A small molecule that targets influenza
Many of us rely on seasonal vaccines for protection against influenza and are only too aware of their limited breadth. Broadly neutralizing antibodies (bnAbs) that target the conserved hemagglutinin (HA) stem of the influenza virus provide hope for the development of universal vaccines and are being evaluated in clinical trials. Van Dongen et al. selected and optimized a small-molecule lead compound that recapitulates key interactions of the bnAb with HA. Like the bnAb, the compound inhibited viral fusion in the endosomes of target cells. The compound protected mice from influenza after oral administration and neutralized virus infection in a 3D cell culture of human bronchial epithelial cells.
Science, this issue p. eaar6221
Structured Abstract
INTRODUCTION
Annual influenza epidemics and episodic pandemics continue to cause widespread illness and mortality. Strategies to prevent and treat acute influenza infection have remained limited to seasonal influenza vaccination and a small arsenal of antiviral drugs. Thus, there is an urgent need for additional prophylactic and therapeutic options, including new targets and mechanisms of action, to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and the emergence of antiviral drug resistance.
RATIONALE
The recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the highly conserved hemagglutinin (HA) stem as a promising target for development of universal vaccines and complementary therapeutics. Even though this spurred several bnAbs to be evaluated as passive immunotherapy in clinical trials, antibodies are large and complex molecules that are generally unsuited for oral delivery. We therefore set out to utilize the structural details of the molecular interactions and mechanisms of HA stem bnAbs to identify an orally active small molecule that mimics bnAb functionality. Influenza A viruses can be separated in group 1 and group 2 on the basis of their HA subtype (H1 to H18), and anti-stem bnAbs usually bind to group 1 or to group 2 viruses, but a few can target both.
RESULTS
We screened a diverse chemical library for compounds that selectively target the group 1 HA epitope of bnAb CR6261 through a binding assay that detects displacement of a CR6261-based designed small protein. Benzylpiperazines were identified as a major hit class, with JNJ7918 being the most promising candidate. Consistent with its binding to the functional HA stem epitope, this compound also neutralized influenza infection in vitro. Key chemical modifications were subsequently introduced to optimize binding and neutralization potency, as well as properties dictating metabolic stability and oral bioavailability, to finally afford JNJ4796. This lead compound binds and neutralizes a broad spectrum of influenza A group 1 viruses in vitro and protects mice against lethal and sublethal influenza challenge after oral administration. The compound also effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Like bnAb CR6261, the mechanism of action of JNJ4796 was demonstrated to be based on inhibition of the pH-sensitive conformational change of HA that triggers fusion of the viral and endosomal membranes and release of the viral genome into the host cell. Cocrystal structures with H1 and H5 HAs reveal that JNJ4796 recapitulates the original CR6261-HA hotspot interactions and provide detailed and valuable information on the minimal epitope in the HA1-HA2 fusion region of the stem for an antiviral small molecule to neutralize influenza A group 1 viruses.
CONCLUSION
We identified an orally active small molecule against influenza A HA that mimics the binding and functionality of the broadly neutralizing antibody CR6261. The small molecule targets the conserved HA stem region, acts as a fusion inhibitor by inhibiting conformational changes that lead to the postfusion HA structure, and neutralizes a broad spectrum of human pandemic, seasonal, and emerging group 1 influenza A viruses. Thus, the compound holds promise as an urgently sought-after therapeutic option offering a complementary mechanism of action to existing antiviral drugs for the treatment of influenza virus infection, and that should further aid development of universal therapeutics that prevent entry of influenza virus in host cells.
(A) Crystal structure of JNJ4796 (red) with H1N1 A/Solomon Islands/3/2006 HA (gray surface). The N-terminal fusion peptide of the HA2 chain (blue ribbon) is highlighted in orange. (B) View along the threefold axis of the HA trimer, with the three identical binding sites of JNJ4796 highlighted (cyan) along with its chemical structure (enlarged view).
Abstract
Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.
The World Health Organization estimates that annual influenza epidemics cause around 3 million to 5 million cases of severe illness and up to 650,000 deaths worldwide (1, 2). Seasonal influenza vaccination still remains the best strategy to prevent infection, but the vaccines that are available now offer a very limited breadth of protection. The discovery of human broadly neutralizing antibodies (bnAbs) to influenza virus provides hope for the development of broad-spectrum, universal vaccines (3–14). Because of the high level of conservation of their epitopes in the hemagglutinin (HA) stem, these bnAbs neutralize a wide range of viruses within and across influenza virus subtypes. Their binding prevents the pH-induced conformational changes in HA that are required for viral fusion in the endosomal compartments of target cells in the respiratory tract (6–11, 13–15). Efforts have therefore been made to develop vaccination modalities aimed at directing the immune response to the HA stem through different vaccination regimens (16, 17), sequential vaccination with different chimeric HA constructs (18, 19), and administration of stem-based immunogens (20–24). In addition, several bnAbs themselves are being evaluated in clinical trials as passive immunotherapy (25). Another recent strategy to prevent influenza infection stems from development of a highly potent multidomain antibody with almost universal breadth against influenza A and B viruses that can be administered intransally in mice using adeno-associated virus–mediated gene delivery (26).
Therapeutic options to treat acute influenza infection also include antiviral drugs directed at blocking virus uncoating during cell entry (M2 proton channel inhibitors) and progeny release from infected cells (neuraminidase inhibitors) (27, 28). However, resistance to antiviral drugs is an emerging problem, owing to the high mutation rate in influenza viruses and their genetic reassembly possibilities (29). New antiviral drugs (30, 31) and combination therapies (32, 33), with alternative mechanisms of action against alternative viral targets are therefore urgently needed. Small-molecule drugs, in contrast to antibodies, offer the advantage of oral bioavailability, high shelf stability, and relatively low production costs.
Influenza A viruses have been classified into 18 hemagglutinin subtypes (H1 to H18), which can be divided phylogenetically into two groups (1 and 2), and 11 neuraminidase subtypes (N1 to N11). Antibody CR6261 broadly neutralizes most group 1 influenza A viruses (7, 9). Cocrystal structures of CR6261 in complex with H1 HA (7, 9) stimulated the design of small-protein ligands of about 10 kDa that target the conserved stem region. These small proteins mimic the antibody interactions with HA and inhibit influenza virus fusion (34–36). Cocrystal structures of bnAbs FI6v3 and CR9114 with HAs (6, 14) further enabled the design of even smaller peptides as influenza fusion inhibitors (37). However, neither small proteins nor peptides generally are orally bioavailable.
Development of small-molecule ligands directed at antibody binding sites is challenging. Antibody epitopes, as for other protein-protein interfaces, are generally flat, large, and undulating (~1000 to 2000 Å2) (38), in stark contrast to the small concave pockets (typically in the 300 to 500 Å2 range), which are common as targets for small-molecule drugs (39). Moreover, to mimic the function of an HA-stem bnAb, a functional small molecule should reproduce the key interactions that lead to fusion inhibition. We have therefore identified and optimized small molecules with such properties through application of a strategy that was guided by detailed knowledge of the binding mode and molecular mechanism of bnAb CR6261 (7, 15) and encouraged by earlier successes in the design of small proteins and peptidic ligands targeted to the HA stem (34, 35, 37).
High-throughput screening and optimization
To identify potent small molecules that mimic group 1 bnAb CR6261, in terms of breadth of binding (7, 9, 35), virus neutralization, and mechanism (Fig. 1A), we screened for compounds that selectively target the CR6261 epitope on HA. We applied the AlphaLISA (amplified luminescent proximity homogeneous assay) technology in competition mode as our high-throughput screening (HTS) method (Fig. 1B). A diverse library of ~500,000 small-molecule compounds was screened for displacing HB80.4, which is a CR6261-based computationally designed small protein with very similar binding mode and fusion inhibition profile (34, 35). HB80.4 was used instead of CR6261, because avidity effects leading to higher apparent affinity of the bivalent antibody would have resulted in a more stringent and thus less sensitive assay. This approach biased the screen toward compounds that act via the desired mechanism of action. About 9000 small molecules with weak to medium binding capacity were initially retrieved; binding of 300 compounds was confirmed through repeated testing and via the Truhit AlphaLISA counter assay that can identify false-positive hits.
(A) Binding mode, breadth of binding, and fusion inhibition profile of influenza HA stem–targeting antibody CR6261. The left panel shows the binding mode of CR6261 to influenza HA, with the HA trimer represented as a gray molecular surface with different shades for the different protomers, CR6261 in green with a molecular surface for the Fab interacting with the HA and a cartoon for the other Fab and Fc of the immunoglobulin G, and the binding epitope on the HA in red. The middle panel illustrates the HA phylogenetic tree, which shows the relationship between the 18 HA subtypes of influenza A virus (group 1 and 2) and the two lineages of influenza B viruses. The breadth of binding of the CR6261 to multiple group 1 subtypes is shown in green, light gray indicates where binding was not tested, and black indicates no binding. The right panel shows the mechanism of action of CR6261 to block the pH-induced HA conformational changes. (B) Our HTS utilized the AlphaLISA technology to identify small molecules targeting the CR6261 epitope. The CR6261-mimicking designed protein HB80.4 was used in the binding competition assay. HB80.4 is represented in blue and the CR6261 epitope on the HA in pink. Small molecules are indicated as illustrative structural formulae.
Benzylpiperazines emerged as a major hit class, with JNJ7918 being the most promising candidate with a median inhibitory concentration (IC50) of 1.39 and 13.06 μM against H1N1 A/California/07/2009 (H1/Cal) and H5N1 A/Vietnam/1203/2004 (H5/Viet) HAs, respectively (Fig. 2). No competition with H1 HA head–binding antibody 2D1 (40) was detected against H1/Cal, further validating the HA-stem specificity (Fig. 2A). Key chemical modifications to increase molecular interactions with the HA stem, that is, introduction of a functional group at the benzylic position and modification of the phenyl with a propargyl moiety (Fig. 2B), greatly improved binding (~30- to 80-fold) to HAs from H1/Cal, H1N1 A/New Caledonia/20/1999 (H1/NCa), H1N1 A/Brisbane/59/2007 (H1/Bris), and H5/Viet, as exemplified by the second-generation compound JNJ6715 (Fig. 2C). Moreover, virus neutralization assays with a panel of H1 and H5 influenza strains, which represent human pandemic, seasonal, and emerging viruses in influenza A group 1, demonstrated improved neutralization (~30- to 500-fold). Like bnAb CR6261, JNJ6715 showed heterosubtypic neutralization of group 1 viruses without measurable cytotoxicity in Madin-Darby canine kidney (MDCK) cells (Fig. 2D and table S1).
(A) Representative dose-dependent competitive binding for compound JNJ7918 that emerged from a HTS that used the AlphaLISA assay. This compound inhibited binding of the designed protein HB80.4 against the HA stem to H1/Cal and H5/Viet HAs with IC50’s of 2.3 and 21.4 μM [−log of median inhibitory concentration (pIC50) = 5.6 and 4.7], respectively, but not to the head binding antibody 2D1, when tested with H1/Cal. The x axis depicts the log molar concentration of the compound, and the y axis depicts the normalized response, relative to the positive (no inhibitor and no HA) and negative (no inhibitor) control. (B) Chemical structures of JNJ7918 and JNJ6715, with key modifications to the latter highlighted in yellow circles. (C) Scatter plot depicting H1/Cal, H1/NCa, H1/Bris, and H5/Viet binding, calculated from AlphaLISA assay as log IC50 versus virus neutralization as log EC50. Each dot represents a tested compound. The dotted line represents the lower limit of quantification. (D) Virus neutralization EC50 values in μM for compounds JNJ7918 and JNJ6715 against the indicated virus strains: H1/Bris; H1/Cal; H1/NCa; H1N1 A/Puerto Rico/8/1934 (H1/PR8); H1/SI06; H5N1 A/Hong Kong/156/1997 (H5/H97); H5N1 A/Vietnam/1194/2004 (H5/Viet; H3N2 A/Brisbane/10/2007 (H3/Bris); H7N7 7:1 reassortant virus with the HA of A/New York/107/2003 (H7/NY) and remaining segments from A/Puerto Rico/8/1934 rescued and propagated on PER.C6 cells; and influenza B B/Brisbane/60/2008 (B/Bris).
In vitro and in vivo pharmacokinetic profiling
Further in vitro profiling of JNJ6715 revealed poor kinetic aqueous solubility of 11.3 μM at pH 7.4 and poor metabolic stability of >346 ml/min per kilogram based on intrinsic clearance in mouse and human liver microsomes, making this compound unsuitable for in vivo testing (Fig. 3A). To enhance its drug-like properties, we replaced the methyl ester functionality at the benzylic position with a 2-methyl oxadiazole group, substituted the central phenyl by pyridine, and replaced the 6-methoxy benzothiazole with a 5-trifluoro-methoxy-benzoxazole group, generating JNJ8897. The compound was further optimized to generate JNJ4796 by replacing the 2-methyl oxadiazole group with 2-methyl tetrazole and the trifluoro-methoxy group with methyl amide (Fig. 3B). These modifications reduced the intrinsic clearance in mouse and human liver microsomes for both compounds while sustaining virus neutralization [median effective concentration (EC50) for H1/Cal and H1/NCa of 0.064 and 0.076 μM for JNJ8897, and 0.066 and 0.038 μM for JNJ4796]. In particular, JNJ4796 demonstrated substantially reduced human and murine intrinsic clearance (Fig. 3, A and C) and increased aqueous solubility, which translated to a favorable in vivo pharmacokinetics profile with compound half-life (t1/2) in mice of 2.4 hours after oral administration. The overall bioavailability for this compound was 30.0%, with a plasma concentration reaching 1152 ng/ml, equivalent to 2.1 μM, after oral administration at 10 mg/kg (mg of JNJ4796 per kg of body weight) (Fig. 3A). Testing JNJ4796 in a diverse panel of pharmacologically relevant receptors, ion channels, and transporters showed that the compound does not substantially inhibit any of these potential off-targets (table S2).
(A) Absorption, distribution, metabolism, and excretion (ADME) data of small-molecule compounds: kinetic solubility at pH 4.0 and 7.4; intrinsic clearance (CLint) in human liver microsomes and murine liver microsomes (hLM/mLM); plasma protein binding (PPB) human and murine (hu/mu); mouse t1/2 (h, hours; iv, intravenous; po, per os); area under the curve (AUC 0−∞); maximum concentration (Cmax) po; time point for maximum concentration (tmax) po; and po bioavailability. ND, not determined. (B) Chemical structures of JNJ6715, JNJ8897, and JNJ4796, with key chemical modifications compared with the previous generation highlighted in orange and red circles. (C) Scatter plots depict CLint of small-molecule compounds in human and mouse liver microsomes versus their average log EC50 neutralization value against H1/Cal and H1/NCa HAs. The individual black dots represent the analyzed small-molecule compounds, and the yellow, orange, and red dots represent JNJ6715 (EC50 = 0.14 μM), JNJ8897 (EC50 = 0.063 μM), and JNJ4796 (EC50 = 0.033 μM), respectively. The shaded area reflects the optimal area of high virus neutralization capacity and low intrinsic clearance. (D) Survival curves and weight loss of mice challenged intranasally (at day 0) with a lethal dose of the mouse-adapted H1N1 A/Puerto Rico/8/1934 virus (25 × LD50) after oral administration of the indicated compounds at days −1 to 5 (twice daily).
Oral efficacy against influenza infection
In line with its in vitro neutralizing activity and acceptable pharmacokinetics data, oral administration of JNJ4796 protected mice from lethal challenge of 25 times the median lethal dose (LD50) of H1N1 A/Puerto Rico/8/1934 virus (Fig. 3D). Doses of 50 and 10 mg/kg of JNJ4796 twice daily, initiated one day before challenge and continuing for 7 days, resulted in 100% survival at day 21 in comparison to the less potent compound JNJ8897, for which less than 50% survival was achieved (day 21 survival was 40% for 50 mg/kg and 25% for 10 mg/kg) (Fig. 3D and tables S3 and S4). JNJ4796 only partly alleviated morbidity in this stringent H1N1 infection model, as demonstrated by animal weight loss. Nevertheless, dose-dependent reversal of weight loss was observed at the study end for both 50 mg/kg and 10 mg/kg of JNJ4796 (Fig. 3D). Oral doses of JNJ4796 also resulted in dose-dependent efficacy after a sublethal viral challenge (LD90), with twice daily administration of 15 and 5 mg/kg of JNJ4796 giving rise to 100% survival and only moderate weight loss effects that were moreover restricted to the period directly after treatment (fig. S1 and tables S3 and S4).
To assess the potential practical utility of JNJ4796 in the context of human airway infection, the neutralization capacity of JNJ4796 was evaluated in a reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells (HBECs) derived from a pool of donors. This model system recapitulates several relevant characteristics of human airway target tissue, such as production of mucus, cilia beating, and local metabolic activity (41), which could potentially limit the compound’s efficacy. Incubation of an HBEC culture, infected with H1N1 A/Puerto Rico/8/1934 virus, with the compound dramatically reduced the viral titers when assessed 96 hours postinfection (fig. S2).
Group 1 binding specificity and mechanism of JNJ4796
Like bnAb CR6261, JNJ4796 further demonstrated heterosubtypic group 1 HA binding and virus neutralization breadth without cytotoxicity (Fig. 4, A and B; fig. S3; and tables S1 and S5). CR6261 neutralizes group 1 influenza A viruses by inhibiting the low-pH-induced HA conformational change, which triggers fusion of the viral and endosomal membranes and release of the viral genome into the host cell (42). Each HA monomer is composed of two subunits (HA1 and HA2). CR6261 and JNJ4796 bind components of both HA1 and HA2 in the trimeric HA stem, thereby stabilizing the prefusion conformation of HA and preventing conformational rearrangements in the HA stem that lead to the postfusion structure. This inhibition is demonstrated in two different experiments. First, in a conformational change inhibition assay (Fig. 4C), JNJ4796 dose-dependently prevents pH-induced transition of HA to the postfusion conformation and subsequent loss of the HA1 subunit after reduction of the interchain HA disulfide. Second, the protease-susceptibility assay indicates that, like the stem-targeting bnAbs, compound JNJ4796 stabilizes the prefusion conformation and blocks the HA conformational change at low pH and the subsequent susceptibility to trypsin (43) (Fig. 4D).
(A) Small molecule JNJ4796 binds multiple group 1 subtypes (green text), as displayed on the HA phylogenetic tree (see Fig. 1A). Gray indicates that binding against H8, bat H17, and bat H18 HA subtypes was not tested, and black indicates no binding. (B) Comparison of breadth of virus neutralization for JNJ4796 and CR6261 against representative influenza viruses: Influenza A H1/Bris; H1/Cal; H1/NCa; H1N1 A/Puerto Rico/8/1934 (H1/PR8); H1/SI06; H5N1 A/Hong Kong/156/1997 (H5/H97); H5N1 A/Vietnam/1194/2004 (H5/Viet); H3N1 A/Brisbane/10/2007 (H3/Bris); H7N7 A/New York/107/2003 (H7/NY) and influenza B B/Brisbane/60/2008 (B/Bris). (C) Conformational-change inhibition assay showing that binding of JNJ4796 to cleaved H1/Bris HA blocks the low-pH-induced conformational change after lowering the pH to 5.25. The log of the molar concentration of JNJ4796 is plotted against the inhibition of conformational change normalized to the full inhibition by CR6261 (positive control). The vertical intersecting line represents the IC50. (D) Small molecule JNJ4796 inhibits the low-pH-induced conformational changes in H1/PR8 HA. In the trypsin susceptibility assay, exposure to low pH renders the HA (H1/PR8) as sensitive to trypsin digestion (lane 7 versus 8), but small molecule JNJ4796 prevents its conversion to a trypsin-susceptible conformation (lane 11 versus 12). The mechanism is similar to that of fusion-inhibiting CR6261 Fab (lane 9 versus 10). Numbers to the left of the gel indicate molecular mass in kDa.
Crystallographic analysis of JNJ4796-HA complexes
To decipher the structural basis for its mechanism of action and broad group 1 specificity, crystal structures of JNJ4796 in complexes with H1N1 A/Solomon Islands/3/2006 (H1/SI06) and H5/Viet HAs were determined at 2.72 and 2.32 Å resolution, respectively (Figs. 5 and 6, fig. S4, and table S6). JNJ4796 binds with a stoichiometry of three binding sites per trimer in a highly conserved hydrophobic groove at the HA1-HA2 interface in the HA stem (Fig. 5A). The JNJ4796 binding site comprises HA1 His18, Thr318, and β-strand residues His38 to Leu42 and HA2 Thr41 to lle56 from helix-A and Gly20 and Trp21 from the N-terminal fusion peptide (HA2 residues are in italics throughout). The epitope recognized by the small molecule is similar to the epitopes of stem-targeting bnAbs CR6261, FI6v3, and CR9114 (Fig. 5, C and F) (6, 7, 14). JNJ4796 occupies the same hydrophobic groove as CR6261, where hydrophobic residues from the heavy-chain complementarity-determining regions (HCDRs) and framework region 3 (HFR3), including HCDR2 signature residues Ile53 and Phe54 encoded by the germline gene VH1-69, insert into the binding site (Fig. 5C).
(A) The crystal structure of JNJ4796 in complex with influenza HA from the group 1 H1/SI06 strain. JNJ4796 is shown in a ball-and-stick representation, with one HA protomer of H1/SI06 rendered as a cartoon and the other two protomers in surface representation. HA1 is in light gray and HA2 in aquamarine. A magnified view of one of three JNJ4796 binding sites in the HA trimer is shown with the C, O, and N atoms of JNJ4796 in yellow, red, and blue, respectively. (B) 2Fo-Fc electron density map (black color mesh) contoured at 1σ is displayed around the bound conformation of JNJ4796 in complex with H1/SI06 HA. (C) Overlay of the structure of JNJ4796 (yellow ball and sticks) in complex with H1/SI06 with the HA-interacting loop residues (green sticks) from CR6261 Fab of the HA-Fab complex [Protein Data Bank (PDB) 3GBN]. JNJ4796 occupies the same conserved hydrophobic groove at the interface of HA1 and HA2 as residues from HFR3, HCDR1, HCDR2, and HCDR3 of Fab CR6261. (D) The molecular structure of JNJ4796 with the A- to E-rings labeled. (E) CH-π and other polar interactions in the JNJ4796-H1/SI06 HA complex, with the interactions of each ring [labeled in (D)] depicted as black dotted lines and measured in Å. The centroid of each of the rings is shown as a red sphere. (F) Comparison of the footprints of small molecule JNJ4796 and stem-targeting bnAbs on the HA. The JNJ4796 footprint on H1/SI06 HA is highlighted in red, with the interacting residues labeled in white. Footprints of Fabs CR6261, FI6v3, and CR9114 in the complex with H1N1 A/Brevig mission/1/1918 (PDB 3GBN), H1N1 A/California/04/2009 (PDB 3ZTN), and H5/Viet (PDB 4FQI), respectively, are depicted in green, with the corresponding interacting residues labeled in white.
(A to C) The binding mode of JNJ4796 on H1/SI06 (A) and H5/Viet (C) HAs. HAs are represented as gray and lavender molecular surfaces and JNJ4796 as yellow and purple sticks. An overlay of the JNJ4796 binding modes in H1/SI06 and H5/Viet is shown in (B). The V40Q (Val40→Gln) mutation and conformations of the E-ring of JNJ4796 are highlighted in the red dotted ellipses. (D to F) Key differences in the binding site of JNJ4796 bound to H1/SI06 and H5/Viet HAs. The average B values of JNJ4796 and its individual rings A and E in the structures of JNJ4796-H1/SI06 and JNJ4796-H5/Viet HAs are shown in (D). Noncovalent interactions of the B-ring of JNJ4796 with H5/Viet HA are shown in (E), where the interactions are represented as black dotted lines and measured in Å. The centroid of the ring is shown in the red sphere. The B-ring is represented in the purple ball-and-stick model and HA in light blue cartoon. An overlay of the conformation of the E-ring of JNJ4796 bound to H1/SI06 (yellow) and H5/Viet (purple) HAs is shown in (F), with nitrogen atoms of the E-ring depicted in blue. The E-ring shows an ~180° flip in the H1 versus H5 HA binding site. (G and H) Superimposition of JNJ4796 (yellow) from its group 1 H1/SI06 complex onto a group 2 apo H3/HK68 (cyan) HA. H3/HK68 residues of interest are shown as cyan sticks with a transparent molecular surface. Potential steric clashes are shown between the A to E rings of JNJ4796 and the glycosylated Asn38 and His18 from HA1 (G) and Trp21, Asn49, and Leu52 from HA2 (H) of H3/HK68 HA. (I) Magnified view of the conformation of residues that have potential steric clashes from group 2 H3/HK68 (cyan sticks) versus the corresponding residues from group 1 H1/SI06 (gray sticks).
JNJ4796 contacts this hydrophobic groove through both hydrophobic and polar interactions (Fig. 5E and fig. S5). The substituted benzoxazole moiety (A-ring; the rings A to E are depicted in Fig. 5D and indicated in bold throughout) of JNJ4796 occupies a small hydrophobic cavity formed by Val40 and Leu42 on HA1 and Val52, Asn53, and IIe56 on helix-A (Fig. 5, A and E). In addition, JNJ4796 makes a polar CH-π interaction with the Cγ1 CH of Val52. Such H-bond interactions can contribute ~1.0 kcal/mol to the binding energy (44, 45). The B-ring of JNJ4796 engages HA1 Thr318 through a direct H bond with its hydroxyl group and a CH-π interaction with Cγ2 CH from Thr318. The edge of the B-ring also makes nonpolar contacts with IIe48 and Thr49 from helix-A. The C- and D-rings of JNJ4796 make CH-π bonds with His18 and His38 from HA1 and Trp21 from HA2, whereas the E-ring makes a CH-π bond with Cδ1 CH from IIe45. This triad of rings, C to E, locks the conformation of Trp21, which is important in the fusion process (46, 47). Overall, JNJ4796 buries ~453 Å2 (on H1) and ~472 Å2 (on H5) of surface at the HA interface, completely enveloping the central hydrophobic groove as compared with more discontinuous coverage by stem-targeting bnAbs (CR6261, FI6v3, and CR9114) (Fig. 5F).
The structural basis for the ~25-fold decreased binding affinity of JNJ4796 to H5 HA compared with H1 HA was elucidated by comparing the crystal structures of JNJ4796 bound to H1/SI06 and H5/Viet HAs (Fig. 6, A to F). The A- and E-rings of JNJ4796 have higher thermal mobility when bound to H5 as compared with H1 HA (Fig. 6D). The presence of Val40 in H1 HA allows for a more stable interaction of the A-ring with the hydrophobic groove, whereas the corresponding Gln40 in H5, with its longer and more flexible side chain, does not interact as tightly, thereby increasing the dynamics of the bound A-ring that contributes to the higher thermal mobility of JNJ4796 (Fig. 6, A to D). The E-ring is relatively solvent-exposed in both HA-JNJ4796 complexes but is rotated ~180° in H5 compared with H1 HA (Fig. 5F). Another key difference pertains to the B-ring, where the pyridine moiety is displaced outward by ~0.4 Å in the H5-JNJ4796 complex, resulting in slightly weaker interactions compared with the H1-JNJ4796 complex (Figs. 5E and 6E).
To rationalize the inability of JNJ4796 to bind with group 2 HAs, the apo structure of group 2 HA H3N2 A/Hong Kong/1/1968 (H3/HK68) was compared with group 1 HA (H1/SI06) bound to JNJ4796. The key differences in the epitope of the small molecule on H1 HA with the corresponding region on H3 HA are the presence of a glycosylation site at HA1 Asn38 in group 2 HAs, the orientations of His18 and Trp21 on HA1 and HA2, respectively, and substitution of Thr49 and Val52 in helix-A of group 1 H1 to larger Asn49 and Leu52 in group 2 H3 HAs (Fig. 6, G to I). The stem-targeting bnAbs FI6v3 and CR9114 are able to reorient the glycan and accommodate these helix-A differences to acquire pan-influenza reactivity, whereas bnAbs CR6261 and F10 are unable to do so and exhibit group 1 specificity. The different orientations of these antibodies on the HA surface also reflect their different breadths for group 1 and group 2 HAs (48). JNJ4796 is probably also unable to reorient the Asn38 glycan and accommodate the helix-A mutations and, therefore, would experience steric clashes with group 2 H3 HA (Fig. 6, G to I) that could account for its group 1 specificity.
Conclusions
Although antibodies are increasingly recognized as effective therapeutics, they may fail to achieve broad applicability in some settings owing to inconvenience in administration and relatively high cost. Therefore, replacing antibodies with small molecules is desirable. Here we present proof of concept for antibody-guided, small-molecule discovery. Starting from a well-characterized antibody with a desired activity profile, we selected and further improved a small-molecule “antibody mimetic” that recapitulates the antibody features in vitro and in vivo. We demonstrated the feasibility of targeting the conserved stem epitope on influenza HA with the orally bioavailable small molecule JNJ4796 with comparable high affinity and breadth of binding to bnAb CR6261. The compound mimics the key interactions observed in the antibody-HA cocrystal structures, inhibits the pH-sensitive conformational change of HA, neutralizes influenza viruses in vitro, and protects mice from lethal viral challenge. The success of this approach demonstrates the advantage of rigorously considering the targeted epitope-specific binding activity in close combination with the associated functional activity and mechanism of action, instead of focusing on potency alone. This strategy allows for the generation of robust structure-activity relationships, thereby yielding a great level of control over the pharmacodynamic and pharmacokinetic properties of the selected ligand classes.
Material and Methods
Expression and purification of the hemagglutinin for binding and x-ray crystallography
Hemagglutinin (HA) proteins for binding and crystallographic studies were expressed using a baculovirus system as described previously (49, 50). Briefly, each HA was fused with gp67 signal peptide at the N terminus and to a BirA biotinylation site, thrombin cleavage site, trimerization domain, and His-tag at the C terminus. The HAs were purified using metal affinity chromatography using Ni-NTA resin. For binding studies, each HA was biotinylated with BirA and purified by gel filtration chromatography. For crystallization studies, each HA was treated with trypsin (New England Biolabs, 5mU trypsin per mg HA, overnight at 4°C) to produce uniformly cleaved HA (HA1/HA2), and to remove the trimerization domain and His-tag. The cleaved material was purified by gel filtration.
Site-specific modification of the HA for conformational change inhibition assay
HA constructs were designed with a Sortase A recognition motif (LPETG) between the trimerization domain and C-terminal His-tag to allow site-specific modification via Sortase A mediated transpeptidation (37). Expi293F cells (Thermo Fischer) were transfected with a pcDNA2004 mammalian expression plasmid encoding the modified HA protein, according to manufacturer’s instructions. The cell culture supernatant was harvested 7 days posttransfection. Soluble HA was purified from clarified supernatant via a three-step purification protocol: HisTrap Excel (GE Healthcare Life Sciences), HisTrap HP (GE Healthcare Life Sciences), and finally Superdex 200 Size Exclusion (GE Healthcare Life Sciences). Purified HA proteins were biotinylated using a peptide-based Sortase A recognition sequence, GGGGGK-Biotin (Pepscan). The labeling reaction was performed as described previously (51). Excess peptide and Sortase A were separated from the modified HA via Superdex 200 Size Exclusion (GE Healthcare Life Sciences). For the conformational change assay, the uncleaved HA (HA0) was cleaved into HA1 and HA2 by incubation with trypsin (Trysin EDTA, Gibco), and the reaction was stopped by addition of Trypsin Inhibitor (Gibco).
Expression and purification of mini-protein HB80.4
DNA encoding HB80.4-His was cloned into pET29b(+) (Novagen) and expressed using Rosetta (DE3) cells (Novagen) and auto-induction Magic Media (ThermoFisher). Bacteria were grown to an optical density at 600 nm (OD600) of ~0.6 at 37°C and then at 25°C overnight. Periplasmic extracts were obtained using BugBuster protein extraction reagent (EMD Millipore) according to the manufacturer’s specifications. HB80.4 was purified from periplasmic extract using HisTrap FF (GE Healthcare Life Sciences) followed by dialysis with PBS pH 7.4 (Gibco). Purity was determined by SDS-PAGE and was >95%.
Human antibodies and influenza viruses for assays
Fully human IgG1 antibodies CR6261 (7), CR9114 (6), and CH65 (52) were expressed and purified as described previously (8). Wild-type influenza viruses A/Brisbane/59/2007 (H1N1), A/California/07/2009 (H1N1), A/New Caledonia/20/1999 (H1N1), A/Puerto Rico/8/1934 (H1N1), A/Solomon Islands/3-2006 IVR-145 (H1N1), A/Hong Kong/156/1997 (H5N1), A/Vietnam/1194/2004 (H5N1), A/Brisbane/10/2007 (H3N2), H7N7 7:1 reassortant virus with the HA of A/New York/107/2003 (H7/NY), and the remaining segments from A/Puerto Rico/8/1934 and influenza B B/Brisbane/60/2008) were propagated and rescued in PER.C6 cells (53) by standard viral culture techniques.
AlphaLISA competition assay
Small-molecule (SM) compounds were dissolved at 5 mM in 100% DMSO and threefold serially diluted in 100% DMSO nine times in 96-well, half-area microtiter plates. The compounds were further diluted 1:40 in assay buffer (PBS, 0.05% BSA, 0.05% Tween-20) and subsequently spun down for 15 min at 1000g to separate any insoluble material. 10 μl of the diluted compounds were incubated for 60 min with 10 μl HA biotinylated with a Lightning Link kit (Innova Biosciences, 2.5 nM in assay buffer) in white 96-well, half-area untreated plates (PerkinElmer), after which 10 μl of His-labeled competitor protein HB80.4 (diluted in assay buffer) was added, followed by another 60-min incubation, addition of 10 μl of anti-His acceptor beads (PerkinElmer; 50 μg/ml in assay buffer), and a third 60-min incubation. Finally, 10 μl of streptavidin donor beads (PerkinElmer; 50 μg/ml in assay buffer) were added, followed by 60-min incubation before the plates were read in a microplate reader at 615 nm (Biotek Synergy Neo). Eight copies of samples containing no compound, with and without HA, were prepared for each plate and served as high and low controls, respectively. The sigmoidal inhibition curves were fitted with a robust four-parameter logistic model to derive IC50 values. For the high-throughput screen, only a single concentration of the SM compounds was tested (final concentration of 30 μM).
Truhit AlphaLISA counter assay
Threefold serially diluted SM compounds dissolved in 100% DMSO were screened using the TruHits kit (PerkinElmer) following the manufacturer’s instructions. The compounds were tested in the same dilutions as described in the AlphaLISA competition assay (assay buffer: PBS, 0.05% BSA, 0.05% Tween-20). Eight copies of samples containing no compound, with and without HA, were prepared for each plate and served as high and low controls, respectively.
Cell toxicity assay
MDCK cells (ATCC CCL-34) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and 2 mM l-glutamine at 37°C, 10% CO2. On the day of the experiment, MDCK cells were seeded in 2× infection medium (DMEM, 1× l-glutamine, 6 μg/ml trypsin-EDTA) at 25,000 cells/well (50 μl) in white opaque 96-well plates (BD Falcon). The 96-well plates containing threefold serially diluted compounds were further diluted 1:10 in incomplete medium (DMEM, 1× l-glutamine) and subsequently spun down (1000g, 15 min). A volume of 12 μl was added to 48 μl incomplete medium in a fresh 96-well plate (96-well sterile cell culture plate, V-bottom, Greiner). Then, 50 μl of the compound solution was added to the MDCK plate followed by incubation for 96 hours at 37°C, 10% CO2. After incubation, cell viability was measured by adding 70 μl of ATPlite 1step reagent (PerkinElmer) to the wells. Luminescence was measured in a Biotek Synergy neo plate reader.
Virus neutralization assay (VNA)
Threefold serial dilutions of the SM compounds were prepared as described above. On the day of the experiment, MDCK cells were seeded in 2× infection medium (DMEM, 1× l-glutamine, 6 μg/ml trypsin-EDTA) in white opaque 96-well plates at 25,000 cells/well. A 10× predilution of the SMs was prepared in incomplete medium (DMEM, 1× l-glutamine). CR6261 was diluted to 15 μM in incomplete medium and serially diluted 1:3 nine times. Subsequently, the SMs and CR6261 were spun down at 1000g for 15 min. After spinning, 12 μl supernatant was added to 48 μl of the respective virus dilutions in incomplete medium in a 96-well plate. Two solvent controls, with and without virus, were setup for each sample. Then, 50 μl of the virus/SM mixture was added to the cells followed by incubation for 4 days at 37°C under 10% CO2. After incubation, 70 μl of ATPlite 1step (PerkinElmer) was added to the wells and the plate read for luminescence.
Conformational change inhibition assay (CCI)
Threefold serial diluted SM compounds were prepared as described in the AlphaLISA competition assay above and further diluted 1:100 in assay buffer (PBS, 1% BSA, 0.1% Tween-20). White, half-area, high-binding, 96-well plates (Perkin Elmer) were coated overnight with 50 μl 0.5 μg/ml streptavidin (Pierce) in PBS (Gibco). The plates were washed (150 μl PBS, 0.05% Tween-20) and blocked by exposing them to assay buffer for 1 hour. Subsequently, plates were incubated for 60 min with 50 μl sortase biotinylated, trypsin-EDTA (Gibco) cleaved HA (0.1 μg/ml in assay buffer). After 60 min, the plates were washed, and 50 μl of the diluted compounds were added to the plates for 60 min while shaking. Then, 10 μl 1M acetate pH 5.25 was added to induce HA conformational changes, followed by 20-min incubation at room temperature on a shaking platform. The plates were washed followed by addition of 2.5 mM DTT (diluted in PBS) to reduce any postfusion HA and remove HA1. To detect the presence or absence of HA1, after 60-min incubation on a shaking platform, plates were washed followed by incubation with 0.5 μg/ml HA1 head-binding antibody CH65-HRP (labeled with Lightning Link HRP, Innova Biosciences) in assay buffer for 60 min. The plates were washed, and 50 μl of POD substrate (Roche) was added followed by luminescence read out on a Biotek Synergy Neo plate reader. Total inhibition of the low-pH induced HA conformational change was achieved by using HA stem-binding bnAbs CR6261 or CR9114 (4 nM) as a positive control. Complete conformational change of the HA was achieved at low pH in presence of a nonbinding IgG1 control (40 nM).
Kinetic solubility
SM compounds were dissolved in DMSO to obtain 5 mM DMSO stock solutions. Stock solutions were diluted in duplicate into the required buffers (pH 4.0 and pH 7.4) to a final maximum concentration of 100 μM in 2% DMSO/buffer. Sample plates were gently shaken for 4 hours at room temperature. The samples were centrifuged and the supernatants were transferred to a new plate and diluted 1:1 with HCL/ACN for UPLC analysis. Reference and analyte samples were analyzed by UPLC/UV using a generic UPLC method. The measured solubilities are presented as mean values of duplicate determinations, with a maximum solubility threshold of 100 μM, and the lower limit of quantitation governed by the UV absorption properties of the compound.
Metabolic stability
SM compounds (1 μM) were incubated with pooled human liver microsomes (BD Ultrapool; pooled male and female) and pooled mouse liver microsomes (male CD mice). Microsomes (final protein concentration 0.5 mg/ml), 0.1 M phosphate buffer pH 7.4 containing 1 mM MgCl2 and SM compound (final substrate concentration 1 μM; final DMSO concentration 0.05%) were preincubated at 37°C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final incubation volume was 500 μl. Three species-specific control compounds were included with each species. All incubations were performed singularly for each SM. At six time points (0, 5, 10, 20, 40, and 60 min), reactions were stopped by transferring 50 μl of the incubation mixture into methanol. SM compound concentrations were analyzed by LC-MS/MS and the resulting data used to determine the half-life and intrinsic clearance of the compound in each species.
Plasma protein binding
The free and bound fractions of the SM compound in mouse and human plasma was determined by rapid equilibrium dialysis (RED device, Thermo Fisher Scientific, Geel, Belgium). The RED device consists of a 48-well plate containing disposable inserts bisected by a semipermeable membrane creating two chambers. A 300-μl aliquot of plasma containing SM compound at 5 μM was placed one side and 500 μl of phosphate buffered saline (PBS) on the other. The plate was sealed and incubated at approximately 37°C for 4.5 hours. Samples were removed from both the plasma and buffer compartment and analyzed for the SM compound using a specific LC-MS/MS method to estimate free and bound concentrations.
In vivo pharmacokinetics
The pharmacokinetic profiles of the SM compounds were evaluated in fed male BALB/c mice (n = 3/group). Mice were i.v. injected with the SM compound at 2.5 mg/kg, formulated as a 0.25 mg/ml solution in 20% w/v hydroxypropyl-beta-cyclodextrin pH 6.5, and blood samples were collected from the saphenous vein at 0.08, 0.17, 0.5, 1, 2, 4, 7, and 24 hours into EDTA-containing microcentrifuge tubes. The compound was administered p.o. at 10 mg/kg, formulated as 1.33 mg/ml solution in 20% w/v hydroxypropyl-beta-cyclodextrin pH 6.5, and blood samples were collected from the saphenous vein at 0.5, 1, 2, 4, 7, 12, and 24 hours into EDTA-containing microcentrifuge tubes. The blood samples were immediately centrifuged at 4°C, and the plasma was stored at −20°C. SM compound concentrations from the plasma samples were analyzed using LC-MS/MS. Individual plasma concentration-time profiles were subjected to a noncompartmental pharmacokinetic analysis (NCA) using Phoenix WinNonlin version 6.3 (Certara, NJ, USA).
In vitro selectivity profiling
The binding selectivity of the SM compounds at concentration of 10 μM was assessed in a panel of 52 radioactive ligand displacement assays (Eurofins Cerep SA) as per the providers’ validated protocols. Results were expressed as percent inhibition of control specific binding. Inhibition values higher than 50% were considered to represent significant effects.
Mouse influenza challenge
Dosing formulations for the SM compounds were freshly prepared on the day before administration by dissolving the SM compound in cyclodextrin (40% hydroxypropyl-beta-cyclodextrin). The SM compound (7.5 ml/kg per animal) was administered twice daily per os (p.o.). Female BALB/cAnNCrl mice (Charles River, Sulzfeld, Germany) were intranasally infected with 2× 25 μl of 25× LD50 or 1× LD90 of H1N1 A/Puerto Rico/8/34 dissolved in sterile phosphate buffered saline (D-PBS). All experiments were in accordance with the general principles governing the use of animals in experiments of the European Communities (Directive 2010/63/EU) and Dutch legislation (The Revised Experiments on Animals Act, 2014). This included licensing of the project by the Central Committee on Animal Experimentation and approval of the study by the Animal Welfare Body.
Viral neutralization assay in HBEC cultures
MucilAir human bronchial epithelial cells (HBECs) (pool of 14 donors) (Epithelix Sàrl) were delivered and maintained at an air-liquid interface according to the manufacturer’s instructions. Each MucilAir insert contained ~500,000 well-differentiated respiratory epithelial cells consisting of ciliated cells, mucus-producing goblet cells, and basal cells. Prior to the start of the experiment, inserts were washed once with PBS (with Ca++ and Mg++) to remove mucus and cell debris. Cells were infected with H1N1 A/Puerto Rico/8/1934 at an MOI of 0.1 and concomitantly treated with a JNJ4796 at different concentrations. Cells were treated both at the apical and basolateral side of the tissue culture. After 1 hour of incubation, virus and compounds administered to the apical compartment were removed to reconstitute the air-liquid interface, whereas cells remained exposed to JNJ4796 through the basolateral compartment. Sham-treated (PBS supplemented with DMSO at a final concentration identical to compound-treated samples) and mock-infected wells were taken along as positive and negative controls, respectively. For each experimental condition, four biological replicates were included. Ninety-six hours postinfection, apical washes (D-PBS, 200μl/insert) of the epithelium were used to determine the amount of released viral RNA by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Trypsin susceptibility (TS) assay
In the TS assay, 5 μM H1/PR8 HA was preincubated separately with 25 μM of JNJ4796 and 10 μM CR6261 Fab for ~30 min at room temperature. Control reactions were incubated with 2% DMSO. The pH of each reaction was lowered using 1 M sodium acetate buffer (pH 5.0). One reaction was retained at pH 7.4 to assess digestion at neutral pH. The reaction solutions were then thoroughly mixed and incubated for about 30 min at 37°C. After incubation, the reaction solutions were equilibrated at room temperature, and the pH was neutralized by addition of 200 mM Tris buffer, pH 8.5. Trypsin-ultra (NEB Inc.) was added at final ratio of 1:50 by mass and the samples were digested for about 40 min at 37°C. After incubation with trypsin, the reaction solutions were equilibrated at room temperature and quenched by addition of nonreducing SDS buffer and boiled for 2 min at 100°C. All samples were analyzed by 4-20% SDS-PAGE gel and imaged using BioRad ChemDoc imaging system.
Surface plasmon resonance (SPR)
All SPR experiments were performed using a Biacore T200 instrument operating at 25°C. Biotinylated HA was covalently immobilized on a streptavidin-coated, carboxymethylated dextran sensor surface (SA chip, GE Healthcare). JNJ4796 was dissolved at 10 mM in 100% DMSO and then diluted in the running buffer [20 mM PBS, 137 mM NaCl, 0.05% P-20 surfactant, pH 7.4 (GE Healthcare), supplemented with 2% DMSO]. Binding constants were obtained from a series of injections of JNJ4796 from 0.1 nM to 1 μM with a flow rate of 30 μl/min. Data from single-cycle kinetics were analyzed using BIAevaluation software. Base lines were adjusted to zero for all curves, and injection start times were aligned. The reference sensorgrams were subtracted from the experimental sensorgrams to yield curves representing specific binding followed by background subtraction (i.e., double-referencing). Binding kinetics was evaluated using a 1:1 binding model (Langmuir) to obtain association rate constants (ka) and dissociation rate constants (kd). Binding affinity (KD) was estimated from the concentration dependence of the observed steady-state responses.
Thermodynamic binding profile
Biotinylated HA was purified using a spin-column to remove excess biotin and covalently immobilized on a streptavidin-coated, carboxymethylated dextran sensor surface (SA chip, GE Healthcare). JNJ4796 was dissolved at 10 mM in 100% DMSO and diluted in the running buffer [20 mM PBS, 137 mM NaCl, 0.05% P-20 surfactant, pH 7.4 (GE Healthcare), supplemented with 2% DMSO]. Binding constants were obtained from a series of injections of JNJ4796 from 0.1 nM to 10 μM in five half-log serial dilutions, individually injected at the following temperatures: 10°, 15°, 25°, 35°, and 40°C, at a flow rate of 30 μl/min. Experiments were performed in duplicate (for H1/NCa) or triplicate (for H1/Bri) to ensure reproducibility. Data from single-cycle kinetics were analyzed using BIAevaluation software (GE Healthcare). Base lines were adjusted to zero for all curves, and injection start times were aligned. The reference sensorgrams were subtracted from the experimental sensorgrams to yield curves representing specific binding followed by background subtraction (i.e., double- referencing). Binding kinetics was evaluated using a 1:1 binding model (Langmuir) to obtain association rate constants (ka) and dissociation rate constants (kd). Binding affinity (KD) was estimated from the concentration dependence of the steady-state responses observed. Changes in thermodynamic parameters enthalpy (ΔH) and entropy (ΔS) were calculated from the slope and intercept, respectively, of the temperature dependence of the dissociation constant using the van’t Hoff approximation: lnKD = −ΔH∕R·T + ΔS∕R, where R is the gas constant and T is the absolute temperature. The binding free energy, ΔG, was derived from the Gibbs-Helmholtz equation (ΔG = ΔH − ΔT · S).
Crystallization and structure determination of the JNJ4796-HA complexes
Gel filtration fractions containing H1/SI06 and H5/Viet HAs were concentrated to ~10 mg/ml in 20 mM Tris, pH 8.0, and 150 mM NaCl. Compound JNJ4796 at ~5 molar excess was incubated with the HAs for ~1 hour at room temperature and centrifuged at 14,000g for ~2 to 3 min before setting up crystallization trials. Crystallization screens used the sitting drop vapor diffusion method with our automated CrystalMation robotic system (Rigaku) at TSRI. Within 3 to 7 days, diffraction quality crystals had formed in 0.2 M disodium hydrogen phosphate, 20% w/v PEG3350 at 20°C (for H1/SI06) and 0.2 M lithium sulfate, 20% w/v PEG3350 at 4°C (for H5/Viet). The resulting crystals were cryoprotected with 5-15% ethylene glycol, flash cooled, and stored in liquid nitrogen until data collection. Diffraction data were collected at 100 K on the Stanford Synchrotron Radiation Lightsource beamline 12-2 and processed with HKL-2000 (54). Initial phases were determined by molecular replacement using Phaser (55, 56) with HA models corresponding to PDB codes 1RU7 (for H1/SI06) and 4FQI (for H5/Viet). Refinement was carried out in Phenix (57), and alternated with manual rebuilding and adjustment in COOT (58). The final coordinates were validated using MolProbity (59). Data collection and refinement statistics are summarized in table S6.
Structural analyses
Surface areas buried on the HA upon binding of JNJ4796 were calculated with the Protein Interfaces, Surfaces and Assemblies (PISA) server at the European Bioinformatics Institute (60). MacPyMol (DeLano Scientific) was used to render structure figures.
Statistics
Inhibition potencies in AlphaLISA, viral neutralization and conformational change inhibition assays were determined by robust four-parameter logistic (4PL) regression routines in SPSS (IBM) or R (www.R-project.org). The inflection point (C value) of the curve is taken as the IC50 value. A transform-both-sides (TBS, square root) approach, robust regression techniques (including Huber’s M) and the optional inclusion of the low and high control values as anchor points were used to stabilize the variance, down-weigh outliers, and to fit incomplete curves, respectively. For reasons of presentation, results and curves were scaled from 0 to 100% that represent the range between the robust averages of the low and high control values. For the H1N1 A/Puerto Rico/8/1934 in vivo challenge studies, the differences in survival (as compared to vehicle control) were tested using a two-sided Fisher’s exact test. The significance of the differences was adjusted for multiple testing by Holm-Bonferroni adjustment for the compounds at their highest dose (within the two studies in which the compounds were investigated), followed by a step-wise approach for lower doses. Statistical analysis of bodyweight was based on area under the curve (AUC) analysis. For this analysis, the last observed bodyweight was carried forward if a mouse died during follow-up of the study. Briefly, the weight per mouse at day 0 was used as baseline, and weight change was determined relative to baseline. The AUC was defined as the summation of the area above and below the baseline. Differences in net AUC’s were tested in a one-way analysis of variance (ANOVA) with the same test strategy and post hoc adjustment as mentioned for survival. Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., USA) and SPSS version 20 (SPSS Inc., USA). Statistical significance level was set at α = 0.05. For studies performed on HBEC cultures, results were statistically analyzed by ANOVA between the log titers and the (categorized) concentrations. The significance is based on the contrasts between the values of each of the three highest concentration with the aggregated results for the concentrations in the lower plateau.
Supplementary Materials
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