Chemical Microarrays to Identify Ligands that Bind ... - Google Sites

DOI: 10.1002/cbic.200600260

Chemical Microarrays to Identify Ligands that Bind Pathogenic Cells Olivia J. Barrett, Jessica L. Childs, and Matthew D. Disney*[a] Cell-surface recognition is a quintessential event in biological systems. Interactions at the cell surface mediate the formation of organs and tissues in multicellular organisms and contribute to pathogenesis. Recognition of cell surfaces by carbohydrates and proteins is a major contributor to disease and often initiates the infection process. Antiadhesion therapeutics have been designed based on the multivalent display of cell-surface ligands that play critical roles in stabilizing pathogen–host interfaces.[1] Such compounds have found uses in treating the influenza virus and anthrax infections.[2] Other important therapeutics target cell surfaces, including antibiotics that inhibit bacterial cell-wall biosynthesis.[3] Therefore, finding new scaffolds that recognize cell surfaces is an important topic in biomedical research. Such compounds can be used in therapeutic and diagnostic applications. One way in which new ligands that interact with cell surfaces can be identified is by microarray-based screening. Herein, we describe the development of a microarray-based platform to study the binding of ligands to the surface of a variety of pathogens. These studies have identified a pathogen “fingerprint” for ligand recognition and give insight into the type of compounds that bind to particular cell types. The use microarrays to study the binding of ligands to whole cells has been reported, by using arrays of carbohydrates that have known affinities for particular cells.[4] Herein, we describe the use of microarrays to identify new ligands for cells by interrogating their binding to a library of organic ligands. The advantages of using microarrays include assay miniaturization and the manner in which ligands are displayed. Microarray-immobilized ligands are displayed in a manner that accommodates multivalent binding because many ligands are displayed in a small area (Figure 1). This display mimics interactions that occur at cell–cell interfaces and amplifies binding affinities. One potential limitation for using microarrays to study binding to pathogenic cells that we have encountered is nonspecific binding of pathogens to array surfaces. Many common array surfaces, such as ones displaying amines, bovine serum albumin (BSA), or amino acids, are functionalized with compounds that nonspecifically bind pathogens.[5] In order to develop a platform that alleviates this limitation, a series of array surfaces [a] O. J. Barrett, Dr. J. L. Childs, Prof. Dr. M. D. Disney Center for Excellence in Bioinformatics & Life Sciences and Department of Chemistry, University at Buffalo Buffalo, NY 14260 (USA) Fax: (+ 1) 716-645-6963 E-mail: [email protected] Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

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Figure 1. Top: multivalent binding that occurs at cell–cell interfaces. Bottom: binding of cells to chemicals on a microarray mimics the interactions that occur at cell–cell interfaces.

was tested for resisting nonspecific binding. In these experiments, we used the pathogenic bacterium Pseudomonas aeruginosa, which forms sticky biofilms,[6] and five different surfaces. Surfaces included amine-, BSA-, and agarose- (aldehyde-) coated glass, plain glass, and glass coated with ethanolamine prepared in a two-step procedure from amine-coated slides.[7, 8] After hybridization of fluorescently labeled P. aeruginosa to the arrays and washing off unbound organisms, slides were scanned with a fluorescent slide reader. Results show that background signals from slides coated with amines were the highest followed by BSA and ethanolamine (Figure 2). The two platforms that gave the least signal were plain glass and glass coated with agarose. This is not surprising given the hydrophilic nature of agarose. The observation that glass slides have a very low background while the amine-functionalized slides have a high background suggests that P. aeruginosa binds to surfaces displaying positive charges whereas it does not bind to ones that are negatively charged. Amino acids have been studied for binding to Candida albicans, a pathogenic fungus, and results show that C. albicans strongly binds to amino acids and proteins, including BSA.[6] Previously, a BSA-coated surface has been described for use in inhibiting nonspecific pathogen binding in protein arrays.[9] Based on this report and our observations that many pathogenic organisms bind to microarrayimmobilized BSA (unpublished data), we believe that agarose slides offer an alternative as a more general arraying substrate

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Figure 2. Testing of cell surfaces for resisting nonspecific binding of P. aeruginosa.

to study the binding of microarray-immobilized ligands to cells. Additionally, Belleville et al. reported that agarose-functionalized slides were optimal for studying the binding of antibodies to small molecules.[10] The advantages of agarose-functionalized slides are numerous: they are easy to prepare, inexpensive, and readily functionalized to amine-reactive aldehydes upon treatment with NaIO4.[8] Moreover, the surface is three dimensional, which enhances loading and potentially limits effects due to steric encumbrance by the slide surface. Therefore, agarose can function as a dendrimeric linker to which compounds are immobilized and displayed on a surface. Atomic force and electron microscopy studies have shown that agarose-coated slides have a porous sheet-like structure with pores ranging in size from 100 to 500 nm and parallel to the surface.[11] We used this platform to interrogate the affinities of a small library of heterocyclic compounds for binding to the pathogens P. aeruginosa, E. coli, and C. albicans. All compounds were chosen such that they contained an amino group for surface immobilization (Scheme 1). Microarrays containing a series of 60 compounds were constructed by spatially delivering 200 nL of a 5 mm organic ligand solution in a buffer containing 5 % Na2CO3 and 10 % glycerol. Slides were then quenched with a solution of NaCNBH3 to reduce all remaining aldehydes. In other array-based platforms, BSA,[12] ethanolamine,[13] or glycine is used to quench slide surfaces; we took care to avoid these steps so as to diminish nonspecific binding. Each organism was stained with the cell-permeable nucleic acid stain SYTO 60 and incubated with the arrays in phosphate-buffered saline (PBS) for 1 h at room temperature. After incubation, unbound organisms were washed from the surface with PBS, and the arrays were air dried and scanned with a microarray scanner. Each organism bound to many of the 60 compounds placed on the surface (Figure 3). Most bound all three organisms. Of the 60 compounds tested, compounds 22, 23, and 34 bound to E. coli, P. aeruginosa, and C. albicans with

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signals above background. Interesting structure–activity relationships are observed upon inspection of the data. The highest affinity ligand for all of the organisms is tryptACHTUNGREamine (23). The 2-amino-5,6-dimethylbenzimidazole (16) gave the second highest signal for P. aeruginosa and C. albicans (Figure 3). Other related compounds arrayed include l-tryptophan (30), which gave the second highest signal for binding E. coli. l-tryptophan has reduced signals for binding to all organisms relative to tryptamine; signals are 61 %, 32 %, and 40 % for E. coli, C. albicans, and P. aeruginosa, respectively. These results suggest that interactions occur with the indole moiety present in both tryptamine and l-tryptophan but binding is affected by placement of substituents around this scaffold. It is of interest that several scaffolds containing the indole side chain of tryptophan have previously been shown to recognize components of bacterial surfaces. For example, a tetratryptophan derivative has been reported to sense lysed Gramnegative bacteria and bind to lipid A,[14] and lipid vesicles displaying tryptophan have been used as a colorimetric sensor for lipopolysaccharide from a variety of sources.[15] Interestingly, there are some compounds that are unique to one organism over another. For example, compound 22 only binds to Pseudomonas, while compounds 1 and 57 give a higher signal for Candida than for E. coli or Pseudomonas. The amino acids lphenylalanine (52) and d-phenylalanine (53) bind to each organism with different affinities. For example, both compounds bind P. aeruginosa with low signals (less than 5 % normalized fluorescence) but both bind at least two times more strongly to C. albicans and E. coli, with 52 giving a slightly higher signal than 53 in both cases. Although there are no ligands that are selective for each organism in this study, we hope that screening a larger and more diverse set of compounds will allow for the discovery of selective binders. Next, we investigated if ligands identified by microarraybased screening could also recognize cells when multivalently displayed on a water-soluble polymer. Poly(ethylene maleic anhydride), a polymer that has been previously used to multivalently display mannose,[16] was functionalized with tryptamine or ethanolamine in 1 mL DMSO overnight (Figure 4). Unreacted anhydrides on the polymer were quenched with water, and uncoupled tryptamine or ethanolamine was removed by dialysis. A UV spectrum of the polymer was taken to confirm coupling of tryptamine (Supporting Information). A solution of polymer (80 mg) and E. coli in PBS was tumbled overnight. An aliquot of the solution was removed, spotted onto slides, and viewed under a microscope. Images showed that the tryptamine-functionalized polymer causes large bacterial aggregates whereas ethanolamine-functionalized polymer did not (Figure 4); similar clustering of bacteria has been observed with sugar-functionalized fluorescent polymers.[17] These results confirm that tryptamine recognizes E. coli when displayed multivalently on a solid surface or on a polymer in solution. In summary, we have developed a microarray-based technique that probes the binding of small organic ligands to a ACHTUNGREvariety of pathogenic cell types ranging from Gram-negative bacteria (P. aeruginosa and E. coli) to fungi (C. albicans). The ligands identified can be used for a variety of therapeutic and

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Scheme 1. The library of structures immobilized onto microarrays and probed for binding to pathogenic cells. Compounds were immobilized by treating amines with aldehydes displayed on the surface.

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diagnostic applications. Our laboratory is pursuing both of these areas, and progress will be reported in due course.

Acknowledgements This work was funded by a New York State Center of Excellence in Bioinformatics & Life Sciences, University at Buffalo Interdisciplinary Research Development Fund grant, and the Camille and Henry Dreyfus New Faculty Award to M.D.D. We would also like to thank Dr. Alice Huang and Prof. Paras Prasad for help with microscopy in the Institute for Laser, Photonics, and Biophotonics at the University at Buffalo. Keywords: biosensors · cell adhesion · microarrays · molecular recognition · pathogens

Figure 3. Microarray-based screening of small-molecule ligands for binding to a variety of pathogenic organisms. A) An image of a chemical microarray after hybridization with E. coli stained with cell-permeable SYTO 60. B) The data from arrays hybridized with three different pathogens. Data are the average of four experiments.

[1] M. Mammen, S. K. Choi, G. M. Whitesides, Angew. Chem. 1998, 110, 2908 – 2953; Angew. Chem. Int. Ed. 1998, 37, 2755 – 2794. [2] J. D. Reuter, A. Myc, M. M. Hayes, Z. Gan, R. Roy, D. Qin, R. Yin, L. T. Piehler, R. Esfand, D. A. Tomalia, J. R. Baker, Jr., Bioconjugate Chem. 1999, 10, 271 – 278; S. K. Choi, M. Mammen, G. M. Whitesides, Chem. Biol. 1996, 3, 97 – 104; G. D. Glick, P. L. Toogood, D. C. Wiley, J. J. Skehel, J. R. Knowles, J. Biol. Chem. 1991, 266, 23 660 – 23 669; M. Mourez, R. S. Kane, J. Mogridge, S. Metallo, P. Deschatelets, B. R. Sellman, G. M. Whitesides, R. J. Collier, Nat. Biotechnol. 2001, 19, 958 – 961. [3] S. J. Projan, Curr. Opin. Pharmacol. 2002, 2, 513 – 522; C. Walsh, Nature 2000, 406, 775 – 781. [4] M. D. Disney, P. H. Seeberger, Chem. Biol. 2004, 11, 1701 – 1707; L. Nimrichter, A. Gargir, M. Gortler, R. T. Altstock, A. Shtevi, O. Weisshaus, E. Fire, N. Dotan, R. L. Schnaar, Glycobiology 2004, 14, 197 – 203. [5] S. P. Hawser, K. Islam, Infect. Immun. 1998, 66, 140 – 144. [6] C. Bavington, C. Page, Respiration 2005, 72, 335 – 344; W. M. Dunne, Jr., Clin. Microbiol. Rev. 2002, 15, 155 – 166. [7] M. D. Disney, P. H. Seeberger, Chem. Eur. J. 2004, 10, 3308 – 3314. [8] V. Afanassiev, V. Hanemann, S. Wolfl, Nucleic Acids Res. 2000, 28, E66. [9] T. T. Huang, J. Sturgis, R. Gomez, T. Geng, R. Bashir, A. K. Bhunia, J. P. Robinson, M. R. Ladisch, Biotechnol. Bioeng. 2003, 81, 618 – 624. [10] E. Belleville, M. Dufva, J. Aamand, L. Bruun, C. B. Christensen, Biotechniques 2003, 35, 1044 – 1051. [11] M. Dufva, S. Petronis, L. B. Jensen, C. Krag, C. B. Christensen, Biotechniques 2004, 37, 286 – 292, 294, 296. [12] E. W. Adams, D. M. Ratner, H. R. Bokesch, J. B. McMahon, B. R. O’Keefe, P. H. Seeberger, Chem. Biol. 2004, 11, 875 – 881; G. MacBeath, S. L. Schreiber, Science 2000, 289, 1760 – 1763. [13] M. D. Disney, S. Magnet, J. S. Blanchard, P. H. Seeberger, Angew. Chem. 2004, 116, 1618 – 1620; Angew. Chem. Int. Ed. 2004, 43, 1591 – 1594. [14] S. Chan, S. R. Horner, P. M. Fauchet, B. L. Miller, J. Am. Chem. Soc. 2001, 123, 11 797 – 11 798; R. D. Hubbard, S. R. Horner, B. L. Miller, J. Am. Chem. Soc. 2001, 123, 5810 – 5811. [15] M. Rangin, A. Basu, J. Am. Chem. Soc. 2004, 126, 5038 – 5039. [16] J. E. Gestwicki, C. W. Cairo, L. E. Strong, K. A. Oetjen, L. L. Kiessling, J. Am. Chem. Soc. 2002, 124, 14 922 – 14 933. [17] M. D. Disney, J. Zheng, T. M. Swager, P. H. Seeberger, J. Am. Chem. Soc. 2004, 126, 13 343 – 13 346. Received: June 27, 2006 Published online on && &&, 2006

Figure 4. Microscopic images of E. coli after hybridization with functionalized polymers. Top: structures of tryptamine- and ethanolamine-functionalized polymers. Bottom: microscopy of E. coli after hybridization with the polymer shown above it. Clusters show that tryptamine recognizes cell surfaces when displayed multivalently on a polymer.

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