Direct affinity screening chromatography–mass spectrometry assay for identification of antibacterial agents from natural product sources

Analytica Chimica Acta 713 (2012) 103–110

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Direct affinity screening chromatography–mass spectrometry assay for identification of antibacterial agents from natural product sources Kevin A. Schug a,∗ , Evelyn Wang a , Sijia Shen a , Sunaina Rao b , Stephanie M. Smith b , Laura Hunt b , Laura D. Mydlarz b,∗∗ a b

Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USA Department of Biology, The University of Texas at Arlington, Arlington, TX, USA

a r t i c l e

i n f o

Article history: Received 26 September 2011 Received in revised form 16 November 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Natural products Noncovalent interactions Electrospray ionization Dynamic titration Pseudoplexaura porosa

a b s t r a c t A direct affinity screening – mass spectrometry assay, coupled to liquid chromatography, is presented as a tool for natural product drug discovery. Using the assay, fractionated extracts from a Caribbean gorgonian coral were shown to contain a new chemical entity (NCE) which binds to a mimic of the Gram positive bacterial cell wall (lysine–d-alanine–d-alanine). Conditions for observation of a specific noncovalent complex between the NCE and the target mimic using electrospray ionization-mass spectrometry were validated in a series of positive and negative control experiments, which featured flow injection analysisbased titrations. While the structural identity of the NCE could not be determined due to limited sample quantities, this work provides proof-of-principle for such an approach to potentially accelerate drug discovery from natural product sources. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Electrospray ionization (ESI) is well known as a soft-ionization source for mass spectrometry (MS). It has been used extensively for the interrogation of noncovalent interactions in a wide variety of small molecule and macromolecule-based molecular recognition systems [1–3]. In this context, ESI-MS is widely recognized for enabling fast and sensitive analysis, as well as direct access to interaction stoichiometry, without the need for labeling or immobilization strategies. The ability to handle complex mixtures and extract quantitative information is further enhanced by coupling ESI-MS with techniques, such as flow injection analysis (FIA) and high performance liquid chromatography (HPLC) [4]. The use of ESI-MS as a versatile detection tool in a variety of direct or indirect affinity screening approaches has been broadly termed affinity selection-mass spectrometry (AS-MS) [5]. Direct AS-MS refers to an experimental set-up where an ionic noncovalent complex of interest is transferred intact into the mass spectrometer for direct analysis and manipulation. Different approaches

∗ Corresponding author at: 700 Planetarium Pl.; Box 19065; Arlington, TX 760190065, USA. Tel.: +1 817 272 3541; fax: +1 817 272 3808. ∗∗ Corresponding author at: 501 S. Nedderman Dr.; Box 19498; Arlington, TX 76019-0498, USA. Tel.: +1 817 272 0397; fax: +1 817 272 2855. E-mail addresses: [email protected] (K.A. Schug), [email protected] (L.D. Mydlarz). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.11.038

including competitive binding, titration, host–guest screening, and gas phase tandem mass spectrometry can be used in this context [3]. In contrast, indirect AS-MS methods involve, initially, the isolation of molecules of interest from a complex matrix by selective affinity extraction, followed by the liberation of these molecules and their detection using mass spectrometry, perhaps in conjunction with HPLC separations. Overall, the various direct and indirect methods are characterized by different advantages and limitations. While indirect methods may be more amenable to high throughput screening, direct methods provide better confidence in the specificity of the observed interaction and allow their further interrogation using full scan and tandem mass spectrometry techniques. Both direct and indirect approaches have found significant use in drug discovery programs, in industry and in academia [5,6]. An active area of drug discovery, in general, is the pursuit of new chemical entities (NCEs) for treating infections caused by bacteria. Drug-resistant bacteria, including the Gram positive vancomycin-resistant Enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) bacteria, have been identified as high priority pathogens due to an increased incidence of infection and the lack of available drugs for effective treatment [7]. The result of such infections are prolonged hospital stays, increased heath care costs, and the use of secondary and tertiary treatment options which may be less effective and more toxic than preferential ones. Yet, discovery of NCEs appears to be impeded by the economicallydriven abandonment of antimicrobial drug discovery efforts by

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large pharmaceutical companies [8,9]. In fact, only two new classes of antibiotics have been approved for use in the past 20 years. Thus, a pressing need has arisen for new methods to rapidly identify NCEs as antibiotic lead compounds against these pathogenic organisms. A common mode of antibacterial activity for treating Gram positive bacteria is the inhibition of cell wall synthesis [10]. There is a wealth of literature that describes the interactions between well-known antibiotics, such as macrocyclic glycopeptides, and specific small peptide target motifs of Gram positive bacterial cell walls [11–14]. Work performed using direct ASMS has also contributed significantly to this body of knowledge [15–17]. The binding of macrocyclic glycopeptide antibiotics to depsipeptide mimics of intermediate peptidoglycan cell-surfacesynthesis structures in these studies has validated models for continuing such work. During peptidoglycan synthesis in normal Gram positive bacteria, ligase enzymes facilitate the presentation of terminal l-lysine–d-alanine–d-alanine (Kaa) motifs which provide coupling and crosslinking points for the construction of the peptidoglycan cell wall, as well as sites for inhibition by some antibiotics. Furthermore, there is good correlation between the results of target models showing binding of diand tripeptides models (N-terminal and ␧-Lys blocked and unblocked variants) with antibiotics such as vancomycin, ristocetin, and teicoplanin and in vitro bacterial growth inhibition data. Pharmaceutical companies once relied heavily on the discovery of new chemical entities (NCEs) from terrestrial and marine natural product sources [18,19]. More recently, efforts have shifted away from the natural products realm to combinatorial synthesis and automated screening technologies, citing better cost effectiveness for these approaches [20]. Traditional methods of “grind-and-find” discovery rely heavily on poorly selective and resource intensive bioassay-guided fractionation schemes that have significant drawbacks. Still, the fact that more than half of approved pharmaceuticals can be linked directly to some natural source, or a derivative thereof, emphasizes a significant supply of chemical diversity which is unlikely to have been fully explored [21]. The development of new methods which can more directly identify promising NCEs have and will continue to be necessary to sustain a resurgence of research into this field [22]. In this work, we report proof-of-principle for a direct affinity on-line chromatography–mass spectrometry screening assay which can be used to screen natural product extracts for the presence of NCEs. A series of positive and negative control experiments demonstrated the potential to detect antibacterial compounds which inhibit cell wall synthesis by an AS-MS technique termed dynamic titration [4,23]. To apply this technique to the discovery of NCEs, we screened crude extracts from a range of Caribbean corals for antibacterial activity against human and marine pathogenic bacteria. As a result of these screens one Caribbean gorgonian coral, Pseudoplexaura porosa, showed promising data and was targeted to apply the AS-MS assay. Data from the HPLC–ESI-MS assay correlated with results of standard growth inhibition assays at different levels of fractionation. The results indicated the presence of a previously unreported compound, identified by selective complexation in the HPLC–ESI-MS assay and by accurate mass analysis in an ion trap-time-of-flight-mass spectrometer (IT-TOF-MS), in a fraction which has been shown to significantly inhibit the growth of normal and drug-resistant bacterial strains. Thus, the reported direct AS-MS assay was demonstrated to be a viable way to bypass or enhance traditionally time-consuming bioassay-guided fractionation approaches and facilitate identification of potential NCEs from natural product sources earlier in the drug discovery workflow.

2. Experimental procedures 2.1. Dynamic titration control experiments A flow injection analysis AS-MS titration analysis was performed to confirm the ability of the instrument to detect noncovalent complexes between a series of macrocyclic glycopeptides antibiotics and the bacterial cell wall target mimic, Acetyllysine(acetate)–d-alanine–d-alanine (Ac2 Kaa; monoisotopic mass = 372.200885 Da) (Bachem, Torrance, CA). The macrocyclic glycopeptides, vancomycin, ristocetin, teicoplanin, and teicoplanin aglycone (Advanced Separation Technologies, Whippany, NJ) are considered positive controls based on their interactions with Gram positive peptidoglycan cell wall synthesis intermediates. As negative controls, antibiotics which should not bind to the target mimic due to their known ribosomal inhibition activity, chloramphenicol, gentamycin, neomycin, spectinomycin, and tetracycline (Duchefa Biochemie, Haarlem, The Netherlands) were evaluated. To perform dynamic titration, the host antibiotic was delivered in a continuous flow by a syringe pump (in 100% water + 5 mM ammonium acetate; 30 ␮L min−1 ) at a constant concentration (20 ␮M). A known amount of guest depsipeptide Ac2 Kaa (1.00 × 10−9 mol) was injected (2 ␮L injection) into the flowing stream. The mixture of host and guest was then passed through a 200 ␮L volume of blue PEEK tubing and allowed to band-broaden prior to reaching a conventional electrospray source interfaced with a LCQ Deca XP ion trap mass spectrometer (Thermo Fisher Scientific, West Palm Beach, FL). The data from the formed temporal compositional gradient of guest and host were extracted from the Xcalibur software (Thermo Fisher Scientific) and analyzed. For systems where a host–guest complex could be observed, a dissociation constant for the interaction was determined using an in-house-built software program. Specific details of the software, including the use of a modified Gaussian distribution function to fit the data, have been described elsewhere [4,23]. LC–MS grade water was from Burdick & Jackson (Muskegon, MI) and ammonium acetate (NH4 OAc) was from Sigma–Aldrich (St. Louis, MO). ESI-MS source conditions were optimized separately for robust observation of complex formation between vancomycin (10 ␮M) and Ac2 Kaa (10 ␮M) by direct infusion. 2.2. Sample collection, preparation, and fractionation The gorgonian coral P. porosa was collected using SCUBA from the Looe Key Reef research site in the Florida Keys, USA in July of 2008 under the specifications of a Florida Fish and Wildlife Conservation Commission nonresident saltwater fishing license. Gorgonian fragments (5 cm each from 5 individual coral colonies) were collected at depths of 5–10 m. All specimens were identified by L.R. Hunt, L.D. Mydlarz, and E. Bartels. Coral fragments were flash frozen in liquid nitrogen and shipped on dry ice to the University of Texas at Arlington, and stored at −80 ◦ C until use. At the University of Texas at Arlington, samples from multiple coral colonies were pooled, lyophilized on a VirTis Benchtop K lyophilizer (The VirTis Company, Gardiner, NY) and ground in a mortar and pestle to a fine powder. 100% ethanol (Decon Labs, Inc., King of Prussia, PA) was added at a ratio of 10 mL to every 0.2 g of homogenized coral. Extracts were then transferred to pre-weighed vials, evaporated to dryness under nitrogen, and final weights determined. All samples were diluted to a stock concentration of 100 mg mL−1 and stored at −20 ◦ C until further analysis. The extract was reconstituted in 20% aqueous ethanol and subjected first to reversed phase solid phase extraction (SPE). A step fractionation/elution procedure was used to reduce the complexity of the sample and obtain samples for testing. SPE was carried out using Bakerbond ODSII C18 (500 mg sorbent, 6 mL solvent

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capacity) (J.T. Baker, Phillipsburg, NJ) reversed phase pre-packed extraction cartridges. The cartridges were first conditioned with 10 mL of methanol and then equilibrated with 10 mL of 99/1 0.5% acetic acid/methanol. The reconstituted extract was split into six portions and applied to six separate SPE cartridges to avoid column overload. The sample was loaded and fractions were collected following sequential elution in 5 mL aliquots by 99/1, 75/25, 50/50, 25/75, and 0/100 0.5% acetic acid/methanol (v/v), followed by 10 mL (2 × 5 mL) of 100% n-propanol. Aligned fractions were recombined following collection. Methanol (MeOH) and glacial acetic acid were from J.T. Baker. n-Propanol (nPrOH) was from Burdick & Jackson. Each fraction was evaporated to dryness under nitrogen and/or by lyophilization (depending on organic solvent content) and dry weight was recorded. The solutions were then reconstituted for analysis by the growth inhibition assay and the HPLC–ESI-MS direct affinity assay, as described below. Semi-preparative HPLC was used to further fractionate fraction 9 to track the presence of a potential NCE identified by the AS-MS assay. A Finnigan Spectrasystem HPLC and UV6000LP photodiode array detector (Thermo-Fisher Scientific, Inc., Waltham, MA) connected on-line to a Foxy, Jr. fraction collector (Teledyne-Isco, Inc., Lincoln, NE) was used to fractionate the sample in discrete elution time segments. A 30-min gradient mobile phase composition (0–3 min, hold 75:25 mobile phase A:mobile phase B; 3–18 min, linear gradient to 1:99 A:B; 18–30 min, hold at 99% B; where mobile phase A was 100% water and mobile phase B was 90% acetonitrile + 10% isopropanol) was used in conjunction with a Luna C18 (10 mm i.d. × 150 mm L, 10 ␮m dp ) at a flow rate of 3 mL min−1 to obtain 30 fractions in 1 min segments. Fractions were evaporated to dryness by lyophilization and dry weight was recorded. Fractions were analyzed for the m/z ion signature of the NCE by HPLC–ESI-MS, and fractions containing the compound of interest were also subjected to the growth inhibition and AS-MS assays. All samples and fractions were contained in amber vials to minimize the exposure of any potentially photo-labile compounds to light. All dry and/or reconstituted samples were stored at −20 ◦ C, until they were used. 2.3. Growth inhibition assay Bacterial growth inhibition activity was assessed using a bacteria turbidity assay and conducted in sterile 96-well flat-bottom microtiter plates (Greiner Bio-one, Monroe, NC). Ethanol extracts of P. porosa were diluted in nutrient media Difco marine broth (Becton, Dikinson and Co, Le Pont de Claix, FR) or Luria Broth (LB, Miller, Novagen, Merck KGaA, Darmstadt, Germany), depending on the bacteria being tested, for a final concentration of 250 ␮g mL−1 per well. All strains were either purchased from the American Type Culture Collection (ATCC, Manassas, VA) and ATCC strain numbers are included, or received as generous gifts from K. Ritchie (Mote Marine Laboratory) and GenBank accession numbers are given for reference. Human pathogenic Gram positive bacteria included in the study were Enterococcus faecalis (ATCC #29212), vancomycinresistant Enterococcus faecium (VRE; ATC #700221), S.. aureus (ATCC #29213), Methicillin-resistant S. aureus (MRSA; ATCC #43300), and Bacillus subtilis (ATCC #6051). Human pathogenic Gram negative bacteria were Escherichia coli (ATCC #10536) and Pseudomonas aeruginosa (PAO-PR1; ATCC #39018). Marine pathogenic Gram negative bacteria were Vibrio alginolyticus (GenBank accession #X744690) and Serratia marcescens (PDL100; ATCC #BAA-632). All bacteria strains were handled in a biological safety cabinet and in accordance with the University’s Biosafety Level 2 policies. Overnight bacteria cultures, in exponential growth, were diluted to an optical density (OD600nm ) of 0.2 and added to wells containing

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extracts and fresh broth for a final assay volume of 200 ␮L per well and a concentration of 250 ␮g mL−1 extract, with a maximum of 0.25% ethanol in each well. Assays were run in triplicate wells and experiments contained bacteria with extract, along with the following controls: bacteria only, bacteria + positive control (commercial antibiotic), or bacteria + ethanol (to control for ethanol effects). Assays were run at appropriate temperatures for the select bacteria (37 ◦ C for human pathogenic strains and 29 ◦ C for marine strains). The plates were gently mixed, read over a period of 5 h at OD600 with a Biotek Synergy 2 spectrophotometer, and analyzed using GEN5 software (Bio-Tek Inc, Winooski, VT). Growth inhibition of bacteria cultures was calculated by comparing the growth rate (GR) of bacteria with coral extracts to bacteria with ethanol controls. The GR was calculated with the following formula: 3.3log (tf /ti ))/n), where, tf is final OD600 , ti is initial OD600 , and n = tf − ti . The linear portion of logarithmic growth was used to select the initial (t = 1 h) and final hour (t = 2 or 3 h) and was kept standard between runs within each bacteria species. Percent growth inhibition was determined by calculating the difference in GR of wells with P. porosa extracts to bacterial growth without extracts and only ethanol as vehicle control. 2.4. Direct affinity screening by HPLC–ESI-MS The direct affinity screening HPLC–ESI-MS assay was performed on a Surveyor HPLC (pump and autosampler; Thermo Fisher Scientific) coupled to the LCQ Deca XP ion trap mass spectrometer equipped with a conventional ESI source. An external syringe pump (KD Scientific, Holliston, MA) was placed in line with the solvent flow to complete the experimental set-up shown in Fig. 1. Coral fractions taken from SPE were diluted 10-fold in 50/50 acetonitrile/water + 5 mM NH4 OAc for injection. The samples (25 ␮L) were injected into the HPLC, operated at 40 ␮L min−1 flow rate with a gradient program starting at 75/25 v/v of mobile phase A/mobile phase B (mobile phase A was water + 5 mM NH4 OAc + 0.5% HOAc; mobile phase component B was acetonitrile + 5 mM NH4 OAc) (held for 3 min) and increasing to 1/99 A/B in 10 min, with a subsequent hold at this composition for 15 min, and then reequilibration back to 75/25 A/B, prior to the next injection. The HPLC column was a Tosoh TSKgel C18 (1.0 × 50 mm, 3 ␮m dp ) (Tosoh Bioscience LLC, King of Prussia, PA). The syringe pump was consistently operated at 10 ␮L min−1 for all experiments, joined into the chromatographic flow path with a zero dead volume t-junction, to provide a total flow of 50 ␮L min−1 into the ESI source. The samples were first injected with no affinity ligand (instead, 50/50 acetonitrile/water) added post-column (“Run 1” in Fig. 1) to ascertain the major ion signals produced from the sample. Major ion signals were recorded by manually searching the total ion chromatogram for signals having an intensity above 5 × 105 ion counts. After Run 1, the sample was injected again, but with 30 ␮M Ac2 Kaa (dissolved in 50/50 acetonitrile/water) added post-column from the syringe pump (“Run 2” in Fig. 1). A manual calculation of expected complexes to be observed based on the sum of masses of the dominant signals from Run 1 with the mass of the ligand (372 Da) were manually searched in Run 2 data to ascertain the presence of formed ionic complexes. The properties of compounds identified, which bound to the affinity ligand, were further interrogated by high resolution ESI-MS and tandem mass spectrometry as described below. 2.5. High resolution HPLC–ESI-MS and MSn To aid in the characterization of sample components, identified by the HPLC–ESI-MS screening assay as potential antibacterial

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Fig. 1. Experimental set-up for a direct AS-MS assay to identify compounds in coral extract which bind to a Gram positive cell wall synthesis target mimic.

agents, relevant fractions were also analyzed using HPLC–ESI-MS on a hybrid ion trap-time-of-flight-mass spectrometer (LCMS-IT TOF) coupled to a Prominence HPLC (Shimadzu Scientific Instruments, Inc., Columbia, MD) to obtain high resolution (R ∼10,000 FWHM), mass accurate (<5 ppm error), full scan and tandem mass spectrometry fragmentation information. Reversed phase HPLC was carried out on a Shimpack XR-ODS C18 column (2.0 mm i.d. × 100 mm L, 2.2 ␮m dp ) (Shimadzu). Fractions were reconstituted to 0.2 mg mL−1 and 20 ␮L was injected by autosampler. A gradient separation was used where mobile phase A was the same as above and mobile phase B was composed of 90/10 acetonitrile/isopropyl alcohol. The flow rate was 250 ␮L min−1 . Compounds eluted from the column entered the ESI source, which was operated in the positive ionization mode. Three acquisition events were incorporated in a repeating duty cycle to first collect full-scan data, followed by MS/MS, and then MS3 , in a software-automated data-dependent fashion. Thus, observed ions were isolated and fragmented to obtain qualitative information throughout the chromatographic run. Priority was given to those ions indicated by the HPLC–ESI-MS screening assay to have formed a stable complex with the affinity ligand. To aid in the assignment of possible elemental formulae for the compounds, Fragment Predictor software (Shimadzu) was used. This software uses high mass accuracy full scan and tandem mass spectrometric data, as well as observed isotope abundances to match possible elemental formulae to the ions of interest. The software also accounts for standard rules [24] in assignment of elemental formulae based on the nitrogen rule, double bond equivalence, and mass accuracy tolerances. 3. Results and discussion Identifying novel NCEs with antibacterial properties from complex mixtures of natural product extracts requires innovative methods, which provide results in a more expedited fashion compared to traditional “grind and find” methodologies. To accomplish this, a direct affinity on-line chromatography–AS-MS screening assay, capable of handling complex mixtures from natural product extracts, was developed and a series of validation control and application experiments were performed. The specificity of ESIMS for monitoring selective complexation between Ac2 Kaa and selected antibacterial compounds (both positive and negative controls) was demonstrated using a series of quantitative binding determinations performed by dynamic titration [4,23]. The basis of this approach is the use of flow injection analysis to create a precisely dispersed zone of the guest compound (e.g. Ac2 Kaa) in the presence of a constant concentration of host (e.g. vancomycin), whereby, if complex formation is observed in the mass spectra, an appropriate model can be applied to determine the magnitude

of binding affinity between the host and guest. The multi-point titration in a continuous flow is facilitated by the temporal variation of guest concentration (a “peak”) in the presence of host, as the mixture enters the mass spectrometer. A clear distinction between commercial antibacterial compounds which were supposed to bind Ac2 Kaa, versus those which were not, allowed us to next evaluate post-column addition of the affinity ligand to HPLC separations of coral extract fractions, to identify compounds in the mixture which might also exhibit antibacterial activity. Once such a compound was identified, it was further interrogated by high resolution HPLC–ESI-MS. Accompanying the results of the AS-MS assay, a traditional spectroscopic growth inhibition assay was used to track the antibacterial activity of fractions containing the potential NCE. Described in detail below, all of the results were consistent with the identification of a potentially new antibacterial agent against normal and drug-resistant Gram positive bacteria from the Caribbean gorgonian coral, P. porosa. 3.1. Dynamic titration control experiments The experimental design for dynamic titration control experiments (Fig. 2A) includes its application for monitoring the formation of complexes between commercial antibacterial compounds and Ac2 Kaa (Fig. 2B) and the calculation of binding affinity (dissociation constants) using an in-house-built software program. The program fits a modified Gaussian function to the flow injection analysis data to determine the concentration of guest at each point on the curve. The signal of the complex recorded at each point for a variable concentration of guest and a constant concentration of host generates a titration profile from which a dissociation constant can be determined by fitting the data with a 1:1 solution phase binding model [4,23]. Results of the control/validation experiments using commercially available antibiotic compounds are presented in Table 1. Five compounds known to inhibit bacterial growth through ribosomal inhibition were used as negative controls. No complex formation was observed with the target ligand. Four macrocyclic antibiotics, known to inhibit cell wall synthesis in Gram positive bacteria were used as positive controls. Significant complex formation was observed with the target mimic, and in each case, a dissociation constant was also extracted. Values of dissociation constants obtained for vancomycin and ristocetin were in good agreement with those reported in the literature, determined using both standard solution phase and traditional mass spectrometric binding constant determination techniques (∼1 ␮M for vancomycin, and ∼2 ␮M for ristocetin, binding Ac2 Kaa under similar solution phase conditions [11–17]. These control experiments indicate that optimized ESI-MS instrumental parameters used in this experiment

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Fig. 2. Determination of binding constants by dynamic titration. (A.) A known amount of guest (N0,G ) was injected in a continuous flow of the host compound (c0,H ) and allowed to band broaden prior to entering the ESI source and mass spectrometer. (B.) Extracted ion chromatograms for the ionic complex formed between host and guest were clearly visualized in three discrete injections over a period of approximately 30 min. (C.) Data for the complex normalized to that of the free host ion response were input into a software program which simultaneous fit a modified Gaussian function and a 1:1 host–guest binding model to determine the dissociation constant for the interaction system.

preserved the highly specific binding interactions between cell wall synthesis inhibitors and Gram positive bacterial cell wall synthesis target mimics, while potential false positives were minimized. 3.2. Broad-spectrum antibacterial activity of P. porosa crude extracts The gorgonian coral P. porosa has been investigated for potential compounds with antitumor properties, mainly small molecule diterpenoids [25]. The potential for antibacterial properties have been limited to examining crude extracts against marine bacteria and marine fungi [26,27]. In this study, 100% ethanol extracts of the lyophilized corals were tested for their inhibition of growth against a suite of Gram positive and Gram negative bacteria, mainly

human and marine pathogens (Fig. 3). P. porosa extracts inhibited the growth of Gram positive human bacteria species more significantly than marine strains, with activity against B. subtilis and S. aureus the highest. The inhibitory activity of the extracts against Enteroccocus species were moderate, but significantly higher in vancomycin-resistent Enterococcus, making E. faecalis and VRE good target strains for continued assays after fractionation of the crude extract since limited compound supply did not permit the use of all strains. 3.3. Targeted fractionation and AS-MS of P. porosa extracts A combination of SPE and HPLC fractionation procedures in conjunction with the direct AS-MS screening assay were used to

Table 1 Positive and negative ESI-MS binding specificity validation experiments. Antibiotic

Inhibition mechanism

Complex with Ac2 Kaa

Kd ± S.E. (␮M) (n = 3)a

Chloramphenicol Gentamycin Neomycin Spectinomycin Tetracycline Ristocetin Teicoplanin Teicoplanin aglcyone Vancomycin

Ribosomal function Ribosomal function Ribosomal function Ribosomal function Ribosomal function Cell wall synthesis Cell wall synthesis Cell wall synthesis Cell wall synthesis

No No No No No Yes Yes Yes Yes

N/A N/A N/A N/A N/A 6.8 ± 0.9 0.4 ± 0.1 6.0 ± 0.8 2.8 ± 1.5

a

In the flow injection analysis format, where complexes are formed, a dissociation constant can be calculated based on dynamic titration methodology [4,23].

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Fig. 3. Broad spectrum antibacterial activity of crude P. porosa extracts against Gram positive and Gram negative bacterial strains. Inset: P. porosa.

identify components in the crude extract which exhibited antibacterial activity against Gram positive bacteria. First, the residue of the crude extract was reconstituted in 20% aqueous ethanol and subjected to SPE. Eleven fractions were collected by increasing the mobile phase strength in a step-wise fashion, as described in Section 2. Each fraction was either dried under N2(gas) or lyophilized (depending on water content of the eluent), and then reconstituted in 100% ethanol to test for antibacterial activity against E. faecalis and vancomycin-resistent Enterococcus. As mentioned above, growth inhibition assays were restricted to these bacterial species due to limited sample quantity and because their inhibition was the most relevant to the incorporation of the Ac2 Kaa target mimic ligand in the AS-MS assay. The fractions each had different antibacterial activity (Fig. 4). Fractions 1–4 were collected during the sample loading stage, and since no significant antibacterial activity was recorded for each of these fractions, only fraction 1 is shown in Fig. 4. Fraction 9 exhibited the highest activity against E. faecalis (∼70% inhibition), significantly greater than that recorded for the crude extract. Significant inhibition of VRE was also recorded for this fraction, although slightly less than the crude extract and of the vancomycin-sensitive strain. For initial evaluation and application of the AS-MS assay, the complete crude extract was deemed to be too complex to start with initially. Therefore, fraction 9 was

Fig. 4. Antibacterial activity, measured by percent growth inhibition, of crude and fractionated P. porosa extracts against E. faecalis and vancomycin-resistant Enterococcus (VRE).

targeted for application of the AS-MS assay, as it exhibited the highest antibacterial activity. Fraction 9 was diluted 10-fold in water for injection into the HPLC system. For the first run, a blank 50/50 acetonitrile mixture was added post-column instead of the Ac2 Kaa ligand to determine the m/z values associated with the major ion signals observed from the separation of the fraction. Expected values that might be recorded if 1:1 complex formation with the Ac2 Kaa ligand occurred for each of the observed m/z values in the first run (m/z +372) were manually calculated. The diluted fraction was then injected a second time with the target mimic ligand added post-column and the presence of signals for potential complex ions were monitored in the ion chromatogram. During the 20–22 min region of the ion chromatogram, a possible complex was observed (m/z 1320–1328) between the target mimic ligand and an envelope of signals with m/z 948–952, which had been observed in the first run (Fig. 5). The analysis was repeated several times and was shown to be consistent. Additionally, collision-induced fragmentation of ions assigned as the noncovalent complex returned the appropriate signals consistent with those observed in Fig. 5A (m/z ∼950). No other complexes were observed between other components, represented as MS ion signals, and the target ligand. The identification of a compound which may be responsible for the observed antibacterial activity of fraction 9, based on a m/z signature and specific binding to Ac2 Kaa in the AS-MS assay, provided a handle for further tracking of the compound of interest in subsequent purification steps. To demonstrate that the compound could be tracked using the AS-MS assay, but understanding that very limited sample quantities were available following SPE fractionation, an additional round of fractionation was performed by semi-preparative HPLC. Approximately 1 mg of dry material was obtained from late eluting fractions. Upon reconstitution and analysis by HPLC–ESI-MS, fractions 25–28 contained the MS ion signature of the compound of interest. Due to very limited amounts, these four fractions along with fractions 23 and 24 (as controls) were subjected to the antibacterial growth inhibition assay against only one bacteria (E. faecalis) and with a concentration of 150 ␮g mL−1 instead of the typical 250 ␮g mL−1 . Of the fractions, fraction 25 exhibited the highest relative inhibition with 41.2 ± 2.7% inhibition of growth. This was significantly higher than fraction 23 and 24 which did not contain the MS ion signature (15.8 ± 0.5% and 9.8 ± 1.6%, respectively). Fractions 26 through 28 did demonstrate some inhibitory effects

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Fig. 5. Components present in an ion envelope from P. porosa SPE fraction 9 (top and middle panel in (A.)) form a noncovalent complex with Ac2 Kaa (middle panel in (B.)) during direct AS-MS. The bottom panels of (A.) and (B.) (as controls) indicate that the signals of interest are confined to a discrete time frame.

against E. faecalis that was less than fraction 25 (range of 26.8 ± 2.6 to 33.2 ± 2.8%) further supporting the HPLC–ESI-MS data that these fractions did contain less of the signal. Since fraction 25 had the highest relative inhibition of growth, it was then subjected to the AS-MS assay, and the results confirmed that a specific complex with Ac2 Kaa was observed, as before, in the expected elution timeframe. The fraction was not fully purified by the HPLC fractionation, as evidenced by additional impurities, which eluted through the chromatographic run (data not shown). The amount of material remaining following HPLC fractionation, antibacterial assay, and AS-MS assay was insufficient for further processing. Nevertheless, the AS-MS provided consistent data through two rounds of fractionation and these results provided proof-of-principle for the viability of such an assay for directed tracking of the potential NCE.

3.4. High resolution HPLC–ESI-MS Purity and quantity of material obtained from fractionation of the P. porosa extract precluded use of spectroscopic structural determination tools, such as NMR, to determine the identity of the NCE of interest. Therefore high resolution mass spectrometry and tandem mass spectrometry was used to gain additional insight. Fraction 9 from the SPE fractionation was injected into a HPLC–ESIion trap–time-of-flight-MS instrument. The analysis indicated that the envelope of signals for the NCE observed during the AS-MS assay could be separated into two discrete compounds (Fig. 6). Each compound exhibited an interesting and atypical mass spectral pattern. The first peak was characterized by dominant ions at m/z = 948.5652 and 953.5196 (m/z = 4.9544), and the second peak was characterized by dominant ions at m/z = 950.5842 and 955.5393 (m/z = 4.9551). A circa 5 mass unit difference in the signals is difficult to explain in terms of the make-up of a single compound, especially since each signal was characterized by its own reasonable isotope distribution and identical retention times. Moreover, a closer inspection of the data related to complex formation with Ac2 Kaa from the AS-MS assay (shown in Fig. 5B) indicated that noncovalent binding occurred with the 950/955 m/z compound (major complex ion signals were observed at m/z 1322 and 1327; m/z = 372). Thus, the two peaks shown in Fig. 6A seem to be related, perhaps simply differing by degree of saturation somewhere in the molecule, however only one of the compounds binds to a significant degree with the target ligand. Tandem mass spectrometry was applied to the 950/955 ion signals, in an attempt to obtain further information which might be

used for elemental formula prediction. MS/MS of m/z 950.5829 yielded three dominant product ion signals at m/z 677.3233 (m/z = 273.2596), 515.2595 (m/z = 435.3234), and 405.1342 (m/z = 545.4487). MS/MS of m/z 955.5377 also yielded three dominant product ions at m/z 679.3242 (m/z = 276.2135), 517.2723 (m/z = 438.2654), and 405.1313 (m/z = 550.4064), as well as three minor product ion signals at m/z 677.3127, 515.2556, and 347.0890. Clearly, similar fragmentation patterns can be envisioned, and again, there seems to be minor differences simply in the degree of saturation involved (circa 2 m/z unit shifts). Both m/z 950.5829 and 955.5377 yielded a product ion of 405.13, indicating that the structural unit which corresponds to this fragment is likely to be identical in each ion. Using the high mass accuracy, fragmentation, and isotope ratio data, Formula Predictor software could be used to predict possible elemental formulae. However, given the large mass of the compound of interest, the software produced many (>40 formulae with mass accuracy error <10 ppm) reasonably ranked possibilities (restricting atoms in the compound to only C, H, N, and O). The top four ranked elemental formulae for m/z 955.5377 were C33 H47 N14 O18 (score 99.03; ppm difference = −0.10; double

Fig. 6. High mass accuracy MS on an HPLC–ESI-IT-TOF-MS instrument. Chromatography resolved two compounds (A.) in the ion envelope of interest, each of which had similar signal character. The mass spectrum for the first peak is shown in (B.) and that for the second peak is shown in. (C.)

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bond equivalents (DBE) = 4.0), C34 H70 N18 O14 (score 98.58, ppm difference = −1.57; DBE = 9.0), C37 H78 N8 O20 (score = 95.17, ppm difference = −2.93, DBE = 3.0), and C38 H74 N12 O16 (score = 91.50, ppm difference = −4.40, DBE = 8.0). Searches based on accurate mass and these elemental formulae in the Dictionary of Natural Products returned no hits. Similar searches in SciFinder Scholar returned some similarly defined peptidic-based structures but no close matches. In the end, without a pure sample of sufficient quantity to apply a larger suite of structural and elemental determination methods, such as NMR, IR, X-ray, and thermogravimetric analysis, it is virtually impossible to discern the identity of a circa 1000 molecular weight compound based on mass spectrometry alone. Unfortunately, the additional purification steps needed were precluded by the size of the coral sample available for extraction. Future attempts will be made beginning with a larger quantity of raw material. 4. Conclusions This work demonstrated the potential of a new direct chromatography–AS-MS assay for identification of antibacterial NCEs from natural product extracts. The assay was validated by a quantitative dynamic titration approach, which served to demonstrate the optimal settings of the instrument for discriminating specific binding events. The assay performed against a natural product extract incorporated a well-characterized target mimic (Ac2 Kaa) as a probe for compounds that bind to the cell walls of Gram positive bacteria to inhibit biosynthesis and bacterial growth. Thus, the assay could be used in direct purification processes and provide some insight into the potential biological mechanism of compounds in the extracts and fractions. The methodology/approach was supported by the successful identification of a novel NCE with broad-spectrum antibacterial activity in ethanol extracts from the Caribbean gorgonian coral P. porosa. Antibacterial activity was preserved and enhanced through a series of fractionation steps where focus was placed predominantly on those fractions which contained a compound shown to bind with the target mimic ligand by AS-MS. Even though this particular study was hampered by limited material, this approach can increase the efficiency of the process related to identifying NCEs from natural product sources by being able to analyze complex mixtures and utilizing far less material than traditional bioassay-guided fraction. Our lab is currently working on expanding synergistic affinity extraction media to increase selectivity in the extraction steps [28]. In the present study, a target mimic ligand relevant to probing antibacterial activity against Gram positive bacteria was incorporated in the assay, but in principle, this approach could be used in

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