Article pubs.acs.org/ac
Anti-Viral Inhibitor Binding to Influenza Neuraminidase by MALDI Mass Spectrometry Kavya Swaminathan and Kevin M. Downard* School of Molecular Bioscience University of Sydney, Sydney, NSW 2006, Australia ABSTRACT: A matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based approach is applied to identify active site domains within influenza neuraminidase that bind the antiviral inhibitors zanamivir (ZANA) and 2deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). Combined data from the tryptic and Glu-C endoproteinase digests of neuraminidase-inhibitor complexes have identified binding peptides that contain the active site residues Arg118, Glu119, Arg156, Glu276, and Tyr406. The binding of these residues was confirmed from the analysis of available X-ray crystal structures. The ability to identify peptides within the active sites of proteins and likely binding residues provides both a rapid and relatively high throughput approach with which to screen protein−drug interactions by MALDI mass spectrometry.
T
DANA, or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, is a transition-state analogue of sialic acid. It was the first inhibitor of influenza neuraminidase to be identified.12 Molecular-based X-ray crystallographic studies of the binding of inhibitors to the neuraminidase active site revealed their isosteric binding properties and allowed for the identification of additional interactions that favor higher affinity binding.13 These studies led to development of more potent inhibitors such as zanamivir (ZANA), or 4-guanidino-2-deoxy-2,3-didehydro-N-acetylneuraminic acid, in which a hydroxyl group of DANA is replaced with a guanidino group (Figure 1). This substitution enables ZANA to adopt an energetically more favorable hydrophobic interaction within the active site that results in its higher binding affinity. The success of the NA inhibitors is a result of their impressive safety profile,14−16 but, similar to the M2 inhibitors, a rise in the population of resistant strains17−20 has been observed with their wider administration. This has prompted the development and clinical testing of new inhibitors from both natural and synthetic sources,6,21−25 which has raised the need to improve and supplement current screening methods. Current screening methods range from the high-throughput virtual screening of libraries of potential candidates,26 to nonmolecular-based neuraminidase inhibition assays (NIAs),27 to the use of high-resolution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallographic approaches,6,28,29 to in silico three-dimensional (3D) molecular modeling methods for de novo design.30 A range of approaches is often used, because each of the above has its own limitations. High-throughput screens have largely identified only low-affinity and/or nonselective viral NA inhibitors, whereas the use of NMR or X-ray crystallography to study many neuraminidase-inhibitor
he influenza virus is a leading cause of death the world over and is responsible for up to 500 000 human deaths each year from seasonal strains alone.1 This death toll can rise to millions following pandemic outbreaks that are associated with the emergence of newly reassorted strains for which the population have little or no protective immunity. While inactivated and attenuated vaccines have been the primary means of controlling influenza infections, they require the prior surveillance and characterization of strains in circulation in order for an effective vaccine to be prepared for the following influenza season.2 When significant antigenic drift or shift occurs among virus strains after formulation, the vaccine is rendered less efficacious.3 Furthermore, the time required to formulate and produce a vaccine in sufficient doses may not enable a halt in the spread of infections to be achieved. This is exacerbated during influenza pandemics when new highly virulent forms emerge.4,5 Antiviral inhibitors have become an important alternate means of containing the spread of influenza.6 Membrane (M2) ion channel inhibitors that belong to the adamantanes class of compounds were the first generation of influenza antiviral agents.7 These inhibitors disrupt the viral uncoating inside the host cell and prevent the budding of progeny viruses.8 However, they are only effective against type A viruses and have been associated with adverse toxic effects, as well as the rapid emergence of resistant strains. Neuraminidase (NA) inhibitors have since been developed and widely administered against the influenza virus.9 These sialidase inhibitors, including zanamivir and osteltamivir, block the active site of the neuraminidase enzyme10 that prevent it from detaching virions from the host’s sialic acid receptor.11 This decreases the viral load by preventing the release of progeny viruses. To be effective, these viral inhibitors must be administered 48−72 h after the onset of symptoms, although they have been promoted as prophylatic drugs. © 2012 American Chemical Society
Received: January 29, 2012 Accepted: March 12, 2012 Published: March 12, 2012 3725
dx.doi.org/10.1021/ac300291c | Anal. Chem. 2012, 84, 3725−3730
Analytical Chemistry
Article
reconstituted in 500 μL of Milli-Q purified water. Zanamivir (ZANA) was acquired from Moravek Biochemicals (Brea, CA, USA) while 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and 5-hydroxymethyl furfural (5HF) were purchased from Sigma−Aldrich (Castle Hill, NSW, Australia). Stock solutions of ZANA and DANA were prepared at 120 μM and 5HF at 250 μM in water. Deglycosylation of Influenza Neuraminidase. Deglycosylation was performed by the addition of 2 μL of recombinant N-glycosidase F (PNGase F) stock solution (250 units in 250 μL) from Roche Diagnostics (Castle Hill, NSW, Australia) to a solution containing 3.5 μg of neuraminidase. The combined solution was incubated overnight at 37 °C. The successful deglycosylation of the neuraminidase was confirmed by SDSPAGE with the protein band detected just above the 50 kDa molecular weight reference band. The average theoretical neuraminidase protein mass is 51596.9. Neuraminidase-Inhibitor Binding. Three samples containing 2.5 μg deglycosylated neuraminidase solution were treated with solutions of ZANA, DANA, and 5HF (at a NA:inhibitor molar ratio of ∼1:5) and incubated at 37 °C for 2 h. An excess of inhibitor was employed to promote its binding to protein. A further sample of NA was kept as an untreated control. The solutions were concentrated to 20 μL in volume before being loaded onto a polyacrylamide gel. Native Gel Electrophoresis of Neuraminidase and Neuraminidase-Inhibitor Complexes. Solutions of untreated NA and inhibitor-treated NA (namely, NA + ZANA, NA + DANA, and NA + 5HF) (20 μL each) were added to 5 μL of native loading buffer solution containing 1 M Tris (pH 8.6) and 20% glycerol. These solutions were loaded into separate wells of a Nu-PAGE 3−8% tris-acetate gel (Invitrogen, Mulgrave, VIC, Australia). A further solution of untreated NA was used as a reference marker through its dilution in the native loading buffer solution containing 0.1% Bromophenol Blue in order to track the location of the protein in a separate stained lane. This stained lane shows two main bands, corresponding to the deglycosylated neuraminidase and residual N-glycosidase F (PNGase F). Electrophoresis was performed in an Invitrogen MiniCell tank at 150 V for 60 min in a running buffer solution containing 25 mM Tris-HCl and 192 mM glycine. The gel section that contained the tracked protein was stained for 30 min with 0.25% (w/v) Coomassie Blue (R-250) solution in water, methanol (40%, v/v) and acetic acid (7%, v/v) and then was destained for 2 h in a solution containing methanol (40%, v/v) and acetic acid (7%, v/v) in water. The unstained gel section containing the samples for MALDI-MS analysis was aligned with the stained tracked protein and the regions corresponding to the deglycosylated NA band of each sample were excised. The reminder of the gel was then stained with the Coomassie Blue solution to ensure that the deglycosylated NA was recovered completely. The excised gel sections of each sample were pulverized into 1 mm3 pieces and transferred into separate 0.6 mL tubes. Limited Proteolysis of Neuraminidase-Inhibitor Complexes with Endoproteinases Trypsin and Glu-C. The gel pieces from each sample were washed with 300 μL Milli-Q filtered water and partially dried in a Labconco Centrivap concentrator (Kansas City, MO, USA). The dried pieces were rehydrated in 25 mM ammonium bicarbonate (20 μL, pH 7.8) containing sequencing-grade-modified trypsin (Promega Corporation, Alexandria, NSW, Australia) to achieve a final trypsin concentration of 150 ng/tube or a 1:16 enzyme:protein (w/w)
Figure 1. Structures of the neuraminidase inhibitors: (a) DANA (2deoxy-2,3-didehydro-N-acetylneuraminic acid), (b) ZANA (zanamivir, 4-guanidino-2-deoxy-2,3-didehydro-N-acetylneuraminic acid and a control compound (c) 5-hydroxymethyl furfural.
complexes is both time- and sample-consuming. NIAs establish the sialidase activity of candidate inhibitors but do not provide any detail of the molecular interactions involved. A matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based approach is described that enables both the binding of neuraminidase inhibitors to be detected and their interaction residues to be identified or localized. This molecular-based approach can be rapidly performed at low sample levels, providing a relatively high-throughput approach for the screening of candidate inhibitors. The approach is based on a reported MALDI-MS immunoassay, 31 previously developed in this laboratory, to monitor the antigenicity of influenza strains by identifying the binding antibodies to the virus’ hemagglutinin antigen responsible for host cell attachment. It achieves this by identifying proteolytic peptides whose release from a protein is impeded by its interaction with a binding partner. This results in their absence or reduced relative abundance in the subsequent mass spectrum versus an untreated protein control sample. The immunoassay has been shown to be able to identify antigenic peptide domains within whole virus and recovered antigen digests32−35 and has even been extended to study their relative binding rates.36 A computer algorithm (PRISM) has been developed37 to automatically identify binding peptides in these and other datasets. This MALDI mass spectrometry-based method is applied here for the first time to detect and study protein−inhibitor complexes, without the immobilization of either component or the recovery of the complex. It incorporates the use of native gel electrophoresis38 to allow proteins to be separated from other contaminants in the sample and has broad applicability to the study of other protein−drug complexes.
■
EXPERIMENTAL SECTION Neuraminidase and Inhibitor Stocks. Recombinant influenza neuraminidase from a 2009 pandemic influenza strain (A/California/04/2009) was purchased from Sino Biologicals, Inc. (Beijing, PRC) as a 100-μg lyophilized protein (73% purity) from a filtered solution of PBS (pH 7.4) and 3726
dx.doi.org/10.1021/ac300291c | Anal. Chem. 2012, 84, 3725−3730
Analytical Chemistry
Article
ratio. The samples were allowed to stand for 5 min, after which additional 25 mM ammonium bicarbonate was added to cover the gel pieces, and then the samples were incubated at 37 °C overnight. GluC (Sigma−Aldrich, Castle Hill, NSW, Australia) digestions were performed in separate experiments in a similar manner in 25 mM ammonium bicarbonate buffer (pH 7.8) at a 1:80 enzyme to protein (w/w) ratio overnight at 37 °C. The solution containing proteolytic peptides was separated from the gel into 0.6 mL tubes and the gel pieces sonicated for 2 × 1 h cycles with additional 25 mM ammonium bicarbonate buffer (50 μL, pH 7.8) with periodic cooling on ice. After each 1-h cycle, the solutions were pooled. Reduction and Alkylation of Extracted Peptides. The extracted peptides were reduced with 10 μM dithiotheritol (Sigma−Aldrich, Castle Hill, NSW, Australia) solution at 60 °C for 30 min and then treated with 10 μM iodoacetamide (Sigma−Aldrich, Castle Hill, NSW, Australia) solution and incubated with shielding from ambient light for 30 min at room temperature. The alkylated peptides were fully dried in a concentrator (Labconco Centrivap, Kansas City, MO, USA) and resuspended in 10 μL of 25 mM ammonium bicarbonate (pH 7.8). The samples were briefly sonicated for 10 min to ensure effective resuspension. The final concentration of the peptides is expected to be 0.25 μg/μL, assuming 100% recovery of the 2.5 μg loaded protein. MALDI-MS Analysis of Bound and Unbound Samples. Reconstituted peptide solution (1 μL) was mixed with a 5 mg/ mL solution of α-cyano-4-hydroxycinnamic acid in 60% acetonitrile, 0.1% trifluoroacetic acid, and 39.9% water (4 μL). A portion of each solution (1.2 μL) was spotted onto a MALDI target plate and high-resolution FT-ICR mass spectra were recorded on a 7T Bruker APEX-Qe instrument (Bruker Daltonics, Billerica, MA, USA) in the positive-ion mode. Ions released with 50−200 laser shots (at 35% laser power) from the MALDI plate, held at 400 V, were accumulated above the plate for 0.2 s, stored in the hexapole for 1.0 s, and then passed to the FT-ICR cell within 1.0 ms, using a sidekick voltage of 0 V and an offset of −1.5 V. Fifty to one hundred (50−100) scans were acquired and averaged into a single mass spectrum. Spectra were acquired through the accumulation of one million (1M) data points across a mass range of m/z 400−4000, using a broadband excitation. A mass resolution of over 100 000 (FWHM) at m/z 1296 was typically achieved. The instrument was mass-calibrated with an external mixture of peptides comprising des-Arg-bradykinin, angiotensin, and adrenocorticotropic hormone (ACTH) fragment comprising residues 18− 39. Analysis of MALDI-MS Data. The theoretical masses for the tryptic and GluC peptides from the influenza neuraminidase A/H1N1/California/04/2009 (GenBank ACP41107) were calculated using the FindPept39 algorithm (ExPASy Proteomics Server). The N2 numbering system was used to designate peptide regions corresponding to the identified ions in the MALDI mass spectra that span 38% of the sequence from combined tryptic and Glu-C digests. The relative peak areas of the neuraminidase ions (relative to the base peak in the spectra) were determined with Data Analysis software (Bruker Daltonics, Billerica, MA, USA). The absolute difference in the relative peak area between each binding and the control sample was then calculated. Peptides having a decrease of >10% (absolute) were deemed to bind inhibitor. The PRISM algorithm37 was used to assist with this data analysis.
Figure 2. MALDI mass spectra obtained from tryptic digestion of (a) untreated NA, (b) NA bound to ZANA, (c) NA bound to DANA, and (d) NA treated with 5HF. The NA peptide ions have been labeled. Those showing a decrease in relative peak area exceeding 10% (absolute) have been highlighted. The hash mark symbol (#) denotes a matrix cluster ion, while the asterisk symbol (*) denotes a trypsin autolysis product.
Visualization and Analysis of X-ray Crystallographic Structures. The X-ray crystallographic structures of unbound influenza neuraminidase (3NSS) and zanamivir bound neuraminidase (3TI5) were obtained from the Protein Data Bank (PDB) database. The structure and contact residues were visualized using Ligand Explorer. The accessible surface areas of individual residues in unbound and ZANA-bound neuraminidase were calculated using the GETAREA algorithm40 with a probe radius of 1.4 Å2 and are expressed as a percentage relative to a random-coil value. Residue side chains are considered to be solvent-exposed if the ratio value exceeds 50% and solventburied if the ratio is less than 20%. Those with ratios in the range of 20%−50% are not defined either way.
■
RESULTS AND DISCUSSION Overview of the Approach. The binding of inhibitors ZANA and DANA (Figure 1) to influenza neuraminidase was studied. In addition, 5-hydroxymethyl furfural (5HF) was used as a negative control. The structure of 5HF lacks important functional groups that aid in the binding of ZANA and DANA, and it has no reported inhibitory activity. Deglycosylation of the neuraminidase was carried out prior to digestion in order to overcome the proteolytic resistance41 it
3727
dx.doi.org/10.1021/ac300291c | Anal. Chem. 2012, 84, 3725−3730
Analytical Chemistry
Article
Table 1. Difference in Relative Peak Areas of the Tryptic Peptide Ions between Unbound and Inhibitor-Treated Neuraminidasea Difference in Relative Peak Areab (Standard Deviation) m/z
residues
peptide sequence
ZANA
DANA
5HF
805.4567 1021.5215 1137.6142 1467.7834 1476.6582 1494.6705 1907.8964 1923.8907
112−118 356−364 207−216 131−143 119−130 119−130 157 −172 157−172
GDVFVIR YGNGVWIGR YGNGIITDTIK TFFLTQGALLNDK pyroEPFISCPLECR EPFISCPLECR TLMSCPIGEVPSPYNSR TLMoxSCPIGEVPSPYNSR
−1.43 (1.51) −5.33 (1.23) 0.03 (2.10) 6.40 (3.39) 10.47 (1.58) 31.43 (0.09) 18.60 (0.21) 26.47 (5.82)
−2.77 (0.73) −6.43 (1.81) −1.40 (1.06) 7.27 (5.33) 9.77 (0.33) 26.00 (0.49) 17.30 (1.48) 18.50 (2.55)
−2.20 (0.78) 2.17 (1.53) −0.37 (0.45) 0.63 (7.19) −2.50 (0.07) 0.00 (0.00) −1.13 (0.73) 4.93 (1.79)
a
The peptide ions that show a relative area decrease of >10% (absolute) in the inhibitor-treated samples are shown in bold font. bThe values represent the mean from three replicate datasets; standard deviations are shown in parentheses.
Table 2. Difference in Relative Peak Areas of the Glu-C Peptide Ions between Unbound and Inhibitor-Treated Neuraminidasea Difference in Relative Peak Areab (Standard Deviation) m/z
residues
peptide sequence
ZANA
DANA
5HF
968.6004 999.5218 1233.4034 1480.6485 1492.649 1765.8376 1837.8547 3501.6042
426−433 143−151 277−286 402−412 376−387 412−425 278−293 294−324
LIRGRPKE KHSNGTIKD ECSCYPDSSE WSGYSGFVQHPE MIWDPNGWTGTD LTGLDCIRPCFWVE CSCYPDSSEITCVCRD NWHGSNRPWVSFNQNLEYQIGYICSGIFGD
0.18 (0.14) 1.46 (0.73) 23.70 (−) 16.70 (1.10) −6.99 (0.35) 5.83 (0.66) 4.45 (0.56) −6.61 (3.30)
2.60 (−) 1.00 (−) 10.6 (−) 13.30 (3.20) −2.49 (−) 0.89 (−) −0.81 (−) −8.12 (−)
−0.44 (0.52) −0.07 (0.03) 1.12 (−) 5.28 (0.35) −0.56 (0.67) −0.68 (2.50) 1.39 (0.05) −8.45 (4.23)
a
The peptide ions that show a relative area decrease of >10% (absolute) in the inhibitor-treated samples are shown in bold font. bThe values represent the mean from three replicate datasets; standard deviations are shown in parentheses.
confers and to aid with the migration of the protein through the native gel. Glycosylation assists with oligomerization of neuraminidase to its tetramer but has been shown to have no effect on its activity.41 Furthermore, antiviral inhibitors have been developed using recombinant neuraminidase in its glycanfree state.6 The neuraminidase enzyme was treated with a 5-fold molar excess of each of the inhibitors and 5HF (nonbinding control) while a portion was left untreated (NA-only). The neuraminidase or neuraminidase-inhibitor complexes were then run in separate lanes by native gel electrophoresis to allow the protein to be separated from other contaminants in the sample. The location of the neuraminidase or neuraminidase−inhibitor complex was identified based on the position of the reference NA-only stained band. Peptide extractions were performed without denaturing organic solvents and with the aid of sonication38 with periodic cooling in order to preserve the protein−inhibitor interactions. The reduction and alkylation of cysteine residues was also performed after the recovery of peptides in order to maintain the native structure of the neuraminidase in the gel. The peptides released by proteolysis from the unstained portion of the gel containing protein and protein−inhibitor complexes were then analyzed by MALDI mass spectrometry. These spectra were compared with the spectra obtained for the digested untreated NA sample. Identification of Inhibitor Binding Peptides. The spectra of tryptic peptides of unbound (control) as well as inhibitor-bound neuraminidase are shown in Figure 2. The spectra from all four samples were found to have common NA peptide ions. The major difference between the spectra of the untreated NA and 5HF treated samples (see Figures 2a and 2d, respectively) and their ZANA- and DANA-treated counterparts
(see Figures 2b and 2c, respectively) is evident by a significant decrease in the intensity of the ions at m/z 1494.6696 and 1923.8908. The ions at m/z 1494.6696, corresponding to residues 119− 130 of the protein, show an absolute decrease in relative peak area of 31.4% and 26.0% in the ZANA and DANA spectra, respectively, compared to the untreated NA (Table 1). A pyroglutamic acid modified form of the same peptide is detected at m/z 1476.6582 and shows an absolute decrease in relative area of 10.43% and 9.77% in the spectra of the ZANAand DANA-treated protein. Although the latter is slightly below the cutoff used to identify binding peptides, it is nonetheless consistent with data for the peptide at m/z 1494.6696. The ions at m/z 1923.8908 are seen to decrease by 26.5% and 18.5% in the spectra of the ZANA- and DANA-treated protein, respectively. These ions represent the carbamidomethylated and methionine-oxidized form of the peptide consisting of NA residues 157−172. A decrease of 18.60% and 17.30% in the relative area of ions at 1907.8965 representing the unoxidized form of this peptide is also evident. The remaining neuraminidase peptides are relatively unaffected by the presence of the inhibitor. This is evident from their relative peak areas across the mass spectra showing variations well within 10% (absolute) of those areas for the NAonly and control-5HF-treated sample. Consistent data from replicate experiments (Table 1) confirm these results and demonstrate that the binding of these regions of the neuraminidase protein to the inhibitors ZANA and DANA has been identified by this approach. Similar mass spectra were obtained for the neuraminidase and neuraminidase−inhibitor complexes treated with endoproteinase GluC. This was performed to improve the sequence 3728
dx.doi.org/10.1021/ac300291c | Anal. Chem. 2012, 84, 3725−3730
Analytical Chemistry
Article
Waals interactions.13 These interactions restrict access and prevent the release of the peptides by trypsin. The peptide consisting of NA residues 157−172 (of sequence TLMSCPIGEVPSPYNSR) is detected at m/z 1907.8964 and 1923.8907 and flanked by the binding residue at Arg156 that forms hydrogen bonds with the hydroxyl group or one of the primary amine nitrogens in the guanidino group attached to the C4 atom of DANA and ZANA, respectively.24 One tryptic peptide comprised of NA residues 112−118 at m/z 805.4567 did not show any significant peak area decrease despite the presence of a known binding residue at Arg118. This is likely associated with a conformational change at the residue Lys111 that is induced when the inhibitor binds to the active site. The total accessible surface areas at the trypsin cleavage sites (Lys111 and Arg118) were calculated using the GETAREA algorithm,32 based upon the PDB structures for unbound (3NSS) and inhibitor-bound neuraminidase (3TI5). The Lys111 residue becomes more exposed when the inhibitor is bound with its total accessible surface area increasing from 57.26 Å2 to 186.40 Å2. However, the solvent accessibility at Arg118 remains relatively unchanged (25.47 Å2 to 26.12 Å2), as is the case for all other tryptic cleavage site residues in the active site (see Table 3). Thus, the greater accessibility of
coverage (to 38%) achieved by tryptic digestion alone and thus identify a larger set of binding residues within the active site. The relative peak areas for all ions detected in the spectra from GluC digests are tabulated in Table 2. The peptide ions at m/z 1233.4034 comprised of NA residues 277−286 shows an absolute decrease of 23.7% and 10.6% in the spectra of the ZANA- and DANA-treated samples, compared to the untreated control. A significant decrease in the relative area of the ions at m/z 1480.6485 in the NA-ZANA (16.70% absolute) and NADANA (13.30% absolute) spectra is also observed. These ions correspond to NA residues 402−412. The relative peak area of both of these ions does not vary significantly in the spectra of the 5HF-treated sample and untreated NA. All other NA peptide ions detected have relative peak area differences of
