Optimization of Small-Molecule Inhibitors of Influenza Virus Polymerase: From Thiophene-3-Carboxamide to Polyamido Scaffolds

Influenza virus infections represent a serious concern to public health, being characterized by high morbidity and significant mortality. To date, compounds targeting the viral ion-channel M2 or the viral neuraminidase are the drugs available for treatment of influenza, but the emergence of drug-resistant viral mutants renders the search for novel targets and their possible inhibitors a major priority. Recently, we demonstrated that the viral RNA-dependent RNA polymerase (RdRP) complex can be an optimal target of protein–protein disruption by small molecules, with thiophene-3-carboxamide derivatives emerging as promising candidates for the development of new anti-influenza drugs with broad-spectrum activity. Here, we report a further dissection of the thiophene-3-carboxamide structure. By using a GRID molecular interaction field (MIF)-based scaffold-hopping approach, more potent and nontoxic polyamido derivatives were identified, highlighting a new space in the chemical variability of RdRP inhibitors. Finally, a possible pharmacophoric model highlighting the key features required for RdRP inhibition is proposed

ボルツマンマシン ノ サイテキカ モンダイ エノ オウヨウ サイテキカ ノ スウリ ト ソノ オウヨウ

Influenza is an infectious disease that represents an important public health burden, with high impact on the global morbidity, mortality, and economy. The poor protection and the need of annual updating of the anti-influenza vaccine, added to the rapid emergence of viral strains resistant to current therapy make the need for antiviral drugs with novel mechanisms of action compelling. In this regard, the viral RNA polymerase is an attractive target that allows the design of selective compounds with reduced risk of resistance. In previous studies we showed that the inhibition of the polymerase acidic protein-basic protein 1 (PA–PB1) interaction is a promising strategy for the development of anti-influenza agents. Starting from the previously identified 3-cyano-4,6-diphenyl-pyridines, we chemically modified this scaffold and explored its structure–activity relationships. Noncytotoxic compounds with both the ability of disrupting the PA–PB1 interaction and antiviral activity were identified, and their mechanism of target binding was clarified with molecular modeling simulations

Sumoylation of UL44 in HCMV-infected cells.

<p>(<b>A</b>) HFFs were either mock-infected or infected with HCMV for the indicated times. Cell lysates were analyzed by western blotting with an anti-UL44 antibody. (<b>B</b>) Blots were analyzed by densitometry and the percentage of sumoylated UL44 bands relative to that of unmodified UL44 at each time p.i. was plotted versus the p.i. time point. Data represent the means ± standard deviations (error bars) of values from three independent experiments such as that shown in (A). (<b>C</b>) Lysates from either mock-infected or HCMV-infected HFF cells were prepared at 120 h p.i. and immunoprecipitated with an anti-UL44 antibody. Immunoprecipitates were analyzed by western blotting with anti-SUMO-1 (left panel) and anti-UL44 (right panel) antibodies. For all panels, the arrowhead indicates the unmodified form of UL44, the arrow indicates the immunoglobulin G heavy chain (IgG hc) and the asterisks indicate the sumoylated UL44 forms.</p

UL44 interacts with human Ubc9 in yeast two-hybrid assays.

1<p>UL44, UL54, and Ubc9 proteins were fused to the C-terminus of LexA protein and/or of GAL4 activation domain (GAD). Fusion proteins were then assayed for interaction by qualitative β-galactosidase (β-gal) filter assays and by quantitative β-gal liquid assays.</p>2<p>β-gal expression was scored as follows +, strong blue color detected within 2 h of incubation; ±, blue color detected after more than 2 h of incubation; −, no signal detected after 16–24 h of incubation. Values within parentheses represent β-gal units ± standard deviation (SD) of 3–4 yeast colonies from at least three independent transformations.</p

Overexpression of SUMO-1 modifies the subnuclear localization of UL44 and promotes virus replication in HCMV-infected cells.

<p>(A) U373 cells that constitutively overexpress SUMO-1 (U373-SUMO-1), control U373-Neo cells, and U373-SUMO-1 cells transduced with lentiviral particles expressing either a <i>Ubc9</i>-silencing shRNA (U373-SUMO-1 shUbc9) or a non-silencing shRNA sequence (U373-SUMO-1 NS) were mock-infected or infected with HCMV at an MOI of 5 or 1 PFU/cell. At 72 h p.i., cells were fixed and stained with a primary antibody against UL44 (upper panels) or against UL57 (lower panels), and successively with a secondary fluorescein-conjugated antibody (green) which contained Evans Blue to counterstain cells (red). Cell samples were then analyzed by CLSM. (B) Western blot analysis of Ubc9 expression in Ubc9-knocked-down and control cells. Cell lysates were prepared from the indicated cell lines and subjected to SDS-PAGE. Filter was then probed with anti-Ubc9 and anti-α-tubulin MAbs. (C) The indicated cell lines were infected with HCMV at an MOI of 1 and viral DNA levels were measured at 72 h p.i. by quantitative real-time PCR. (D) The indicated cell lines were infected with HCMV at an MOI of 1 and the titers of infectious virus progeny produced at 120 h p.i. were determined by plaque assays. In both (C) and (D), the data shown represent the means ± standard deviations (error bars) of two independent experiments performed in duplicate or triplicate. The asterisks denote a statistically significant difference (<i>P<</i>0.05) between the values relative to U373-SUMO-1 and U373-SUMO-1 NS and the values relative to control U373-Neo cells.</p

HCMV UL44 interacts with human Ubc9.

<p>(<b>A</b>) GST-pulldown assays showing interaction of purified GST or GST-UL44 with <i>in vitro</i>-expressed, radiolabeled Ubc9 (right panels), HCMV UL54 (central panels, positive control), and influenza A virus PB1 (left panels, negative control). I, input; E, eluted by glutathione. Arrowheads indicate the proteins of interest. (<b>B</b>) COS-1 cells were transfected to express the indicated GFP and DsRed2 fusion proteins and imaged by CLSM. Merged images of the green (GFP) and red (DsRed2) channels are shown on the right, with yellow coloration indicative of co-localization. (<b>C</b>) Phoenix cells were transfected to express a UL44-FLAG fusion. At 48 h post-transfection, cell lysates were analyzed by western blotting with anti-FLAG, anti-Ubc9, anti-CycD1, and anti-vinculin antibodies. Cell lysates were also incubated with anti-FLAG-M2-Agarose beads and the immunoprecipitated samples were analyzed by western blotting with anti-FLAG, anti-Ubc9, and anti-CycD1 antibodies. Asterisks indicate the IgG heavy chains, while arrowheads indicate the proteins of interest. (<b>D</b>) Several UL44 mutants were created by deleting different portions of <i>UL44</i> coding sequence and expressed as fusions with LexA. A diagram of the full-length and truncated UL44 proteins, with functional domains indicated, is reported. Numbers refer to remaining amino acid residues of UL44. Blue bars, residues important for dimerization; CL, connector loop; FL, flexible loop; grey boxes, glycine strings; red box, Nuclear Localization Signal. The ability of the mutants to physically interact with Ubc9 was determined by β-gal filter assays and scored as follows: +, strong blue color detected within 2 h of incubation; ±, blue color detected after more than 2 h of incubation; −, no signal detected after 16–24 h of incubation. Values within parentheses represent β-gal units ± standard deviation (SD) of 3–4 yeast colonies from at least three independent transformations, determined by quantitative β-gal assays.</p

Sumoylation sites in UL44 as identified by mass spectrometry.

<p>Tryptic peptides derived from UL44 conjugated to SUMO-1. The table lists the measured <i>m/z</i> values, the experimentally determined (Exp.) molecular weights (M.W.) in Da, the calculated (Cal.) M.W. in Da as determined by database search, and the mass difference between calculated and experimental M.W. in Da. The amino acid sequences are listed with their positions within UL44 protein. Underlined bold amino acid indicates the SUMO-1-conjugated lysine in UL44, that is also listed in the far right column. Amino acids are in one-letter code. Asterisks indicate that the specific sumoylation site was predicted with the SUMOplot software.</p

Sumoylation of UL44 <i>in vitro</i>.

<p>(<b>A</b>) To analyze UL44 sumoylation <i>in vitro</i>, purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either wild-type SUMO-1 (SUMO-1 wt) or a mutant form of SUMO-1 (SUMO-1 mut) which cannot be covalently linked to substrates. The reaction products were analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. As a positive control, <i>in vitro</i> sumoylation of p53 was also analyzed. (<b>B</b>) Purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either SUMO-2 or SUMO-3 and analyzed by western blotting with anti-UL44 and anti-SUMO-2/−3 antibodies. For all panels, the arrowhead indicates the unmodified form of UL44 or p53 and the asterisks indicate the respective sumoylated forms.</p

UL44 sumoylation is stimulated by DNA.

<p>(<b>A</b>) Purified 6His-UL44 protein was incubated with sumoylation proteins in the absence or presence of dsDNA (left panel) or ssDNA (right panel). Samples were analyzed by western blotting with an anti-UL44 antibody. (<b>B, C</b>) Phoenix cells were transfected to express wild-type UL44 or the FLAG-UL44Δloop or FLAG-UL44L86A/L87A mutant, which are defective for DNA binding. At 48 h post-transfection, cell lysates were analyzed by western blotting with anti-FLAG, anti-HA, anti-Ubc9, and anti-GAPDH antibodies. (<b>D</b>) Phoenix cells were transfected to express the indicated proteins. At 48 h post-transfection, cell lysates were analyzed by western blotting with anti-UL44, anti-Ubc9, and anti-vinculin antibodies (left panel). Cell lysates were incubated with anti-FLAG-M2-Agarose beads and the immunoprecipitated samples were analyzed by western blotting with anti-UL44 and anti-Ubc9 antibodies (right panel). (<b>E</b>) The sumoylation <i>in vitro</i> of wild-type 6His-UL44 and mutant 6His-UL44Δloop and 6His-UL44L86A/L87A proteins was carried out as in (A) and analyzed by western blotting with an anti-UL44 antibody. (<b>F</b>) The sumoylation <i>in vitro</i> of a UL44 mutant bearing the K167R substitution in the flexible loop of UL44 involved in DNA binding was carried out in the presence of DNA and compared to that of wild-type UL44. For all panels, the arrowhead indicates the unmodified form of UL44 or free SUMO-1 and the asterisks indicate the sumoylated forms.</p

A Broad Anti-influenza Hybrid Small Molecule That Potently Disrupts the Interaction of Polymerase Acidic Protein–Basic Protein 1 (PA-PB1) Subunits

none13nomixedMassari, Serena; Nannetti, Giulio; Desantis, Jenny; Muratore, Giulia; Sabatini, Stefano; Manfroni, Giuseppe; Mercorelli, Beatrice; Cecchetti, Violetta; Palù, Giorgio; Cruciani, Gabriele; Loregian, Arianna; Goracci, Laura; Tabarrini, OrianaMassari, Serena; Nannetti, Giulio; Desantis, Jenny; Muratore, Giulia; Sabatini, Stefano; Manfroni, Giuseppe; Mercorelli, Beatrice; Cecchetti, Violetta; Palu', Giorgio; Cruciani, Gabriele; Loregian, Arianna; Goracci, Laura; Tabarrini, Orian