path between loop 1 and the RNA groove.

<p>K113 located at the edge of β-sheet 1 strongly interacts with loop 1, in particular E73. The other extremity of the β-sheet had multiple contacts with residues of the RNA grove, in particular hydrophobic interactions between Y97 and M371, interactions between R106 and the linker backbone (residues 360–373 shown in magenta) and electrostatic interactions between K103 and E372. The linker itself was stabilized by salt bridges between E369 and R361 and E369 and R317 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030038#pone-0030038-t001" target="_blank">Table 1</a>).</p

Structure-based design of novel naproxen derivatives targeting monomeric nucleoprotein of Influenza A virus

<div><p>The nucleoprotein (NP) binds the viral RNA genome as oligomers assembled with the polymerase in a ribonucleoprotein complex required for transcription and replication of influenza A virus. Novel antiviral candidates targeting the nucleoprotein either induced higher order oligomers or reduced NP oligomerization by targeting the oligomerization loop and blocking its insertion into adjacent nucleoprotein subunit. In this study, we used a different structure-based approach to stabilize monomers of the nucleoprotein by drugs binding in its RNA-binding groove. We recently identified naproxen as a drug competing with RNA binding to NP with antiinflammatory and antiviral effects against influenza A virus. Here, we designed novel derivatives of naproxen by fragment extension for improved binding to NP. Molecular dynamics simulations suggested that among these derivatives, naproxen A and C0 were most promising. Their chemical synthesis is described. Both derivatives markedly stabilized NP monomer against thermal denaturation. Naproxen C0 bound tighter to NP than naproxen at a binding site predicted by MD simulations and shown by competition experiments using wt NP or single-point mutants as determined by surface plasmon resonance. MD simulations suggested that impeded oligomerization and stabilization of monomeric NP is likely to be achieved by drugs binding in the RNA grove and inducing close to their binding site conformational changes of key residues hosting the oligomerization loop as observed for the naproxen derivatives. Naproxen C0 is a potential antiviral candidate blocking influenza nucleoprotein function.</p></div

Analysis of the interactions observed in four domains of the wt NP and R361A mutant.

<p>Loop 1 (residues 73–90), loop2 (residues 200–211), β-sheet 1 (residues 91–112), and the linker (residues 360–373).</p><p>These interactions define a path between loop 1 and the residue R361 of the linker, located in the RNA binding groove (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030038#pone-0030038-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030038#pone-0030038-g004" target="_blank">4</a>).</p>1<p>HB stands for hydrogen bond.</p>2<p>HPh stands for hydrophobic interaction.</p><p>See the Experimental section for definitions of the contact domains and the two contact types.</p>a)<p><u>Loop 1-loop 2 contact</u>: In R361A, hydrophobic interactions between L79 and loop 2 drove loop 1 to contact loop2; transient salt-bridges between R204 or R208, on the one hand, and E80 or E81, on the other hand, stabilized the interaction between loop 1 and loop 2 at short distances. Such loop-loop interactions were not found in wt NP.</p>b)<p><u>Loop 1 stability (base</u>): The side-chain of K113 was engaged in a strong hydrogen bond with the C-terminus of the loop 1; the guanidinium moiety of K113 formed a salt bridge with E73 at the N-terminus of loop 1, contributing to the stability of loop 1 in wt NP. The R361A mutation drastically reduced the interactions of the K113 with the loop 1, increasing loop 1 flexibility.</p>c)<p><u>Linker-β sheet 1 contacts</u>: The linker was connected to the β sheet 1 through conserved hydrophobic interaction between M371 and the ring of Y97, stable hydrogen bonds between R106 and the linker backbone oxygen atoms. The R361A mutation decreased the population of the solvated K103-E372 salt-bridge.</p>d)<p><u>Inter-linker contacts</u>: In NP, E369 interacted with both R361 and R317. In R361A, the R317-E369 contact population increased to 93% as compared to 73% in wt NP and the R361-E369 interaction was canceled by the mutation.</p

Influence of mutations in loop 1 of NP on RNA binding.

<p><b>A:</b> Effect of the mutations R361A (green circles) and R361A-E80A-E81A (blue squares) compared to wt NP (black triangles) binding to RNA; Inset: comparison of the SPR signals obtained in the presence of Flu1-RNA with 300 nM C-terminal His-tagged NP, R361A or R361A-E80A-E81A. Due to its low affinity for RNA, the signal of the R361A-RNA complex (green) is ca four times smaller than NP-RNA (black), while the signal of the triple mutant (blue) is intermediate between them. The binding of NP or mutants to the surface-bound Flu1-RNA oligonucleotide followed a saturation curve with maximal RU at large protein concentration; the signal deduced from the plateau of the association kinetics as a function of NP concentration was used to obtain the Kd, taken as the concentration at which the RU is 50% of the maximal RU. <b>B:</b> Binding to Flu1-RNA of the double and triple mutants, wt-E80A-E81A (blue squares), R361A-E80A-E81A (red circles) respectively.</p

Comparison of the NP and R361A proteins by molecular modelling.

<p><b>A:</b> Comparison the flexibility of loops 1 and 2 in the trimer (black) and monomer (red) forms of NP quantified by their backbone root-mean-square fluctuations during 4 ns and 50 ns simulation time, respectively. Loop 1 (73–90) and loop 2 (200–214) remained flexible in both NP forms; in contrast, a large difference is seen in the oligomerization loop 3 (402–428) of NP monomer and trimer. <b>B:</b> Root-mean-square fluctuations of the NP (red) and R361A (green) monomers during the simulated trajectories: one can see a reduced flexibility in loop 2 and a small increase of the flexibility of loop 1 of the R361A mutant.</p

Size of NP oligomers in the presence of RNA monitored by Dynamic Light Scattering.

<p><b>A</b> - Size distribution of monomeric wt NP (5 µM) alone (black, 6.8 nm, 93%, 5.0 µ, 7%) and 1 hour (dotted blue, 13.8 nm 91%, 760 nm 9%) or 3 hours after addition of 1.8 µM RNA (16.3 nm, 100%); <b>B</b>- Size distribution of monomeric R361A (5 µM) alone (black, 7.8 nm, 100%) and 4 hours after addition of 1.8 µM RNA (dashed green, 9.85 nm, 75%, 279 nm, 25%) <b>C</b>: Comparison of the oligomerization kinetics of R361A (violet squares) and R361A-E80A-E81A (green stars) (10 µM) after addition of RNA (3 µM). Note the large difference in the final size of the protein-RNA oligomers being 10±1 nm and 16±1 nm for R361A and R361A-E80A-E81A, the latter resembling the size of oligomeric NP-RNA complexes.</p

Distribution of the minimal distance between loops 1 and 2 in NP and the R361A mutant.

<p>It characterized the differences in loops interactions between wt NP and R361A.</p

Influence of mutations in loop 2 of NP on RNA binding and RNA-induced oligomerization.

<p><b>A:</b> Comparison of the association to and dissociation from RNA of R361A-R204A-R208A (full triangles) and wt-R204A-R208A (open triangles); <b>B:</b> Comparison of the oligomerization kinetics of 10 µM proteins after addition of RNA (3 µM): wt NP (full squares), R361A (open squares), wt-R204A-R208A (open circles) and R361A-R204A-R208A (full triangles) (10 µM). The lines represent single exponential fits.</p

Comparison of representative structures of NP (red) and the R361A mutant (green) proteins.

<p>The position of the mutated residue R361 is highlighted in CPK representation; the salt bridge between residues E80 and R208 and hydrophobic interactions between L79 and W207 stabilized the relative positions of the two loops at shorter distance in R361A than in NP (insert).</p

From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives <b>2</b>, <b>3</b>, and <b>4</b> targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative <b>2</b> presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative <b>4</b> also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives <b>2</b> and <b>4</b> inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions