The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.

To evaluate the spectrum of antiviral activity of MBX2329 and MBX2546, both compounds were investigated for inhibition of entry of Lassa virus (LASV) and Ebola virus (EBOV), both of which also bear type 1 envelope proteins similar to HA. The pseudotype platform was used because it provided a direct comparison of the activities of MBX2329 and MBX2546 against HIV/LASV-GP, HIV/EBOV-GP, and HIV/HA(H5). Compounds MBX2329 and MBX2546 displayed little inhibitory activity against HIV/LASV-GP (IC90 of ∼100 μM), HIV/EBOV-GP (IC90 of >100 μM), or HIV/VSV-G (IC90 of 85 to >100 μM) (Table 2), suggesting that they specifically inhibit the entry of influenza viruses.
MBX2329 and MBX2546 are potent subtype-specific inhibitors.
MBX2329 inhibited influenza A H1N1 virus strains A/PR/8/34 (H1N1) , A/Florida/21/2008 (H1N1-H275Y) (oseltamivir-resistant strain) , A/Washington/10/2008 (H1N1), and A/California/10/2009 (H1N1) (2009 pandemic strain) with IC50s of between 0.29 μM and 0.53 μM. Similarly, MBX2546 inhibited influenza A H1N1 virus strains A/PR/8/34 (H1N1) and A/Florida/21/2008 (H1N1-H275Y) (oseltamivir-resistant strain) with IC50s of 0.3 μM and 5.8 μM , respectively. MBX2546 also inhibited other H1N1 strains, including A/California/10/2009/H1N1 (2009 pandemic strain), with IC50s of between 0.55 μM and 1.5 μM. Both MBX2329 and MBX2546 inhibited HPAI H5N1 virus strain A/Hong Kong/H5N1 with IC50s of 5.9 μM and 3.6 μM, respectively.

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MBX2329 and MBX2546 bind to the group 1 HA-specific conformational epitope in the HA stem region.
Specific inhibition of influenza viruses with group 1 HA by MBX2329 and MBX2546 suggests that they interact with group 1 HA. To verify that HA is the target of the compounds, we investigated the binding of MBX2329 and MBX2546 to recombinant H5 HA (a group 1 HA) using WaterLOGSY NMR spectroscopy, which is designed to detect binding of small molecules to high-molecular-mass targets . Recombinant NA was used as the specificity control. In Fig. 3A, the top spectrum corresponds to the 1D NMR spectrum of the downfield region of MBX2329, with the aromatic resonances of the compound being denoted by red arrows. The second spectrum corresponds to the WaterLOGSY spectrum observed for MBX2329 in the absence of HA (i.e., a control experiment), and the third spectrum corresponds to the WaterLOGSY spectrum observed for MBX2329 in the presence of H5 HA. The relatively strong positively phased resonances of MBX2329 in the presence of H5 HA indicate that it is binding to HA. Conversely, the absence of the signals in the fourth spectrum, which corresponds to the WaterLOGSY experiment in the presence of NA, suggests that MBX2329 is not binding to NA.

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The conformational antigenic epitope (amino acid positions 318 to 322 in the HA1 subunit and positions 47 to 58 in HA2) recognized by MAb C179 is in the stem region and is specific for influenza virus with group 1 HA. To further explore the potential roles of amino acids in the group 1 HA-specific region in binding to MBX2329 and MBX2546, we generated HIV/HA(H5) carrying single-amino-acid substitutions by alanine scanning mutagenesis and examined the sensitivity of these mutants to MBX2329 and MBX2546. As shown in Fig. 4E, at 6.25 μM, the HIV/HA(H5) mutants bearing either a K51A mutation in HA1 or a G16A mutation in HA2 were less susceptible to inhibition by MBX2329, suggesting that MBX2329 interacts with amino acid residues K51 in HA1 and G16 in HA2. Interestingly, none of the mutants was resistant to MBX2546 at the same concentration, further suggesting that they bind at different sites near the conformational epitope recognized by C179. Therefore, taken together, we conclude that (i) both MBX2329 and MBX2546 bind to HA near the group 1 HA-specific conformational epitope in the HA stem region and (ii) the binding sites are not overlapping in the stem region of trimeric HA. The results are consistent with the notion that these inhibitors block HA-mediated membrane fusion (see below).
MBX2329 and MBX2546 inhibit HA-mediated fusion.
Based on the results described above, both MBX2329 and MBX2546 bind to the HA stem region, which is the target for group 1 HA-specific antibodies that disrupt the HA-mediated membrane fusion process (50–55). To investigate the role of these inhibitors in HA-mediated fusion, we performed hemagglutination and hemolysis assays.
The hemagglutination assay was performed to determine whether MBX2329 and MBX2546 prevented the binding of virus with cell surface receptors containing sialic acid (SA). Briefly, 10-fold serial dilutions of concentrated influenza A/PR/8/34 (H1N1) virus particles were mixed with chicken red blood cells (cRBCs) using virus-only wells without an inhibitor as the positive control and wells lacking both virus and the inhibitor as the negative control. In addition, we used the antiserum to influenza virus H1 HA (ATCC V-301-501-552) at two different dilutions (1:10 and 1:25) as controls. The results of the hemagglutination experiment in the presence of either MBX2329 or MBX2546 were similar to those of the positive control (without any compound), as shown in Fig. 5A. Therefore, the results suggest that neither MBX2329 nor MBX2546 inhibits the binding of influenza virus to cRBCs.

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The hemolysis assay was performed by using influenza A virus A/PR/8/34 (H1N1) to determine the effect of MBX2329 and MBX2546 on fusion. To trigger hemolysis, the virus-cell suspension was acidified (pH 5.2) briefly to initiate HA conformational changes that lyse cRBCs to release hemoglobin. Wells lacking the virus were used as controls to determine the effect of compounds on cRBCs. Both MBX2329 and MBX2546 inhibited acid-induced hemolysis in a dose-dependent manner (Fig. 5B), with IC50s of 2.1 μM and 1.56 μM for MBX2329 and MBX2546, respectively. Therefore, taken together, the results indicate that MBX2329 and MBX2546 inhibit fusion of the virus with the endosomal membrane. Here we also used antiserum to influenza virus H1 HA as a control (data not shown).
MBX2329 and MBX2546 exhibit strong synergy with oseltamivir.
Finally, the synergistic efficacy of MBX2329 and MBX2546 in combination with oseltamivir or amantadine was evaluated by using influenza A(H1N1) virus strain A/California/10/2009 according to previously described methods (45, 46). Both MBX2329 and MBX2546 in combination with oseltamivir displayed marked synergistic inhibition of influenza virus infection (331 ± 112 μM2% for MBX2329 and 36 ± 2.8 μM2% for MBX2546), as shown by plotting the concentration versus synergy . The large volumes of synergy produced by the combination were statistically significant, as indicated by the values at the 95% confidence level (Table 4). Cytotoxicity was also evaluated with the same experimental design used for the combined efficacy study to evaluate synergistic cytotoxicity. These studies used the same MDCK cell monolayers and the same drug exposures as those used for the antiviral studies. At the concentrations used in these studies, no significant cytotoxicity was observed with either oseltamivir, MBX2329, or MBX2546 (data not shown). Strikingly, the observed synergy was restricted to the combination of the HA inhibitors and oseltamivir; no significant synergy was observed with the combination of HA inhibitors and amantadine.

[1]. New small molecule entry inhibitors targeting hemagglutinin-mediated influenza a virus fusion. J Virol. 2014;88(3):1447-1460.

C16H26CLNO

283.836743831635

283.17

1438272-42-4

71526742

White to off-white solid powder

0

1

2

5

19

211

0

0

JMIAUPZSIMYFEG-UHFFFAOYSA-N

InChI=1S/C16H25NO.ClH/c1-2-15-9-5-6-10-16(15)18-14-13-17-11-7-3-4-8-12-17;/h5-6,9-10H,2-4,7-8,11-14H2,1H3;1H

1-[2-(2-ethylphenoxy)ethyl]azepane;hydrochloride

MBX2329; CHEMBL4077590; SCHEMBL14969476; EX-A4615; AKOS032954055; HY-131069A; BS-52166

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~880.78 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.33 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.08 mg/mL (7.33 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)