RESEARCH ARTICLE SUMMARY◥

DRUG DESIGN

A small-molecule fusion inhibitor ofinfluenza virus is orally active in miceMaria J. P. van Dongen*†, Rameshwar U. Kadam*, Jarek Juraszek, Edward Lawson,Boerries Brandenburg, Frederike Schmitz, Wim B. G. Schepens, Bart Stoops,Harry A. van Diepen, Mandy Jongeneelen, Chan Tang, Jan Vermond,Alida van Eijgen-Obregoso Real, Sven Blokland, Divita Garg, Wenli Yu, Wouter Goutier,Ellen Lanckacker, Jaco M. Klap, Daniëlle C. G. Peeters, Jin Wu, Christophe Buyck,Tim H. M. Jonckers, Dirk Roymans, Peter Roevens, Ronald Vogels, Wouter Koudstaal,Robert H. E. Friesen, Pierre Raboisson, Dashyant Dhanak, Jaap Goudsmit, Ian A. Wilson†

INTRODUCTION: Annual influenza epidem-ics and episodic pandemics continue to causewidespread illness and mortality. Strategies toprevent and treat acute influenza infection haveremained limited to seasonal influenza vaccina-tion and a small arsenal of antiviral drugs. Thus,there is an urgent need for additional prophy-lactic and therapeutic options, including newtargets and mechanisms of action, to addressthe considerable challenges posed by the rapidevolution of influenza viruses that limit theeffectiveness of vaccines and the emergenceof antiviral drug resistance.

RATIONALE: The recent characterizationof broadly neutralizing antibodies (bnAbs)against influenza virus identified the highly

conserved hemagglutinin (HA) stem as apromising target for development of univer-sal vaccines and complementary therapeutics.Even though this spurred several bnAbs to beevaluated as passive immunotherapy in clin-ical trials, antibodies are large and complexmolecules that are generally unsuited fororal delivery. We therefore set out to utilizethe structural details of the molecular inter-actions and mechanisms of HA stem bnAbsto identify an orally active small moleculethat mimics bnAb functionality. InfluenzaA viruses can be separated in group 1 andgroup 2 on the basis of their HA subtype(H1 to H18), and anti-stem bnAbs usuallybind to group 1 or to group 2 viruses, but afew can target both.

RESULTS: We screened a diverse chemicallibrary for compounds that selectively targetthe group 1HAepitope of bnAbCR6261 througha binding assay that detects displacement of aCR6261-based designed small protein. Benzyl-piperazines were identified as a major hit class,with JNJ7918 being the most promising can-didate. Consistent with its binding to the func-tional HA stem epitope, this compound alsoneutralized influenza infection in vitro. Keychemical modifications were subsequently in-troduced to optimize binding and neutral-ization potency, as well as properties dictating

metabolic stability andoral bioavailability, to fin-ally afford JNJ4796. Thislead compound binds andneutralizes a broad spec-trum of influenza A group1 viruses in vitro and pro-

tects mice against lethal and sublethal influ-enza challenge after oral administration. Thecompound also effectively neutralizes virus in-fection in reconstituted three-dimensional cellculture of fully differentiated human bron-chial epithelial cells. Like bnAb CR6261, themechanism of action of JNJ4796 was dem-onstrated to be based on inhibition of the pH-sensitive conformational change of HA thattriggers fusion of the viral and endosomalmembranes and release of the viral genome intothe host cell. Cocrystal structures with H1and H5 HAs reveal that JNJ4796 recapit-ulates the original CR6261-HA hotspot inter-actions and provide detailed and valuableinformation on the minimal epitope in theHA1-HA2 fusion region of the stem for anantiviral small molecule to neutralize influ-enza A group 1 viruses.

CONCLUSION: We identified an orally activesmall molecule against influenza A HA thatmimics the binding and functionality of thebroadly neutralizing antibody CR6261. Thesmall molecule targets the conserved HA stemregion, acts as a fusion inhibitor by inhibitingconformational changes that lead to the post-fusion HA structure, and neutralizes a broadspectrum of human pandemic, seasonal, andemerging group 1 influenza A viruses. Thus,the compound holds promise as an urgentlysought-after therapeutic option offering a com-plementary mechanism of action to existingantiviral drugs for the treatment of influenzavirus infection, and that should further aid de-velopment of universal therapeutics that pre-vent entry of influenza virus in host cells.▪

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The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: mvandon@its.jnj.com(M.J.P.v.D.); wilson@scripps.edu (I.A.W.)Cite this article as M. J. P. van Dongen et al., Science 363,eaar6221 (2019). DOI: 10.1126/science.aar6221

A B

90°

Fusion peptide

Influenzahemagglutinin

Small moleculeJNJ4796

N O

NH

NO

N

N

N NN

N

CH3

O

H3C

Influenza A virus HA in complex with small-molecule fusion inhibitor JNJ4796. (A) Crystalstructure of JNJ4796 (red) with H1N1 A/Solomon Islands/3/2006 HA (gray surface). TheN-terminal fusion peptide of the HA2 chain (blue ribbon) is highlighted in orange. (B) View alongthe threefold axis of the HA trimer, with the three identical binding sites of JNJ4796 highlighted(cyan) along with its chemical structure (enlarged view).

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RESEARCH ARTICLE◥

DRUG DESIGN

A small-molecule fusion inhibitor ofinfluenza virus is orally active in miceMaria J. P. van Dongen1,2*†, Rameshwar U. Kadam3*, Jarek Juraszek1,Edward Lawson4, Boerries Brandenburg1,5, Frederike Schmitz1, Wim B. G. Schepens2,Bart Stoops2, Harry A. van Diepen1, Mandy Jongeneelen1,5, Chan Tang1,5,Jan Vermond1, Alida van Eijgen-Obregoso Real1, Sven Blokland1,5, Divita Garg6,Wenli Yu3, Wouter Goutier1, Ellen Lanckacker7, Jaco M. Klap1, Daniëlle C. G. Peeters2,Jin Wu7, Christophe Buyck2, Tim H. M. Jonckers7, Dirk Roymans7, Peter Roevens2,Ronald Vogels1,5, Wouter Koudstaal1‡, Robert H. E. Friesen1§, Pierre Raboisson7¶,Dashyant Dhanak2,4#, Jaap Goudsmit1,8, Ian A. Wilson3,9†

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virusidentified the conserved hemagglutinin (HA) stem as a target for development of universalvaccines and therapeutics. Although several stem bnAbs are being evaluated in clinicaltrials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge ofthe interactions and mechanism of anti-stem bnAb CR6261, we selected and optimizedsmall molecules that mimic the bnAb functionality. Our lead compound neutralizesinfluenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice againstlethal and sublethal influenza challenge after oral administration, and effectivelyneutralizes virus infection in reconstituted three-dimensional cell culture of fullydifferentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAsreveal that the lead compound recapitulates the bnAb hotspot interactions.

The World Health Organization estimatesthat annual influenza epidemics causearound 3 million to 5 million cases of se-vere illness and up to 650,000 deaths world-wide (1, 2). Seasonal influenza vaccination

still remains the best strategy to prevent infection,but the vaccines that are available now offer avery limited breadth of protection. The dis-covery of human broadly neutralizing antibodies(bnAbs) to influenza virus provides hope for thedevelopment of broad-spectrum, universal vac-cines (3–14). Because of the high level of con-

servation of their epitopes in the hemagglutinin(HA) stem, these bnAbs neutralize a wide rangeof viruses within and across influenza virus sub-types. Their binding prevents the pH-inducedconformational changes in HA that are requiredfor viral fusion in the endosomal compartmentsof target cells in the respiratory tract (6–11, 13–15).Efforts have therefore been made to developvaccination modalities aimed at directing theimmune response to theHA stem through differ-ent vaccination regimens (16, 17), sequential vac-cination with different chimeric HA constructs(18, 19), and administration of stem-based im-munogens (20–24). In addition, several bnAbsthemselves are being evaluated in clinical trialsas passive immunotherapy (25). Another recentstrategy to prevent influenza infection stems fromdevelopment of a highly potent multidomainantibody with almost universal breadth againstinfluenza A and B viruses that can be admin-istered intransally inmice using adeno-associatedvirus–mediated gene delivery (26).Therapeutic options to treat acute influenza

infection also include antiviral drugs directed atblocking virus uncoating during cell entry (M2proton channel inhibitors) and progeny releasefrom infected cells (neuraminidase inhibitors)(27, 28). However, resistance to antiviral drugs isan emerging problem, owing to the high muta-tion rate in influenza viruses and their geneticreassembly possibilities (29). New antiviral drugs(30, 31) and combination therapies (32, 33), withalternative mechanisms of action against alter-native viral targets are therefore urgently needed.

Small-molecule drugs, in contrast to antibod-ies, offer the advantage of oral bioavailability,high shelf stability, and relatively low produc-tion costs.Influenza A viruses have been classified into

18 hemagglutinin subtypes (H1 to H18), whichcan be divided phylogenetically into two groups(1 and 2), and 11 neuraminidase subtypes (N1 toN11). Antibody CR6261 broadly neutralizes mostgroup 1 influenza A viruses (7, 9). Cocrystal struc-tures of CR6261 in complex with H1 HA (7, 9)stimulated the design of small-protein ligands ofabout 10 kDa that target the conserved stem re-gion. These small proteins mimic the antibodyinteractions with HA and inhibit influenza virusfusion (34–36). Cocrystal structures of bnAbsFI6v3 and CR9114 with HAs (6, 14) further en-abled the design of even smaller peptides as in-fluenza fusion inhibitors (37). However, neithersmall proteins nor peptides generally are orallybioavailable.Development of small-molecule ligands directed

at antibody binding sites is challenging. Anti-body epitopes, as for other protein-protein inter-faces, are generally flat, large, and undulating(~1000 to 2000 Å2) (38), in stark contrast to thesmall concave pockets (typically in the 300 to500 Å2 range), which are common as targets forsmall-molecule drugs (39). Moreover, to mimicthe function of an HA-stem bnAb, a functionalsmall molecule should reproduce the key inter-actions that lead to fusion inhibition. We havetherefore identified and optimized small mole-cules with such properties through application ofa strategy that was guided by detailed knowledgeof the binding mode and molecular mechanismof bnAb CR6261 (7, 15) and encouraged by earliersuccesses in the design of small proteins and pep-tidic ligands targeted to the HA stem (34, 35, 37).

High-throughput screeningand optimization

To identify potent small molecules that mimicgroup 1 bnAb CR6261, in terms of breadth ofbinding (7, 9, 35), virus neutralization, andmech-anism (Fig. 1A), we screened for compounds thatselectively target the CR6261 epitope on HA. Weapplied the AlphaLISA (amplified luminescentproximity homogeneous assay) technology in com-petition mode as our high-throughput screening(HTS) method (Fig. 1B). A diverse library of~500,000 small-molecule compoundswas screenedfor displacing HB80.4, which is a CR6261-basedcomputationally designed small protein with verysimilar binding mode and fusion inhibition pro-file (34, 35). HB80.4 was used instead of CR6261,because avidity effects leading to higher appar-ent affinity of the bivalent antibody would haveresulted in a more stringent and thus less sen-sitive assay. This approach biased the screentoward compounds that act via the desiredmechanism of action. About 9000 small mole-cules with weak to medium binding capacitywere initially retrieved; binding of 300 com-pounds was confirmed through repeated test-ing and via the Truhit AlphaLISA counter assaythat can identify false-positive hits.

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1Janssen Prevention Center, Janssen Pharmaceutical Companiesof Johnson & Johnson, Archimedesweg 6, Leiden, Netherlands.2Discovery Sciences, Janssen Research & Development,Turnhoutseweg 30, Beerse, Belgium. 3Department of IntegrativeStructural and Computational Biology, The Scripps ResearchInstitute, La Jolla, CA, USA. 4Discovery Sciences, JanssenResearch & Development, 1400 McKean Rd., Spring House, PA,USA. 5Janssen Infectious Diseases and Vaccines, JanssenResearch & Development, Archimedesweg 4-6, Leiden,Netherlands. 6Department of Molecular Medicine, The ScrippsResearch Institute, La Jolla, CA, USA. 7Janssen InfectiousDiseases and Vaccines, Janssen Research & Discovery,Turnhoutseweg 30, Beerse, Belgium. 8Department ofEpidemiology, Harvard T.H. Chan School of Public Health,Boston, MA, USA. 9The Skaggs Institute for Chemical Biology,The Scripps Research Institute, La Jolla, CA, USA.*These authors contributed equally to this work.†Corresponding author. Email: mvandon@its.jnj.com (M.J.P.v.D.);wilson@scripps.edu (I.A.W.) ‡Present address: Lucidity BiomedicalConsulting, Calle Emir 11, Granada, Spain. §Present address: KiadisPharma, Paasheuvelweg 25A, Amsterdam, Netherlands. ¶Presentaddress: Aligos bvba, Bio-Incubator Leuven NV, Gaston Geenslaan 1,Leuven, Belgium. #Present address: Incyte Corporation, 1801Augustine Cut-Off, Wilmington, DE, USA.

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Benzylpiperazines emerged as a major hitclass, with JNJ7918 being the most promisingcandidate with a median inhibitory concentra-tion (IC50) of 1.39 and 13.06 mM against H1N1 A/California/07/2009 (H1/Cal) andH5N1 A/Vietnam/1203/2004 (H5/Viet) HAs, respectively (Fig. 2).No competition with H1 HA head–binding anti-body 2D1 (40) was detected against H1/Cal, fur-ther validating the HA-stem specificity (Fig. 2A).Key chemical modifications to increase molecu-lar interactions with the HA stem, that is, intro-duction of a functional group at the benzylicposition and modification of the phenyl with apropargylmoiety (Fig. 2B), greatly improved bind-ing (~30- to 80-fold) toHAs fromH1/Cal, H1N1 A/New Caledonia/20/1999 (H1/NCa), H1N1 A/Brisbane/59/2007 (H1/Bris), and H5/Viet, asexemplified by the second-generation compoundJNJ6715 (Fig. 2C). Moreover, virus neutraliza-tion assays with a panel of H1 and H5 influenza

strains, which represent human pandemic, sea-sonal, and emerging viruses in influenza Agroup 1, demonstrated improved neutralization(~30- to 500-fold). Like bnAb CR6261, JNJ6715showed heterosubtypic neutralization of group1 viruses without measurable cytotoxicity inMadin-Darby canine kidney (MDCK) cells (Fig.2D and table S1).

In vitro and in vivopharmacokinetic profiling

Further in vitro profiling of JNJ6715 revealed poorkinetic aqueous solubility of 11.3 mM at pH 7.4and poor metabolic stability of >346 ml/min perkilogram based on intrinsic clearance in mouseand human liver microsomes, making this com-pound unsuitable for in vivo testing (Fig. 3A). Toenhance its drug-like properties, we replaced themethyl ester functionality at the benzylic positionwith a 2-methyl oxadiazole group, substituted the

central phenyl by pyridine, and replaced the6-methoxy benzothiazole with a 5-trifluoro-methoxy-benzoxazole group, generating JNJ8897.The compoundwas further optimized to generateJNJ4796 by replacing the 2-methyl oxadiazolegroup with 2-methyl tetrazole and the trifluoro-methoxy group with methyl amide (Fig. 3B).These modifications reduced the intrinsic clear-ance in mouse and human liver microsomes forboth compounds while sustaining virus neutral-ization [median effective concentration (EC50)forH1/Cal andH1/NCa of 0.064 and 0.076 mMforJNJ8897, and 0.066 and 0.038 mM for JNJ4796].In particular, JNJ4796 demonstrated substan-tially reduced human andmurine intrinsic clear-ance (Fig. 3, A and C) and increased aqueoussolubility, which translated to a favorable in vivopharmacokinetics profilewith compoundhalf-life(t1/2) in mice of 2.4 hours after oral administra-tion. The overall bioavailability for this compoundwas 30.0%, with a plasma concentration reach-ing 1152 ng/ml, equivalent to 2.1 mM, after oraladministration at 10 mg/kg (mg of JNJ4796 perkg of body weight) (Fig. 3A). Testing JNJ4796in a diverse panel of pharmacologically relevantreceptors, ion channels, and transporters showedthat the compound does not substantially inhibitany of these potential off-targets (table S2).

Oral efficacy against influenza infection

In line with its in vitro neutralizing activity andacceptable pharmacokinetics data, oral adminis-tration of JNJ4796 protected mice from lethalchallenge 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 twicedaily, initiated one day before challenge and con-tinuing for 7 days, resulted in 100% survival atday 21 in comparison to the less potent com-pound JNJ8897, for which less than 50% survivalwas achieved (day 21 survival was 40% for 50mg/kg and 25% for 10 mg/kg) (Fig. 3D and tables S3and S4). JNJ4796 only partly alleviated morbid-ity in this stringent H1N1 infection model, as de-monstrated by animal weight loss. Nevertheless,dose-dependent reversal of weight loss was ob-served at the study end for both 50 mg/kg and10 mg/kg of JNJ4796 (Fig. 3D). Oral doses ofJNJ4796 also resulted in dose-dependent effi-cacy after a sublethal viral challenge (LD90), withtwice daily administration of 15 and 5 mg/kg ofJNJ4796 giving rise to 100% survival and onlymoderate weight loss effects that were moreoverrestricted 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 infec-tion, the neutralization capacity of JNJ4796 wasevaluated in a reconstituted three-dimensionalcell culture of fully differentiated human bron-chial epithelial cells (HBECs) derived from apool of donors. This model system recapitulatesseveral relevant characteristics of human airwaytarget tissue, such as production of mucus, ciliabeating, and local metabolic activity (41), whichcould potentially limit the compound’s efficacy.Incubation of an HBEC culture, infected with

van Dongen et al., Science 363, eaar6221 (2019) 8 March 2019 2 of 10

Fig. 1. Approach for small-molecule discovery through mimicking the binding mode andfunctionality of broadly neutralizing group 1 influenza antibody CR6261. (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 representedas a gray molecular surface with different shades for the different protomers, CR6261 in greenwith a molecular surface for the Fab interacting with the HA and a cartoon for the other Fab andFc of the immunoglobulin G, and the binding epitope on the HA in red. The middle panel illustratesthe HA phylogenetic tree, which shows the relationship between the 18 HA subtypes of influenzaA virus (group 1 and 2) and the two lineages of influenza B viruses. The breadth of binding of theCR6261 to multiple group 1 subtypes is shown in green, light gray indicates where binding wasnot tested, and black indicates no binding. The right panel shows the mechanism of action ofCR6261 to block the pH-induced HA conformational changes. (B) Our HTS utilized the AlphaLISAtechnology to identify small molecules targeting the CR6261 epitope. The CR6261-mimicking designedprotein HB80.4 was used in the binding competition assay. HB80.4 is represented in blue and theCR6261 epitope on the HA in pink. Small molecules are indicated as illustrative structural formulae.

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H1N1 A/Puerto Rico/8/1934 virus, with the com-pound dramatically reduced the viral titers whenassessed 96 hours postinfection (fig. S2).

Group 1 binding specificity andmechanism of JNJ4796

Like bnAb CR6261, JNJ4796 further demonstra-ted heterosubtypic group 1 HA binding and virusneutralization breadth without cytotoxicity (Fig.4, A and B; fig. S3; and tables S1 and S5). CR6261neutralizes group 1 influenza A viruses by in-

hibiting the low-pH-induced HA conformationalchange, which triggers fusion of the viral andendosomal membranes and release of the viralgenome into the host cell (42). Each HA mono-mer is composed of two subunits (HA1 andHA2).CR6261 and JNJ4796 bind components of bothHA1 and HA2 in the trimeric HA stem, therebystabilizing the prefusion conformation of HAand preventing conformational rearrangementsin the HA stem that lead to the postfusion struc-ture. This inhibition is demonstrated in two dif-

ferent experiments. First, in a conformationalchange inhibition assay (Fig. 4C), JNJ4796 dose-dependently prevents pH-induced transition ofHA to the postfusion conformation and subse-quent loss of the HA1 subunit after reductionof the interchain HA disulfide. Second, theprotease-susceptibility assay indicates that, likethe stem-targeting bnAbs, compound JNJ4796stabilizes the prefusion conformation and blocksthe HA conformational change at low pH and thesubsequent susceptibility to trypsin (43) (Fig. 4D).

Crystallographic analysis ofJNJ4796-HA complexes

To decipher the structural basis for its mecha-nism of action and broad group 1 specificity,crystal structures of JNJ4796 in complexes withH1N1 A/Solomon Islands/3/2006 (H1/SI06) andH5/Viet HAs were determined at 2.72 and 2.32 Åresolution, respectively (Figs. 5 and 6, fig. S4, andtable S6). JNJ4796 binds with a stoichiometry ofthree binding sites per trimer in a highly con-served hydrophobic groove at the HA1-HA2 in-terface in the HA stem (Fig. 5A). The JNJ4796binding site comprises HA1 His18, Thr318, andb-strand residuesHis38 to Leu42 andHA2 Thr41 tolle56 from helix-A and Gly20 and Trp21 from theN-terminal fusion peptide (HA2 residues are initalics throughout). The epitope recognized bythe small molecule is similar to the epitopesof stem-targeting bnAbs CR6261, FI6v3, andCR9114 (Fig. 5, C and F) (6, 7, 14). JNJ4796 oc-cupies the same hydrophobic groove as CR6261,where hydrophobic residues from the heavy-chain complementarity-determining regions(HCDRs) and framework region 3 (HFR3), includ-ing HCDR2 signature residues Ile53 and Phe54

encoded by the germline gene VH1-69, insert intothe binding site (Fig. 5C).JNJ4796 contacts this hydrophobic groove

through both hydrophobic and polar interactions(Fig. 5E and fig. S5). The substituted benzoxazolemoiety (A-ring; the rings A to E are depicted inFig. 5D and indicated in bold throughout) ofJNJ4796 occupies a small hydrophobic cavityformed by Val40 and Leu42 on HA1 and Val52,Asn53, and IIe56 on helix-A (Fig. 5, A and E). Inaddition, JNJ4796 makes a polar CH-p interac-tion with the Cg1 CH of Val52. Such H-bond in-teractions can contribute ~1.0 kcal/mol to thebinding energy (44, 45). The B-ring of JNJ4796engages HA1 Thr318 through a direct H bondwith its hydroxyl group and a CH-p interactionwith Cg2 CH from Thr318. The edge of the B-ringalso makes nonpolar contacts with IIe48 and Thr49

from helix-A. The C- and D-rings of JNJ4796make CH-p bondswithHis18 andHis38 fromHA1and Trp21 fromHA2,whereas theE-ringmakes aCH-p bond with Cd1 CH from IIe45. This triad ofrings, 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 hydrophobicgroove as compared with more discontinuouscoverage by stem-targeting bnAbs (CR6261, FI6v3,and CR9114) (Fig. 5F).

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Fig. 2. Optimization of in vitro HA stem–binding and virus neutralization. (A) Representativedose-dependent competitive binding for compound JNJ7918 that emerged from a HTS that used theAlphaLISA assay. This compound inhibited binding of the designed protein HB80.4 against the HAstem to H1/Cal and H5/Viet HAs with IC50’s of 2.3 and 21.4 mM [−log of median inhibitoryconcentration (pIC50) = 5.6 and 4.7], respectively, but not to the head binding antibody 2D1, whentested with H1/Cal. The x axis depicts the log molar concentration of the compound, and the y axisdepicts the normalized response, relative to the positive (no inhibitor and no HA) and negative (noinhibitor) control. (B) Chemical structures of JNJ7918 and JNJ6715, with key modifications to thelatter highlighted in yellow circles. (C) Scatter plot depicting H1/Cal, H1/NCa, H1/Bris, and H5/Vietbinding, calculated from AlphaLISA assay as log IC50 versus virus neutralization as log EC50. Eachdot represents a tested compound. The dotted line represents the lower limit of quantification.(D) Virus neutralization EC50 values in mM for compounds JNJ7918 and JNJ6715 against theindicated 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; H3N2A/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 onPER.C6 cells; and influenza B B/Brisbane/60/2008 (B/Bris).

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The structural basis for the ~25-fold decreasedbinding affinity of JNJ4796 to H5 HA comparedwith H1 HA was elucidated by comparing thecrystal structures of JNJ4796 bound to H1/SI06and H5/Viet HAs (Fig. 6, A to F). The A- and E-rings of JNJ4796 have higher thermal mobilitywhen bound toH5 as comparedwithH1HA (Fig.6D). The presence of Val40 in H1 HA allows for amore stable interaction of the A-ring with thehydrophobic groove, whereas the correspondingGln40 in H5, with its longer and more flexibleside chain, does not interact as tightly, therebyincreasing the dynamics of the bound A-ringthat contributes to the higher thermal mobilityof JNJ4796 (Fig. 6, A to D). The E-ring is rela-tively solvent-exposed in both HA-JNJ4796 com-plexes but is rotated ~180° in H5 compared withH1 HA (Fig. 5F). Another key difference pertainsto the B-ring, where the pyridine moiety is dis-placed outward by ~0.4 Å in the H5-JNJ4796complex, resulting in slightly weaker interactionscompared 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 2HA H3N2 A/Hong Kong/1/1968 (H3/HK68) was

compared with group 1 HA (H1/SI06) bound toJNJ4796. The key differences in the epitope ofthe small molecule on H1 HA with the corre-sponding region on H3 HA are the presence of aglycosylation site at HA1 Asn38 in group 2 HAs,the orientations of His18 and Trp21 on HA1 andHA2, respectively, and substitution of Thr49 andVal52 in helix-A of group 1 H1 to larger Asn49

and Leu52 in group 2 H3HAs (Fig. 6, G to I). Thestem-targeting bnAbs FI6v3 and CR9114 areable to reorient the glycan and accommodatethese helix-A differences to acquire pan-influenzareactivity, whereas bnAbs CR6261 and F10 areunable to do so and exhibit group 1 specificity.The different orientations of these antibodies ontheHA surface also reflect their different breadthsfor group 1 and group 2 HAs (48). JNJ4796 isprobably also unable to reorient the Asn38 glycanand accommodate the helix-A mutations and,therefore, would experience steric clashes withgroup 2 H3 HA (Fig. 6, G to I) that could accountfor its group 1 specificity.

Conclusions

Although antibodies are increasingly recognizedas effective therapeutics, they may fail to achieve

broad applicability in some settings owing to in-convenience in administration and relatively highcost. Therefore, replacing antibodies with smallmolecules is desirable. Here we present proof ofconcept for antibody-guided, small-molecule dis-covery. Starting from a well-characterized anti-body with a desired activity profile, we selectedand further improved a small-molecule “anti-body mimetic” that recapitulates the antibodyfeatures in vitro and in vivo. We demonstratedthe feasibility of targeting the conserved stemepitope on influenza HA with the orally bio-available small molecule JNJ4796 with compa-rable high affinity and breadth of binding tobnAb CR6261. The compound mimics the keyinteractions observed in the antibody-HA co-crystal structures, inhibits the pH-sensitive con-formational change of HA, neutralizes influenzaviruses in vitro, and protects mice from lethalviral challenge. The success of this approachdemonstrates the advantage of rigorously con-sidering the targeted epitope-specific bindingactivity in close combination with the asso-ciated functional activity and mechanism ofaction, instead of focusing on potency alone.This strategy allows for the generation of robust

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Fig. 3. Lead compound JNJ4796 is orally bioavailable and effectivein vivo. (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 livermicrosomes (hLM/mLM); plasma protein binding (PPB) human andmurine (hu/mu); mouse t1/2 (h, hours; iv, intravenous; po, per os); areaunder the curve (AUC 0−1); maximum concentration (Cmax) po; timepoint for maximum concentration (tmax) po; and po bioavailability. ND,not determined. (B) Chemical structures of JNJ6715, JNJ8897, andJNJ4796, with key chemical modifications compared with the previousgeneration highlighted in orange and red circles. (C) Scatter plots depict

CLint of small-molecule compounds in human and mouse liver micro-somes versus their average log EC50 neutralization value against H1/Caland H1/NCa HAs. The individual black dots represent the analyzed small-molecule compounds, and the yellow, orange, and red dots representJNJ6715 (EC50 = 0.14 mM), JNJ8897 (EC50 = 0.063 mM), and JNJ4796(EC50 = 0.033 mM), respectively. The shaded area reflects the optimalarea 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 indicatedcompounds at days −1 to 5 (twice daily).

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structure-activity relationships, thereby yielding agreat level of control over the pharmacodynamicand pharmacokinetic properties of the selectedligand classes.

Material and MethodsExpression and purificationof the hemagglutinin for bindingand x-ray crystallography

Hemagglutinin (HA) proteins for binding andcrystallographic studies were expressed usinga baculovirus system as described previously(49, 50). Briefly, each HA was fused with gp67signal peptide at the N terminus and to a BirAbiotinylation site, thrombin cleavage site, trimer-ization domain, and His-tag at the C terminus.The HAs were purified using metal affinity chro-matography using Ni-NTA resin. For bindingstudies, each HA was biotinylated with BirA andpurified by gel filtration chromatography. For

crystallization studies, each HA was treated withtrypsin (New England Biolabs, 5mU trypsin permg HA, overnight at 4°C) to produce uniformlycleaved HA (HA1/HA2), and to remove the tri-merization domain and His-tag. The cleavedmaterial was purified by gel filtration.

Site-specific modification of the HA forconformational change inhibition assay

HA constructs were designed with a Sortase Arecognition motif (LPETG) between the trimer-ization domain and C-terminal His-tag to allowsite-specific modification via Sortase A mediatedtranspeptidation (37). Expi293F cells (ThermoFischer) were transfected with a pcDNA2004mammalian expression plasmid encoding themodified HA protein, according to manufac-turer’s instructions. The cell culture supernatantwas harvested 7 days posttransfection. SolubleHA was purified from clarified supernatant via a

three-step purification protocol: HisTrap Excel(GE Healthcare Life Sciences), HisTrap HP (GEHealthcare Life Sciences), and finally Superdex200 Size Exclusion (GE Healthcare Life Sciences).Purified HA proteins were biotinylated using apeptide-based Sortase A recognition sequence,GGGGGK-Biotin (Pepscan). The labeling reac-tion was performed as described previously (51).Excess peptide and Sortase A were separatedfrom the modified HA via Superdex 200 SizeExclusion (GE Healthcare Life Sciences). For theconformational change assay, the uncleaved HA(HA0) was cleaved into HA1 and HA2 by incu-bation with trypsin (Trysin EDTA, Gibco), andthe reaction was stopped by addition of TrypsinInhibitor (Gibco).

Expression and purification ofmini-protein HB80.4

DNA encoding HB80.4-His was cloned intopET29b(+) (Novagen) and expressed using Rosetta(DE3) cells (Novagen) and auto-induction MagicMedia (ThermoFisher). Bacteria were grown toan optical density at 600 nm (OD600) of ~0.6at 37°C and then at 25°C overnight. Periplasmicextracts were obtained using BugBuster proteinextraction reagent (EMDMillipore) according tothe manufacturer’s specifications. HB80.4 waspurified from periplasmic extract using HisTrapFF (GE Healthcare Life Sciences) followed bydialysis with PBS pH 7.4 (Gibco). Purity was de-termined by SDS-PAGE and was >95%.

Human antibodies and influenza virusesfor assays

Fully human IgG1 antibodies CR6261 (7), CR9114(6), and CH65 (52) were expressed and purifiedas described previously (8). Wild-type influenzaviruses A/Brisbane/59/2007 (H1N1), A/California/07/2009 (H1N1), A/NewCaledonia/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/Bris-bane/10/2007 (H3N2), H7N7 7:1 reassortant viruswith the HA of A/New York/107/2003 (H7/NY),and the remaining segments fromA/Puerto Rico/8/1934 and influenza B B/Brisbane/60/2008) werepropagated and rescued in PER.C6 cells (53) bystandard viral culture techniques.

AlphaLISA competition assay

Small-molecule (SM) compounds were dissolvedat 5 mM in 100% DMSO and threefold seriallydiluted in 100% DMSO nine times in 96-well,half-area microtiter plates. The compounds werefurther diluted 1:40 in assay buffer (PBS, 0.05%BSA, 0.05% Tween-20) and subsequently spundown for 15 min at 1000g to separate any in-soluble material. 10 ml of the diluted compoundswere incubated for 60 min with 10 ml HA bio-tinylated with a Lightning Link kit (InnovaBiosciences, 2.5 nM in assay buffer) in white 96-well, half-area untreated plates (PerkinElmer),after which 10 ml of His-labeled competitor pro-tein HB80.4 (diluted in assay buffer) was added,followed by another 60-min incubation, additionof 10 ml of anti-His acceptor beads (PerkinElmer;

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Fig. 4. Group 1 binding specificity and mechanism of JNJ4796. (A) Small molecule JNJ4796 bindsmultiple group 1 subtypes (green text), as displayed on the HA phylogenetic tree (see Fig. 1A). Grayindicates that binding against H8, bat H17, and bat H18 HA subtypes was not tested, and black indicatesno binding. (B) Comparison of breadth of virus neutralization for JNJ4796 and CR6261 againstrepresentative 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 ofJNJ4796 to cleaved H1/Bris HA blocks the low-pH-induced conformational change after lowering the pHto 5.25.The log of the molar concentration of JNJ4796 is plotted against the inhibition of conformationalchange normalized to the full inhibition by CR6261 (positive control).The vertical intersecting linerepresents the IC50. (D) Small molecule JNJ4796 inhibits the low-pH-induced conformational changes inH1/PR8 HA. In the trypsin susceptibility assay, exposure to low pH renders the HA (H1/PR8) as sensitiveto trypsin digestion (lane 7 versus 8), but small molecule JNJ4796 prevents its conversion to a trypsin-susceptible conformation (lane 11 versus 12).Themechanism is similar to that of fusion-inhibiting CR6261Fab (lane 9 versus 10). Numbers to the left of the gel indicate molecular mass in kDa.

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50 mg/ml in assay buffer), and a third 60-minincubation. Finally, 10 ml of streptavidin donorbeads (PerkinElmer; 50 mg/ml in assay buffer)were added, followed by 60-min incubation be-fore the plates were read in amicroplate readerat 615 nm (Biotek Synergy Neo). Eight copies ofsamples containing no compound, with andwithout HA, were prepared for each plate andserved as high and low controls, respectively.The sigmoidal inhibition curves were fitted witha robust four-parameter logistic model to deriveIC50 values. For the high-throughput screen, onlya single concentration of the SM compoundswastested (final concentration of 30 mM).

Truhit AlphaLISA counter assay

Threefold serially diluted SM compounds dis-solved in 100% DMSO were screened using theTruHits kit (PerkinElmer) following the manu-facturer’s instructions. The compounds weretested in the same dilutions as described in theAlphaLISA competition assay (assay buffer: PBS,0.05% BSA, 0.05% Tween-20). Eight copies ofsamples containing no compound, with andwithout HA, were prepared for each plate andserved as high and low controls, respectively.

Cell toxicity assay

MDCK cells (ATCC CCL-34) were maintained inDulbecco’s Modified Eagle Medium (DMEM,Gibco) supplementedwith 10% fetal bovine serumand 2 mM L-glutamine at 37°C, 10% CO2. On theday of the experiment, MDCK cells were seededin 2× infectionmedium (DMEM, 1× L-glutamine,6 mg/ml trypsin-EDTA) at 25,000 cells/well(50 ml) inwhite opaque 96-well plates (BD Falcon).The 96-well plates containing threefold seriallydiluted compounds were further diluted 1:10 inincomplete medium (DMEM, 1× L-glutamine)and subsequently spun down (1000g, 15 min). Avolume of 12 ml was added to 48 ml incompletemedium in a fresh 96-well plate (96-well sterilecell culture plate, V-bottom, Greiner). Then, 50 mlof the compound solution was added to theMDCK plate followed by incubation for 96 hoursat 37°C, 10% CO2. After incubation, cell viabilitywas measured by adding 70 ml of ATPlite 1step re-agent (PerkinElmer) to the wells. Luminescencewasmeasured in aBiotek Synergyneoplate reader.

Virus neutralization assay (VNA)

Threefold serial dilutions of the SM compoundswere prepared as described above. On the day ofthe experiment, MDCK cells were seeded in 2×infectionmedium (DMEM, 1× L-glutamine, 6 mg/ml trypsin-EDTA) in white opaque 96-well platesat 25,000 cells/well. A 10× predilution of the SMswas prepared in incompletemedium (DMEM, 1×L-glutamine). CR6261 was diluted to 15 mM inincomplete medium and serially diluted 1:3 ninetimes. Subsequently, the SMs and CR6261 werespun down at 1000g for 15 min. After spinning,12 ml supernatant was added to 48 ml of the re-spective virus dilutions in incomplete mediumin a 96-well plate. Two solvent controls, with andwithout virus, were setup for each sample. Then,50 ml of the virus/SM mixture was added to the

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Fig. 5. Structural characterization of the JNJ4796 binding site on influenza HA. (A) Thecrystal structure of JNJ4796 in complex with influenza HA from the group 1 H1/SI06 strain. JNJ4796is shown in a ball-and-stick representation, with one HA protomer of H1/SI06 rendered as acartoon and the other two protomers in surface representation. HA1 is in light gray and HA2 inaquamarine. A magnified view of one of three JNJ4796 binding sites in the HA trimer is shown withthe C, O, and N atoms of JNJ4796 in yellow, red, and blue, respectively. (B) 2Fo-Fc electron densitymap (black color mesh) contoured at 1s is displayed around the bound conformation of JNJ4796in complex with H1/SI06 HA. (C) Overlay of the structure of JNJ4796 (yellow ball and sticks) incomplex with H1/SI06 with the HA-interacting loop residues (green sticks) from CR6261 Fab of theHA-Fab complex [Protein Data Bank (PDB) 3GBN]. JNJ4796 occupies the same conservedhydrophobic groove at the interface of HA1 and HA2 as residues from HFR3, HCDR1, HCDR2, andHCDR3 of Fab CR6261. (D) The molecular structure of JNJ4796 with the A- to E-rings labeled. (E) CH-pand 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 isshown as a red sphere. (F) Comparison of the footprints of small molecule JNJ4796 and stem-targetingbnAbs on the HA. The JNJ4796 footprint on H1/SI06 HA is highlighted in red, with the interactingresidues labeled in white. Footprints of Fabs CR6261, FI6v3, and CR9114 in the complex with H1N1A/Brevig mission/1/1918 (PDB 3GBN), H1N1 A/California/04/2009 (PDB 3ZTN), and H5/Viet (PDB4FQI), respectively, are depicted in green, with the corresponding interacting residues labeled in white.

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cells followed by incubation for 4 days at 37°Cunder 10% CO2. After incubation, 70 ml of ATPlite1step (PerkinElmer) was added to the wells andthe plate read for luminescence.

Conformational change inhibitionassay (CCI)

Threefold serial diluted SM compounds wereprepared as described in the AlphaLISA compe-tition assay above and further diluted 1:100 inassay buffer (PBS, 1% BSA, 0.1% Tween-20).White, half-area, high-binding, 96-well plates(Perkin Elmer) were coated overnight with 50 ml0.5 mg/ml streptavidin (Pierce) in PBS (Gibco).The plates were washed (150 ml PBS, 0.05%Tween-20) and blocked by exposing them toassay buffer for 1 hour. Subsequently, plateswere incubated for 60 min with 50 ml sortasebiotinylated, trypsin-EDTA (Gibco) cleaved HA(0.1 mg/ml in assay buffer). After 60 min, theplates were washed, and 50 ml of the dilutedcompounds were added to the plates for 60 minwhile shaking. Then, 10 ml 1M acetate pH 5.25was added to induce HA conformational changes,followed by 20-min incubation at room temper-ature on a shaking platform. The plates werewashed followed by addition of 2.5 mM DTT(diluted in PBS) to reduce any postfusion HAand remove HA1. To detect the presence or ab-sence of HA1, after 60-min incubation on ashaking platform, plates were washed followedby incubation with 0.5 mg/ml HA1 head-bindingantibody CH65-HRP (labeledwith Lightning LinkHRP, Innova Biosciences) in assay buffer for60 min. The plates were washed, and 50 ml ofPOD substrate (Roche) was added followed byluminescence read out on a Biotek SynergyNeo plate reader. Total inhibition of the low-pHinduced HA conformational change was achievedby using HA stem-binding bnAbs CR6261 orCR9114 (4 nM) as a positive control. Completeconformational change of the HA was achievedat low pH in presence of a nonbinding IgG1control (40 nM).

Kinetic solubility

SM compounds were dissolved in DMSO to ob-tain 5 mM DMSO stock solutions. Stock solu-tions were diluted in duplicate into the requiredbuffers (pH 4.0 and pH 7.4) to a final maximumconcentration of 100 mM in 2% DMSO/buffer.Sample plates were gently shaken for 4 hours atroom temperature. The samples were centrifugedand the supernatants were transferred to a newplate and diluted 1:1 with HCL/ACN for UPLCanalysis. Reference and analyte samples wereanalyzed by UPLC/UV using a generic UPLCmethod. Themeasured solubilities are presentedasmean values of duplicate determinations, witha maximum solubility threshold of 100 mM, andthe lower limit of quantitation governed by theUV absorption properties of the compound.

Metabolic stability

SM compounds (1 mM) were incubated withpooled human liver microsomes (BD Ultrapool;pooled male and female) and pooled mouse liver

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Fig. 6. Structural basis for the group-specific binding of JNJ4796 on influenza HA. (A to C) Thebindingmode of JNJ4796 onH1/SI06 (A) and H5/Viet (C)HAs. HAs are represented as gray and lavendermolecular surfaces and JNJ4796 as yellow and purple sticks. An overlay of the JNJ4796 binding modesin H1/SI06 and H5/Viet is shown in (B).The V40Q (Val40→Gln) mutation and conformations of theE-ring of JNJ4796 are highlighted in the red dotted ellipses. (D to F) Key differences in the binding siteof JNJ4796 bound to H1/SI06 and H5/Viet HAs.The average B values of JNJ4796 and its individualrings 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 theinteractions are represented as black dotted lines and measured in Å.The centroid of the ring is shownin the red sphere.TheB-ring is represented in the purple ball-and-stickmodel 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 ofinterest are shown as cyan sticks with a transparent molecular surface. Potential steric clashes areshown 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 conformationof residues that have potential steric clashes from group 2 H3/HK68 (cyan sticks) versus thecorresponding residues from group 1 H1/SI06 (gray sticks).

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microsomes (male CD mice). Microsomes (finalprotein concentration 0.5 mg/ml), 0.1 M phos-phate buffer pH 7.4 containing 1 mMMgCl2 andSMcompound (final substrate concentration 1 mM;final DMSO concentration 0.05%) were preincu-bated at 37°C prior to the addition of NADPH(final concentration 1 mM) to initiate the reac-tion. The final incubation volume was 500 ml.Three species-specific control compounds wereincluded with each species. All incubations wereperformed singularly for each SM. At six timepoints (0, 5, 10, 20, 40, and 60 min), reactionswere stopped by transferring 50 ml of the incu-bation mixture into methanol. SM compoundconcentrations were analyzed by LC-MS/MS andthe resulting data used to determine the half-lifeand intrinsic clearance of the compound in eachspecies.

Plasma protein binding

The free and bound fractions of the SM com-pound in mouse and human plasma was deter-mined by rapid equilibriumdialysis (REDdevice,Thermo Fisher Scientific, Geel, Belgium). TheRED device consists of a 48-well plate containingdisposable inserts bisected by a semipermeablemembrane creating two chambers. A 300-mlaliquot of plasma containing SM compound at5 mM was placed one side and 500 ml of phos-phate buffered saline (PBS) on the other. Theplate was sealed and incubated at approxi-mately 37°C for 4.5 hours. Samples were removedfrom both the plasma and buffer compartmentand analyzed for the SM compound using aspecific LC-MS/MS method to estimate free andbound concentrations.

In vivo pharmacokinetics

The pharmacokinetic profiles of the SM com-pounds were evaluated in fed male BALB/c mice(n = 3/group). Mice were i.v. injected with theSM compound at 2.5 mg/kg, formulated as a0.25 mg/ml solution in 20% w/v hydroxypropyl-beta-cyclodextrin pH 6.5, and blood sampleswere 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 compoundwas administered p.o. at 10 mg/kg, formulatedas 1.33 mg/ml solution in 20% w/v hydroxypropyl-beta-cyclodextrin pH 6.5, and blood samples werecollected from the saphenous vein at 0.5, 1, 2,4, 7, 12, and 24 hours into EDTA-containingmicrocentrifuge tubes. The blood samples wereimmediately centrifuged at 4°C, and the plasmawas stored at −20°C. SM compound concentra-tions from the plasma samples were analyzedusing LC-MS/MS. Individual plasma concentration-time profiles were subjected to a noncompartmen-tal pharmacokinetic analysis (NCA) using PhoenixWinNonlin version 6.3 (Certara, NJ, USA).

In vitro selectivity profiling

The binding selectivity of the SM compoundsat concentration of 10 mM was assessed in apanel of 52 radioactive ligand displacement as-says (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 con-sidered to represent significant effects.

Mouse influenza challenge

Dosing formulations for the SM compounds werefreshly prepared on the day before administra-tion by dissolving the SM compound in cyclo-dextrin (40% hydroxypropyl-beta-cyclodextrin).The SM compound (7.5 ml/kg per animal) wasadministered twice daily per os (p.o.). FemaleBALB/cAnNCrl mice (Charles River, Sulzfeld,Germany) were intranasally infected with 2×25 ml of 25× LD50 or 1× LD90 of H1N1 A/PuertoRico/8/34 dissolved in sterile phosphate bufferedsaline (D-PBS). All experiments were in accor-dance with the general principles governing theuse of animals in experiments of the EuropeanCommunities (Directive 2010/63/EU) and Dutchlegislation (The Revised Experiments on AnimalsAct, 2014). This included licensing of the pro-ject by the Central Committee on Animal Exper-imentation and approval of the study by theAnimal Welfare Body.

Viral neutralization assayin HBEC cultures

MucilAir human bronchial epithelial cells (HBECs)(pool of 14 donors) (Epithelix Sàrl) were de-livered and maintained at an air-liquid interfaceaccording to the manufacturer’s instructions.Each MucilAir insert contained ~500,000 well-differentiated respiratory epithelial cells consist-ing of ciliated cells, mucus-producing gobletcells, and basal cells. Prior to the start of theexperiment, inserts were washed once with PBS(with Ca++ and Mg++) to remove mucus and celldebris. Cells were infected with H1N1 A/PuertoRico/8/1934 at an MOI of 0.1 and concomitantlytreated with a JNJ4796 at different concentra-tions. Cells were treated both at the apical andbasolateral side of the tissue culture. After 1 hourof incubation, virus and compounds admin-istered to the apical compartment were removedto reconstitute the air-liquid interface, whereascells remained exposed to JNJ4796 through thebasolateral compartment. Sham-treated (PBS sup-plemented with DMSO at a final concentrationidentical to compound-treated samples) andmock-infected wells were taken along as pos-itive and negative controls, respectively. Foreach experimental condition, four biologicalreplicates were included. Ninety-six hours post-infection, apical washes (D-PBS, 200ml/insert)of the epithelium were used to determine theamount of released viral RNA by quantitativereverse transcriptase polymerase chain reaction(qRT-PCR).

Trypsin susceptibility (TS) assay

In the TS assay, 5 mM H1/PR8 HA was pre-incubated separately with 25 mMof JNJ4796 and10 mM CR6261 Fab for ~30 min at room temper-ature. Control reactions were incubated with 2%DMSO. The pH of each reaction was loweredusing 1 M sodium acetate buffer (pH 5.0). Onereaction was retained at pH 7.4 to assess di-

gestion at neutral pH. The reaction solutionswere then thoroughly mixed and incubatedfor about 30 min at 37°C. After incubation, thereaction solutions were equilibrated at room tem-perature, and the pH was neutralized by additionof 200 mM Tris buffer, pH 8.5. Trypsin-ultra(NEB Inc.) was added at final ratio of 1:50 bymass and the samples were digested for about40 min at 37°C. After incubation with trypsin,the reaction solutions were equilibrated at roomtemperature and quenched by addition of non-reducing SDSbuffer and boiled for 2min at 100°C.All samples were analyzed by 4-20% SDS-PAGEgel and imaged using BioRad ChemDoc imagingsystem.

Surface plasmon resonance (SPR)

All SPR experiments were performed using aBiacore T200 instrument operating at 25°C.Biotinylated HA was covalently immobilized ona streptavidin-coated, carboxymethylated dex-tran sensor surface (SA chip, GE Healthcare).JNJ4796 was dissolved at 10 mM in 100% DMSOand then diluted in the running buffer [20 mMPBS, 137mMNaCl, 0.05% P-20 surfactant, pH 7.4(GEHealthcare), supplementedwith 2%DMSO].Binding constants were obtained from a series ofinjections of JNJ4796 from 0.1 nM to 1 mMwith aflow rate of 30 ml/min. Data from single-cyclekinetics were analyzed using BIAevaluation soft-ware. Base lines were adjusted to zero for allcurves, and injection start times were aligned.The reference sensorgramswere subtracted fromthe experimental sensorgrams to yield curves re-presenting specific binding followed by back-ground subtraction (i.e., double-referencing).Binding kinetics was evaluated using a 1:1 bind-ing model (Langmuir) to obtain association rateconstants (ka) and dissociation rate constants(kd). Binding affinity (KD) was estimated fromthe concentration dependence of the observedsteady-state responses.

Thermodynamic binding profile

BiotinylatedHAwas purified using a spin-columnto remove excess biotin and covalently immobi-lized on a streptavidin-coated, carboxymethylateddextran sensor surface (SA chip, GE Healthcare).JNJ4796 was dissolved at 10 mM in 100% DMSOand diluted in the running buffer [20 mM PBS,137 mMNaCl, 0.05% P-20 surfactant, pH 7.4 (GEHealthcare), supplemented with 2% DMSO].Binding constants were obtained from a seriesof injections of JNJ4796 from 0.1 nM to 10 mMin five half-log serial dilutions, individuallyinjected at the following temperatures: 10°, 15°,25°, 35°, and 40°C, at a flow rate of 30 ml/min.Experiments were performed in duplicate (forH1/NCa) or triplicate (for H1/Bri) to ensure re-producibility. Data from single-cycle kineticswere analyzed using BIAevaluation software (GEHealthcare). Base lines were adjusted to zero forall curves, and injection start times were aligned.The reference sensorgramswere subtracted fromthe experimental sensorgrams to yield curves re-presenting specific binding followed by back-ground subtraction (i.e., double- referencing).

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Binding kinetics was evaluated using a 1:1 bind-ing model (Langmuir) to obtain association rateconstants (ka) and dissociation rate constants(kd). Binding affinity (KD) was estimated fromthe concentration dependence of the steady-state responses observed. Changes in thermo-dynamic parameters enthalpy (DH) and entropy(DS) were calculated from the slope and inter-cept, respectively, of the temperature depend-ence of the dissociation constant using the van’tHoff approximation: lnKD = −DH/R·T + DS/R,where R is the gas constant and T is the absolutetemperature. The binding free energy, DG, wasderived from theGibbs-Helmholtz equation (DG=DH − DT · S).

Crystallization and structuredetermination of theJNJ4796-HA complexes

Gel filtration fractions containing H1/SI06 andH5/Viet HAs were concentrated to ~10 mg/mlin 20 mM Tris, pH 8.0, and 150 mM NaCl. Com-pound JNJ4796 at ~5 molar excess was in-cubated with the HAs for ~1 hour at roomtemperature and centrifuged at 14,000g for~2 to 3 min before setting up crystallizationtrials. Crystallization screens used the sittingdrop vapor diffusion method with our auto-mated CrystalMation robotic system (Rigaku)at TSRI. Within 3 to 7 days, diffraction qualitycrystals had formed in 0.2 M disodium hydro-gen phosphate, 20% w/v PEG3350 at 20°C (forH1/SI06) and 0.2 M lithium sulfate, 20% w/vPEG3350 at 4°C (for H5/Viet). The resultingcrystals were cryoprotected with 5-15% eth-ylene glycol, flash cooled, and stored in liquidnitrogen until data collection. Diffraction datawere collected at 100 K on the Stanford Syn-chrotron Radiation Lightsource beamline 12-2and processed with HKL-2000 (54). Initial phaseswere determined by molecular replacementusing Phaser (55, 56) with HA models corre-sponding to PDB codes 1RU7 (for H1/SI06) and4FQI (for H5/Viet). Refinement was carried outin Phenix (57), and alternated with manual re-building and adjustment in COOT (58). The finalcoordinates were validated using MolProbity(59). Data collection and refinement statisticsare summarized in table S6.

Structural analyses

Surface areas buried on the HA upon binding ofJNJ4796 were calculated with the Protein Inter-faces, Surfaces and Assemblies (PISA) serverat the European Bioinformatics Institute (60).MacPyMol (DeLano Scientific) was used to renderstructure figures.

Statistics

Inhibition potencies in AlphaLISA, viral neutral-ization and conformational change inhibition as-says were determined by robust four-parameterlogistic (4PL) regression routines in SPSS (IBM) orR (www.R-project.org). The inflection point (Cvalue) of the curve is taken as the IC50 value. Atransform-both-sides (TBS, square root) ap-proach, robust regression techniques (includ-

ing Huber’s M) and the optional inclusion of thelow and high control values as anchor pointswere used to stabilize the variance, down-weighoutliers, and to fit incomplete curves, respec-tively. For reasons of presentation, results andcurves were scaled from0 to 100% that representthe range between the robust averages of the lowand high control values. For the H1N1 A/PuertoRico/8/1934 in vivo challenge studies, the differ-ences in survival (as compared to vehicle control)were tested using a two-sided Fisher’s exact test.The significance of the differences was adjustedfor multiple testing by Holm-Bonferroni adjust-ment for the compounds at their highest dose(within the two studies in which the compoundswere investigated), followedbya step-wise approachfor lower doses. Statistical analysis of body-weight was based on area under the curve (AUC)analysis. For this analysis, the last observedbodyweight was carried forward if a mouse diedduring follow-up of the study. Briefly, the weightper mouse at day 0 was used as baseline, andweight change was determined relative to base-line. The AUC was defined as the summation ofthe area above and below the baseline. Differ-ences in net AUC’s were tested in a one-wayanalysis of variance (ANOVA) with the same teststrategy and post hoc adjustment as mentionedfor survival. Statistical analyses were performedusing SAS version 9.4 (SAS Institute Inc., USA)and SPSS version 20 (SPSS Inc., USA). Statisticalsignificance level was set at a = 0.05. For studiesperformed on HBEC cultures, results were stat-istically analyzed by ANOVA between the logtiters and the (categorized) concentrations. Thesignificance is based on the contrasts betweenthe values of each of the three highest concen-tration with the aggregated results for the con-centrations in the lower plateau.

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ACKNOWLEDGMENTS

We thank P. Vermeulen for setting up the high-throughput screenand for analysis of the data; B. Shook and P. Jackson for sharingexpertise in medicinal chemistry; S. Ceyhan for execution of invitro activity assays; M. Seijsener-Peeters and A. McCreary forscientific guidance on in vitro assay execution, in vivo testing, anddata analysis; T. Kwaks, J. Kolkman, D. Zuijdgeest, M. van der NeutKolfschoten, and M. Bujny for helpful discussions on antibody-related activities; H. Tien for help in setting up automatedcrystallization screens; J. P. Verenini for help with manuscriptformatting; and R. Stanfield, X. Zhu, and X. Dai for helpfuldiscussions on structure refinement. Funding: This work issupported in part by NIH grants R56 AI117675 and R56 AI127371(to I.A.W.). R.U.K is grateful to the Swiss National ScienceFoundation for an Early Postdoc.Mobility fellowship. X-ray datasetswere collected at the Stanford Synchrotron Radiation Lightsource(SSRL beamline 12-2). Use of the SSRL, SLAC National AcceleratorLaboratory, is supported by the U.S. Department of Energy(DOE), Office of Science, Office of Basic Energy Sciences, undercontract no. DE-AC02-76SF00515. The SSRL Structural MolecularBiology Program is supported by the DOE Office of Biologicaland Environmental Research and by the National Institutes ofHealth, National Institute of General Medical Sciences (includingP41GM103393). Author contributions: M.J.P.v.D., R.U.K., J.J.,B.B., R.V., P.Ro., R.H.E.F., P.Ra., D.D., J.G., and I.A.W. designed theproject; R.U.K., J.J., M.J., C.T., J.V., A.v.E.-O.R., S.B., D.G., W.Y., W.G.,E.Lan., and J.W. performed experiments; M.J.P.v.D., R.U.K., J.J.,E.Law., B.B., W.B.G.S., B.S., H.A.v.D., J.M.K., D.C.G.P., J.W., C.B.,T.H.M.J., D.R., P.Ro., R.V., W.K., P.Ra., and I.A.W. provided scientificguidance on experimental setup and execution and performed dataanalysis and interpretation; M.J.P.v.D., R.U.K., F.S., W.K., and I.A.W.wrote the manuscript; and all authors provided comments andsuggestions on the manuscript. Competing interests: A patentapplication related to this work has been filed by some of the authors(application number WO2018EP52537; publication number WO2018141854). The content of this manuscript is solely theresponsibility of the authors and does not necessarily represent theofficial view of Janssen Pharmaceutical Companies of Johnson &Johnson or the official views of NIAID, NIGMS, or NIH. This ismanuscript 29584 from The Scripps Research Institute. Data andmaterials availability: All data and code to understand and assessthe conclusions of this research are available in the main text,supplementary materials, and the Protein Data Bank via accessioncodes 6CF7 and 6CFG. The sharing of materials described in this workwill be subject to standard material transfer agreements.

SUPPLEMENTARY MATERIALS

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7 December 2017; resubmitted 9 October 2018Accepted 29 January 201910.1126/science.aar6221

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A small-molecule fusion inhibitor of influenza virus is orally active in mice

Robert H. E. Friesen, Pierre Raboisson, Dashyant Dhanak, Jaap Goudsmit and Ian A. WilsonPeeters, Jin Wu, Christophe Buyck, Tim H. M. Jonckers, Dirk Roymans, Peter Roevens, Ronald Vogels, Wouter Koudstaal, Eijgen-Obregoso Real, Sven Blokland, Divita Garg, Wenli Yu, Wouter Goutier, Ellen Lanckacker, Jaco M. Klap, Daniëlle C. G.Wim B. G. Schepens, Bart Stoops, Harry A. van Diepen, Mandy Jongeneelen, Chan Tang, Jan Vermond, Alida van Maria J. P. van Dongen, Rameshwar U. Kadam, Jarek Juraszek, Edward Lawson, Boerries Brandenburg, Frederike Schmitz,

DOI: 10.1126/science.aar6221 (6431), eaar6221.363Science 

, this issue p. eaar6221Scienceafter oral administration and neutralized virus infection in a 3D cell culture of human bronchial epithelial cells.bnAb, the compound inhibited viral fusion in the endosomes of target cells. The compound protected mice from influenza

theselected and optimized a small-molecule lead compound that recapitulates key interactions of the bnAb with HA. Like et al.provide hope for the development of universal vaccines and are being evaluated in clinical trials. Van Dongen

virusbreadth. Broadly neutralizing antibodies (bnAbs) that target the conserved hemagglutinin (HA) stem of the influenza Many of us rely on seasonal vaccines for protection against influenza and are only too aware of their limited

A small molecule that targets influenza

ARTICLE TOOLS http://science.sciencemag.org/content/363/6431/eaar6221

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