- Email: [email protected]
Fluorescent sialic derivatives for the specific detection of influenza viruses
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Fluorescent sialic derivatives for the specific detection of influenza viruses Dandan Liua, Xikai Cuia, Abasaheb N. Dhawanea, Vasanta Chivukulab, Suri S. Iyera,
⁎
a b
788 Petit Science Center, Department of Chemistry, Centre for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, United States Atlanta Metropolitan State College, 1630 Metropolitan Parkway, Atlanta, GA 30310, United States
ARTICLE INFO
ABSTRACT
Keywords: Influenza Streptococcus pneumoniae Neuraminidase Sialic acid Glycan
Early and accurate diagnosis of influenza viruses can decrease its harmful impact. Here, we have synthesized fluorescent sialic acid derivatives that are cleaved by influenza neuraminidases (NAs) and not by Streptococcus pneumoniae that also inhabits the human olfactory. We have also attempted to develop assays that could differentiate between influenza virus and S. pneumoniae by taking advantage of the structural differences between NAs from these pathogens.
Respiratory infections caused by influenza cause considerable health and economic harm.1,2 Seasonal influenza leads to millions of infections worldwide leading to > 960,000 hospitalizations when it causes increased respiratory distress.3 Vaccinations are highly recommended for senior citizens, children and immunocompromised people, however, it is not feasible or realistic to vaccinate everyone. Antivirals are most efficacious when administered 24–48 h prior to infection in a prophylactic manner. Additionally, the initial infection caused by the virus often leads to secondary infection caused by bacterial pathogens.4 It is very important for a physician to ascertain if the infection is viral or bacterial, because the treatment is different. For influenza infections, antiviral drugs like Tamiflu or Relenza are prescribed;5,6 in contrast, for bacteria, antibiotics such as Azithromycin Zpak, is prescribed.7 Secondary infections can increase burden, for example,8,9 infections caused by Streptococcus pneumoniae, can lead to otitis media, especially in immunocompromised people and children five years old or younger.10 It takes 1–4 days for symptoms of influenza related sickness to appear after exposure to the virus. The time when secondary infection due to S. pneumoniae or other bacteria occurs is not very clear, as the interplay between viral, bacterial and the host immune system is complex11,12 and to compound the issue further, overall morphological symptoms due to viral/bacteria are quite similar. Thus, by the time the patient enters the clinic, it is unclear if the patient is suffering from viral, bacterial or coinfections. Indeed, postmortem analysis of the 1918 and the 1957 influenzas pandemics demonstrated that a significant number of patients had a secondary bacterial illness, up to 80% of the latter pandemic severe and fatal cases had bacterial infections;4 29% of the 2009 H1N1 swine influenza pandemic had secondary bacterial infections.9 Typically, primary care physicians
⁎
focus on patient history and underlying morbidity, the longevity of the infection (which is often subjective), the inflammation in the throat/ ears to start antiviral/antibiotic treatment. However, these physical examinations are far from ideal for a variety of reasons that include misdiagnosis, increased pathogen resistance due to overuse of antibiotic13 or antiviral therapy,14,15 side effects of antibiotics, etc. Clearly, it is very important to accurately detect influenza early in the infection process for appropriate treatment. We recently developed an electrochemical assay to detect enzymes using a repurposed glucose meter (Fig. 1). Briefly, we developed substrates bearing a glucose molecule and expose it to samples containing the enzyme.16 If enzyme is present and is active, glucose is released, which can be quantified using a glucose meter and correlated to the activity of the enzyme. Our first target was influenza virus; we synthesized sialic acid bearing molecules that release glucose.16 These molecules, when exposed to NA (or sialidase) from any source resulted in release of glucose, which was detected using a glucose meter. In further iterations, we demonstrated that molecules like galactose17 and paracetamol18 can also be detected using glucose meters; and we synthesized sialic acid derivatives that were highly specific for influenza viruses. Similar biochemiluminescent molecules, when exposed to influenza or S. pneumoniae, were cleaved only by influenza viral strains with high specificity.19 By using a combination of natural and modified sialic acid derivatives, we could differentiate between influenza and S. pneumoniae.17 While electrochemical detection using repurposed glucometers are very valuable in diagnostics, fluorescence based detection of pathogens cannot be understated, especially when it is integrated with smartphones.20,21 Here, we report a fluorescence based assay that could lead to the accurate detection of influenza virus by exploiting
Corresponding author. E-mail address: [email protected] (S.S. Iyer).
https://doi.org/10.1016/j.bmcl.2019.126773 Received 11 August 2019; Received in revised form 19 October 2019; Accepted 19 October 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Dandan Liu, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126773
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 1. Electrochemical assay to detect enzymes using glucose meter. The synthesized substrate can be cleaved by enzyme and release glucose, which can be detected by glucose meter.
Fig. 2. The binding pocket of NA complexed with sialic acid downloaded from NIH PDB database and PyMOL was used to show the surface and binding pocket. The carbon atom is represented in green color, the oxygen atom element in red color, the nitrogen atom in blue color. a) The binding pocket of influenza NA (PDB: 4gzq). b) The binding pocket of S. pneumoniae NA (PDB: 3 h72). These figures shows the binding pocket of influenza NA is larger than S. pneumoniae NA.
structural differences between NAs from influenza and S. pneumoniae. To differentiate between influenza virus and S. pneumoniae, we use the modified substrate, i.e. MUNANA(4,7OMe), because the binding pocket of influenza is considerably larger than any other NA (Fig. 2), especially at the 4,7 positions.22,23 To this end, we synthesized a fluorescent sialic acid derivative bearing OMe groups at the 4 and 7 positions. The synthesis is shown in Scheme 1. The known compound, 1,24,25 is reacted with 4-methylumbelliferone to yield 3 in high yield. The α linkage was confirmed by the H3eq peak at 2.8 ppm in the 1H NMR spectroscopy. Removal of the acetate and ester groups resulted in the desired compound, which was characterized extensively by NMR and mass spectroscopies. The natural substrate, MUNANA (Fig. 3) and the modified sialic acid derivative, MUNANA(4,7OMe), (Fig. 3) was exposed to different influenza strains and S. pneumoniae and the fluorescence spectra was recorded after 2 h. (Fig. 4) NA, if present, will
cleave the compounds to yield 4-methylumbelliferone, which can be quantified and correlated to the NA activity. When the natural substrate is used, NAs from all three viral strains and S. pneumoniae cleave the sialic acid to release 4-methylumbelliferone. (Fig. 4a) However, when the modified sialic acid was used, S. pneumoniae does not cleave the compound as the binding pocket is smaller, demonstrating high specificity of the compounds. (Fig. 4b). These results clearly demonstrate that the new derivative MUNANA(4,7OMe) can detect influenza virus with high specificity. Our results are similar to previous reports that introduction of larger groups at the 4 and 7 positions of sialic acid make the compounds highly specific towards influenza virus.24–27 Next, we attempted to identify conditions where fluorescence would be realized upon exposure to S. pneumoniae and not influenza. To this end, we tested the compounds at different pH conditions since it was reported that some viral strains lose activity at higher pH and therefore, S.
Scheme 1. Reagents and conditions: a) EtOAc, TBAHS, Na2CO3, RT, 16 h, 60%; b) i) NaOMe, MeOH, RT, 30 min; ii) 0.05 M NaOH in H2O, RT, 2 h, 84% yield after two steps. 2
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 3. Sialic acid derivatives for influenza and S. pneumoniae. When the substrate MUNANA is cleaved by influenza or S. pneumoniae, 4-MU is released and detected at 360/460 nm. The substrate MUNANA (4,7 OMe) only can be cleaved by influenza and relaese 4-MU.
Fig. 4. Detection of influenza or S. pneumoniae by fluorogenic substrate. A. Detection of influenza virus or S. pneumoniae by MUNANA at different pH. Inactivated A/ Aichi/2/1968 H3N2 (sample AC), A/Winsconsin/15/2009 H3N2 (sample WS), A/Netherlands/2629/2009 H1N1 (sample NL), S. pneumoniae B (sample SB) were incubated with different buffers (pH = 5.5, pH = 7, pH = 8.5, pH = 10) for 30 min. Then MUNANA was added, the fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 2 h. Then 1 M, 100 µL tris solution was added, fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 10 min. Both influenza virus and S. pneumoniae can cleave MUNANA and release fluorescence in different pH. B. Detection of influenza or S. pneumoniae by MUNANA (4,7 OMe) at different pH. The influenza or S. pneumoniae samples were incubated in MUNANA (4, 7 OMe) solution with different buffers (pH = 5.5, pH = 7, pH = 8.5, pH = 10), the fluorescence intensity was detected. Only influenza virus can cleave MUNANA (4,7 OMe) release fluorescence. C. The influence of DTT to the activity of influenza virus and S. pneumoniae. The influenza or S. pneumoniae samples were incubated in MUNANA solution with or without DTT. D. The results for the influence of DTT. Influenza or S. pneumoniae samples were incubated in MUNANA solution with or without DTT. After 2 h, 1 M, 100 µL tris solution was added, fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 10 min. 3
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 5. The disulfide bonds in NA monomer from S. pneumoniae and influenza virus. The structures are downloaded from NIH PDB database. The surface is showed in blue color, the disulfide bond is showed in red color. A. The structure of S. pneumoniae Nan A monomer (PDB: 2ya5) which shows no disulfide bond. B. The structure of H1N1 NA monomer (PDB: 3ti6) which shows eight disulfide bonds, four of them are buried inside, the other four are exposed on the surface. C. The structure of H3N2 NA monomer (PDB: 4gzp) which shows nine disulfide bonds, four of them are buried inside, the other five are exposed on the surface.
pneumonia could potentially be detected specifically at high pH conditions.19 Unfortunately, we found that, while activity of all NAs decreased at higher pH, NAs from these influenza viral strains still retain their activity. Therefore, we cannot differentiate between coinfections using these assay conditions. Next, we used a reducing agent, Dithiothreitol (DTT), which is a reducing agent that reduce disulfide bonds, in addition to testing pH changes. Briefly, the X-ray crystal structure of NA’s from H1N1 and H3N2 strains reveal that there are eight disulfide bonds in one monomer of H1N1 influenza NA, nine disulfide bonds in one monomer of H3N2 NA.22,23,28,29 (Fig. 5) Four of these disulfide bonds are buried inside the enzyme, whereas the others are exposed on the surface. DTT is expected to reduce the disulfide bonds, and potentially disrupt the quarternary structure of influenza NA leading to enzyme deactivation. Since bacterial NA does not contain disulfide linkages, our rationale was that DTT will not affect S. pneumoniae NA. We observed that exposure of the sample to DTT in buffered condition, followed by introduction of the natural substrate, MUNANA, results in minimal or no activity of influenza viral strains (Fig. 4c). In contrast, S. pneumoniae cleaves the natural substrate under the same conditions (Fig. 4d). However, the specificity is not flawless, while A/Wisconsin/15/2009 H3N2 (sample WS) and A/Netherlands/2629/2009 H1N1 strains lose activity under these conditions, the A/Aichi/2/1968 H3N2 has residual activity. To summarize, we have designed fluorescent molecules specific for influenza virus. We have also attempted to exploit structural differences between S. pneumoniae and influenza neuraminidases to develop assay conditions to specifically detect S. pneumoniae with limited success.
References 1. Herold S, Becker C, Ridge KM, Budinger GRS. Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J. 2015;45:1463–1478. 2. von Itzstein M. The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov. 2007;6:967–974. 3. Chow EJ, Davis CT, Abd Elal AI, et al. Update: influenza activity - United States and Worldwide, May 20-October 13, 2018. Mmwr-Morbid Mortal W. 2018;67:1178–1185. 4. Morris DE, Cleary DW, Clarke SC. Secondary bacterial infections associated with influenza pandemics. Front Microbiol. 2017;8. 5. Moscona A. Drug therapy - neuraminidase inhibitors for influenza. New Engl J Med. 2005;353:1363–1373. 6. De Clercq E. Antiviral agents active against influenza A viruses. Nat Rev Drug Discov. 2006;5:1015–1025. 7. Leophonte P. Azithromycin and lower respiratory-tract infections. Pathol Biol. 1995;43:534–541. 8. Sharma-Chawla N, Sender V, Kershaw O, et al. Influenza A virus infection predisposes hosts to secondary infection with different streptococcus pneumoniae serotypes with similar outcome but serotype-specific manifestation. Infect Immun. 2016;84:3445–3457. 9. MacIntyre CR, Chughtai AA, Barnes M, et al. The role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(H1N1) pdm09. BMC Infect Dis. 2018;18. 10. Tan TQ, Mason EO, Wald ER, et al. Clinical characteristics of children with complicated pneumonia caused by Streptococcus pneumoniae. Pediatrics. 2002;110. 11. Nuutila J, Lilius EM. Distinction between bacterial and viral infections. Curr Opin Infect Dis. 2007;20:304–310. 12. Kash JC, Walters KA, Davis AS, et al. Lethal synergism of 2009 pandemic H1N1 influenza virus and Streptococcus pneumoniae coinfection is associated with loss of murine lung repair responses. MBio. 2011;2. 13. Adam HJ, Golden AR, Karlowsky JA, et al. Analysis of multidrug resistance in the predominant Streptococcus pneumoniae serotypes in Canada: the SAVE study, 2011–15. J Antimicrob Chemother. 2018;73 vii12–vii19. 14. McKimm-Breschkin JL. Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. Influenza Other Respir Viruses. 2013;7:25–36. 15. Thorlund K, Awad T, Boivin G, Thabane L. Systematic review of influenza resistance to the neuraminidase inhibitors. BMC Infect Dis. 2011;11:134. 16. Zhang XH, Dhawane AN, Sweeney J, He Y, Vasireddi M, Iyer SS. Electrochemical assay to detect influenza viruses and measure drug susceptibility. Angew Chem Int Edit. 2015;54:5929–5932. 17. Cui XK, Das A, Dhawane AN, et al. Highly specific and rapid glycan based amperometric detection of influenza viruses. Chem Sci. 2017;8:3628–3634. 18. Das A, Cui XK, Chivukula V, Iyer SS. Detection of enzymes, viruses, and bacteria using glucose meters. Anal Chem. 2018;90:11589–11598. 19. Yang W, Liu XY, Peng XX, et al. Synthesis of novel N-acetylneuraminic acid derivatives as substrates for rapid detection of influenza virus neuraminidase. Carbohyd Res. 2012;359:92–96. 20. Shrivastava S, Lee WI, Lee NE. Culture-free, highly sensitive, quantitative detection of bacteria from minimally processed samples using fluorescence imaging by
Acknowledgement This work was supported by NIH-NIAID (RO1-A1089450). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126773. 4
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al. smartphone. Biosens Bioelectron. 2018;109:90–97. 21. Wang S, Zheng L, Cai G, et al. A microfluidic biosensor for online and sensitive detection of Salmonella typhimurium using fluorescence labeling and smartphone video processing. Biosens Bioelectron. 2019;140:111333. 22. Gut H, Xu G, Taylor GL, Walsh MA. Structural basis for Streptococcus pneumoniae NanA inhibition by influenza antivirals zanamivir and oseltamivir carboxylate. J Mol Biol. 2011;409:496–503. 23. Hsiao YS, Parker D, Ratner AJ, Prince A, Tong L. Crystal structures of respiratory pathogen neuraminidases. Biochem Biophys Res Commun. 2009;380:467–471. 24. Liav A, Hansjergen JA, Achyuthan KE, Shimasaki CD. Synthesis of bromoindolyl 4,7di-O-methyl-Neu5Ac: specificity toward influenza A and B viruses. Carbohydr Res. 1999;317:198–203. 25. Cui X, Das A, Dhawane AN, et al. Highly specific and rapid glycan based
amperometric detection of influenza viruses. Chem Sci. 2017;8:3628–3634. 26. Achyuthan KE, Pence LM, Appleman JR, Shimasaki CD. ZstatFlu-II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics. Luminescence. 2003;18:131–139. 27. Shimasaki CD, Achyuthan KE, Hansjergen JA, Appleman JR. Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets. Philos Trans R Soc Lond B Biol Sci. 2001;356:1925–1931. 28. Vavricka CJ, Li Q, Wu Y, et al. Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition. PLoS Pathog. 2011;7:e1002249. 29. Zhu X, McBride R, Nycholat CM, Yu W, Paulson JC, Wilson IA. Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors. J Virol. 2012;86:13371–13383.
5
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Fluorescent sialic derivatives for the specific detection of influenza viruses Dandan Liua, Xikai Cuia, Abasaheb N. Dhawanea, Vasanta Chivukulab, Suri S. Iyera,
⁎
a b
788 Petit Science Center, Department of Chemistry, Centre for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, United States Atlanta Metropolitan State College, 1630 Metropolitan Parkway, Atlanta, GA 30310, United States
ARTICLE INFO
ABSTRACT
Keywords: Influenza Streptococcus pneumoniae Neuraminidase Sialic acid Glycan
Early and accurate diagnosis of influenza viruses can decrease its harmful impact. Here, we have synthesized fluorescent sialic acid derivatives that are cleaved by influenza neuraminidases (NAs) and not by Streptococcus pneumoniae that also inhabits the human olfactory. We have also attempted to develop assays that could differentiate between influenza virus and S. pneumoniae by taking advantage of the structural differences between NAs from these pathogens.
Respiratory infections caused by influenza cause considerable health and economic harm.1,2 Seasonal influenza leads to millions of infections worldwide leading to > 960,000 hospitalizations when it causes increased respiratory distress.3 Vaccinations are highly recommended for senior citizens, children and immunocompromised people, however, it is not feasible or realistic to vaccinate everyone. Antivirals are most efficacious when administered 24–48 h prior to infection in a prophylactic manner. Additionally, the initial infection caused by the virus often leads to secondary infection caused by bacterial pathogens.4 It is very important for a physician to ascertain if the infection is viral or bacterial, because the treatment is different. For influenza infections, antiviral drugs like Tamiflu or Relenza are prescribed;5,6 in contrast, for bacteria, antibiotics such as Azithromycin Zpak, is prescribed.7 Secondary infections can increase burden, for example,8,9 infections caused by Streptococcus pneumoniae, can lead to otitis media, especially in immunocompromised people and children five years old or younger.10 It takes 1–4 days for symptoms of influenza related sickness to appear after exposure to the virus. The time when secondary infection due to S. pneumoniae or other bacteria occurs is not very clear, as the interplay between viral, bacterial and the host immune system is complex11,12 and to compound the issue further, overall morphological symptoms due to viral/bacteria are quite similar. Thus, by the time the patient enters the clinic, it is unclear if the patient is suffering from viral, bacterial or coinfections. Indeed, postmortem analysis of the 1918 and the 1957 influenzas pandemics demonstrated that a significant number of patients had a secondary bacterial illness, up to 80% of the latter pandemic severe and fatal cases had bacterial infections;4 29% of the 2009 H1N1 swine influenza pandemic had secondary bacterial infections.9 Typically, primary care physicians
⁎
focus on patient history and underlying morbidity, the longevity of the infection (which is often subjective), the inflammation in the throat/ ears to start antiviral/antibiotic treatment. However, these physical examinations are far from ideal for a variety of reasons that include misdiagnosis, increased pathogen resistance due to overuse of antibiotic13 or antiviral therapy,14,15 side effects of antibiotics, etc. Clearly, it is very important to accurately detect influenza early in the infection process for appropriate treatment. We recently developed an electrochemical assay to detect enzymes using a repurposed glucose meter (Fig. 1). Briefly, we developed substrates bearing a glucose molecule and expose it to samples containing the enzyme.16 If enzyme is present and is active, glucose is released, which can be quantified using a glucose meter and correlated to the activity of the enzyme. Our first target was influenza virus; we synthesized sialic acid bearing molecules that release glucose.16 These molecules, when exposed to NA (or sialidase) from any source resulted in release of glucose, which was detected using a glucose meter. In further iterations, we demonstrated that molecules like galactose17 and paracetamol18 can also be detected using glucose meters; and we synthesized sialic acid derivatives that were highly specific for influenza viruses. Similar biochemiluminescent molecules, when exposed to influenza or S. pneumoniae, were cleaved only by influenza viral strains with high specificity.19 By using a combination of natural and modified sialic acid derivatives, we could differentiate between influenza and S. pneumoniae.17 While electrochemical detection using repurposed glucometers are very valuable in diagnostics, fluorescence based detection of pathogens cannot be understated, especially when it is integrated with smartphones.20,21 Here, we report a fluorescence based assay that could lead to the accurate detection of influenza virus by exploiting
Corresponding author. E-mail address: [email protected] (S.S. Iyer).
https://doi.org/10.1016/j.bmcl.2019.126773 Received 11 August 2019; Received in revised form 19 October 2019; Accepted 19 October 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Dandan Liu, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126773
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 1. Electrochemical assay to detect enzymes using glucose meter. The synthesized substrate can be cleaved by enzyme and release glucose, which can be detected by glucose meter.
Fig. 2. The binding pocket of NA complexed with sialic acid downloaded from NIH PDB database and PyMOL was used to show the surface and binding pocket. The carbon atom is represented in green color, the oxygen atom element in red color, the nitrogen atom in blue color. a) The binding pocket of influenza NA (PDB: 4gzq). b) The binding pocket of S. pneumoniae NA (PDB: 3 h72). These figures shows the binding pocket of influenza NA is larger than S. pneumoniae NA.
structural differences between NAs from influenza and S. pneumoniae. To differentiate between influenza virus and S. pneumoniae, we use the modified substrate, i.e. MUNANA(4,7OMe), because the binding pocket of influenza is considerably larger than any other NA (Fig. 2), especially at the 4,7 positions.22,23 To this end, we synthesized a fluorescent sialic acid derivative bearing OMe groups at the 4 and 7 positions. The synthesis is shown in Scheme 1. The known compound, 1,24,25 is reacted with 4-methylumbelliferone to yield 3 in high yield. The α linkage was confirmed by the H3eq peak at 2.8 ppm in the 1H NMR spectroscopy. Removal of the acetate and ester groups resulted in the desired compound, which was characterized extensively by NMR and mass spectroscopies. The natural substrate, MUNANA (Fig. 3) and the modified sialic acid derivative, MUNANA(4,7OMe), (Fig. 3) was exposed to different influenza strains and S. pneumoniae and the fluorescence spectra was recorded after 2 h. (Fig. 4) NA, if present, will
cleave the compounds to yield 4-methylumbelliferone, which can be quantified and correlated to the NA activity. When the natural substrate is used, NAs from all three viral strains and S. pneumoniae cleave the sialic acid to release 4-methylumbelliferone. (Fig. 4a) However, when the modified sialic acid was used, S. pneumoniae does not cleave the compound as the binding pocket is smaller, demonstrating high specificity of the compounds. (Fig. 4b). These results clearly demonstrate that the new derivative MUNANA(4,7OMe) can detect influenza virus with high specificity. Our results are similar to previous reports that introduction of larger groups at the 4 and 7 positions of sialic acid make the compounds highly specific towards influenza virus.24–27 Next, we attempted to identify conditions where fluorescence would be realized upon exposure to S. pneumoniae and not influenza. To this end, we tested the compounds at different pH conditions since it was reported that some viral strains lose activity at higher pH and therefore, S.
Scheme 1. Reagents and conditions: a) EtOAc, TBAHS, Na2CO3, RT, 16 h, 60%; b) i) NaOMe, MeOH, RT, 30 min; ii) 0.05 M NaOH in H2O, RT, 2 h, 84% yield after two steps. 2
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 3. Sialic acid derivatives for influenza and S. pneumoniae. When the substrate MUNANA is cleaved by influenza or S. pneumoniae, 4-MU is released and detected at 360/460 nm. The substrate MUNANA (4,7 OMe) only can be cleaved by influenza and relaese 4-MU.
Fig. 4. Detection of influenza or S. pneumoniae by fluorogenic substrate. A. Detection of influenza virus or S. pneumoniae by MUNANA at different pH. Inactivated A/ Aichi/2/1968 H3N2 (sample AC), A/Winsconsin/15/2009 H3N2 (sample WS), A/Netherlands/2629/2009 H1N1 (sample NL), S. pneumoniae B (sample SB) were incubated with different buffers (pH = 5.5, pH = 7, pH = 8.5, pH = 10) for 30 min. Then MUNANA was added, the fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 2 h. Then 1 M, 100 µL tris solution was added, fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 10 min. Both influenza virus and S. pneumoniae can cleave MUNANA and release fluorescence in different pH. B. Detection of influenza or S. pneumoniae by MUNANA (4,7 OMe) at different pH. The influenza or S. pneumoniae samples were incubated in MUNANA (4, 7 OMe) solution with different buffers (pH = 5.5, pH = 7, pH = 8.5, pH = 10), the fluorescence intensity was detected. Only influenza virus can cleave MUNANA (4,7 OMe) release fluorescence. C. The influence of DTT to the activity of influenza virus and S. pneumoniae. The influenza or S. pneumoniae samples were incubated in MUNANA solution with or without DTT. D. The results for the influence of DTT. Influenza or S. pneumoniae samples were incubated in MUNANA solution with or without DTT. After 2 h, 1 M, 100 µL tris solution was added, fluorescence intensity was detected at 37 °C, 360 nm/460 nm for 10 min. 3
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al.
Fig. 5. The disulfide bonds in NA monomer from S. pneumoniae and influenza virus. The structures are downloaded from NIH PDB database. The surface is showed in blue color, the disulfide bond is showed in red color. A. The structure of S. pneumoniae Nan A monomer (PDB: 2ya5) which shows no disulfide bond. B. The structure of H1N1 NA monomer (PDB: 3ti6) which shows eight disulfide bonds, four of them are buried inside, the other four are exposed on the surface. C. The structure of H3N2 NA monomer (PDB: 4gzp) which shows nine disulfide bonds, four of them are buried inside, the other five are exposed on the surface.
pneumonia could potentially be detected specifically at high pH conditions.19 Unfortunately, we found that, while activity of all NAs decreased at higher pH, NAs from these influenza viral strains still retain their activity. Therefore, we cannot differentiate between coinfections using these assay conditions. Next, we used a reducing agent, Dithiothreitol (DTT), which is a reducing agent that reduce disulfide bonds, in addition to testing pH changes. Briefly, the X-ray crystal structure of NA’s from H1N1 and H3N2 strains reveal that there are eight disulfide bonds in one monomer of H1N1 influenza NA, nine disulfide bonds in one monomer of H3N2 NA.22,23,28,29 (Fig. 5) Four of these disulfide bonds are buried inside the enzyme, whereas the others are exposed on the surface. DTT is expected to reduce the disulfide bonds, and potentially disrupt the quarternary structure of influenza NA leading to enzyme deactivation. Since bacterial NA does not contain disulfide linkages, our rationale was that DTT will not affect S. pneumoniae NA. We observed that exposure of the sample to DTT in buffered condition, followed by introduction of the natural substrate, MUNANA, results in minimal or no activity of influenza viral strains (Fig. 4c). In contrast, S. pneumoniae cleaves the natural substrate under the same conditions (Fig. 4d). However, the specificity is not flawless, while A/Wisconsin/15/2009 H3N2 (sample WS) and A/Netherlands/2629/2009 H1N1 strains lose activity under these conditions, the A/Aichi/2/1968 H3N2 has residual activity. To summarize, we have designed fluorescent molecules specific for influenza virus. We have also attempted to exploit structural differences between S. pneumoniae and influenza neuraminidases to develop assay conditions to specifically detect S. pneumoniae with limited success.
References 1. Herold S, Becker C, Ridge KM, Budinger GRS. Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J. 2015;45:1463–1478. 2. von Itzstein M. The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov. 2007;6:967–974. 3. Chow EJ, Davis CT, Abd Elal AI, et al. Update: influenza activity - United States and Worldwide, May 20-October 13, 2018. Mmwr-Morbid Mortal W. 2018;67:1178–1185. 4. Morris DE, Cleary DW, Clarke SC. Secondary bacterial infections associated with influenza pandemics. Front Microbiol. 2017;8. 5. Moscona A. Drug therapy - neuraminidase inhibitors for influenza. New Engl J Med. 2005;353:1363–1373. 6. De Clercq E. Antiviral agents active against influenza A viruses. Nat Rev Drug Discov. 2006;5:1015–1025. 7. Leophonte P. Azithromycin and lower respiratory-tract infections. Pathol Biol. 1995;43:534–541. 8. Sharma-Chawla N, Sender V, Kershaw O, et al. Influenza A virus infection predisposes hosts to secondary infection with different streptococcus pneumoniae serotypes with similar outcome but serotype-specific manifestation. Infect Immun. 2016;84:3445–3457. 9. MacIntyre CR, Chughtai AA, Barnes M, et al. The role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(H1N1) pdm09. BMC Infect Dis. 2018;18. 10. Tan TQ, Mason EO, Wald ER, et al. Clinical characteristics of children with complicated pneumonia caused by Streptococcus pneumoniae. Pediatrics. 2002;110. 11. Nuutila J, Lilius EM. Distinction between bacterial and viral infections. Curr Opin Infect Dis. 2007;20:304–310. 12. Kash JC, Walters KA, Davis AS, et al. Lethal synergism of 2009 pandemic H1N1 influenza virus and Streptococcus pneumoniae coinfection is associated with loss of murine lung repair responses. MBio. 2011;2. 13. Adam HJ, Golden AR, Karlowsky JA, et al. Analysis of multidrug resistance in the predominant Streptococcus pneumoniae serotypes in Canada: the SAVE study, 2011–15. J Antimicrob Chemother. 2018;73 vii12–vii19. 14. McKimm-Breschkin JL. Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. Influenza Other Respir Viruses. 2013;7:25–36. 15. Thorlund K, Awad T, Boivin G, Thabane L. Systematic review of influenza resistance to the neuraminidase inhibitors. BMC Infect Dis. 2011;11:134. 16. Zhang XH, Dhawane AN, Sweeney J, He Y, Vasireddi M, Iyer SS. Electrochemical assay to detect influenza viruses and measure drug susceptibility. Angew Chem Int Edit. 2015;54:5929–5932. 17. Cui XK, Das A, Dhawane AN, et al. Highly specific and rapid glycan based amperometric detection of influenza viruses. Chem Sci. 2017;8:3628–3634. 18. Das A, Cui XK, Chivukula V, Iyer SS. Detection of enzymes, viruses, and bacteria using glucose meters. Anal Chem. 2018;90:11589–11598. 19. Yang W, Liu XY, Peng XX, et al. Synthesis of novel N-acetylneuraminic acid derivatives as substrates for rapid detection of influenza virus neuraminidase. Carbohyd Res. 2012;359:92–96. 20. Shrivastava S, Lee WI, Lee NE. Culture-free, highly sensitive, quantitative detection of bacteria from minimally processed samples using fluorescence imaging by
Acknowledgement This work was supported by NIH-NIAID (RO1-A1089450). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126773. 4
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
D. Liu, et al. smartphone. Biosens Bioelectron. 2018;109:90–97. 21. Wang S, Zheng L, Cai G, et al. A microfluidic biosensor for online and sensitive detection of Salmonella typhimurium using fluorescence labeling and smartphone video processing. Biosens Bioelectron. 2019;140:111333. 22. Gut H, Xu G, Taylor GL, Walsh MA. Structural basis for Streptococcus pneumoniae NanA inhibition by influenza antivirals zanamivir and oseltamivir carboxylate. J Mol Biol. 2011;409:496–503. 23. Hsiao YS, Parker D, Ratner AJ, Prince A, Tong L. Crystal structures of respiratory pathogen neuraminidases. Biochem Biophys Res Commun. 2009;380:467–471. 24. Liav A, Hansjergen JA, Achyuthan KE, Shimasaki CD. Synthesis of bromoindolyl 4,7di-O-methyl-Neu5Ac: specificity toward influenza A and B viruses. Carbohydr Res. 1999;317:198–203. 25. Cui X, Das A, Dhawane AN, et al. Highly specific and rapid glycan based
amperometric detection of influenza viruses. Chem Sci. 2017;8:3628–3634. 26. Achyuthan KE, Pence LM, Appleman JR, Shimasaki CD. ZstatFlu-II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics. Luminescence. 2003;18:131–139. 27. Shimasaki CD, Achyuthan KE, Hansjergen JA, Appleman JR. Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets. Philos Trans R Soc Lond B Biol Sci. 2001;356:1925–1931. 28. Vavricka CJ, Li Q, Wu Y, et al. Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition. PLoS Pathog. 2011;7:e1002249. 29. Zhu X, McBride R, Nycholat CM, Yu W, Paulson JC, Wilson IA. Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors. J Virol. 2012;86:13371–13383.
5