The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Click and Virus-Based Chemistry for Biological Discovery

M.G. Finn, M. Baksh, D. Banerjee, J. Fiedler, V. Hong, A. Kislukhin, A. Udit

Virus capsids, the protein shells of virus particles that self-assemble in host cells, are a readily available biological material that we use for a variety of health-related applications. During the past several years, with crucial support from the Skaggs Institute, we have developed methods for the chemical derivatization of these structures, enabling us to bring the full power of both chemistry and molecular biology to bear in the creation of biologically active particles. The chemical methods are derived from click chemistry, the field of highly reliable synthetic methods developed and popularized by scientists in the laboratories of K.B. Sharpless, the Skaggs Institute, V.V. Fokin, Scripps Research, and in our laboratory. The following applications were pursued in the past year.

Fundamental Conjugation Reactions of Proteins and Virus Particles

We continue to develop new chemistry that allows the facile connections of biologically active molecules to capsid surfaces. Recently, we focused on connectors that become fluorescent when making bonds to the desired compounds and that can break those bonds at a predetermined rate (Fig. 1). This type of copper-free click chemistry is useful for the synthesis of protein conjugates in vivo and for the construction of delivery agents that release their cargoes at the desired site of action, such as a tumor.

Fig. 1. Top, Thiol-selective reagents that efficiently label proteins and peptides, providing a fluorescent signal upon bond formation. Bottom, Example of the generation of fluorescence upon the addition of a protein containing a free cysteine residue to a 100-μM solution of the thiol-selective reagent.

Polyvalent Carbohydrate Conjugates

We have extended our investigation of the immune response to carbohydrates displayed on the exterior surface of virus capsids. We found that the immunogenicity previously detected in chickens also occurs in mice and that both IgM and IgG antibodies are produced that are highly selective for the displayed glycans. This finding is important because many pathogens and disease states are marked by the display of unusual surface glycans. If immune responses can be raised against these glycans, the possibility exists to create novel vaccines.

Cationic Particles

Polycations are of interest as cell-penetrating agents and as molecules that bind tightly to polyanions such as DNA and RNA. The polyanion heparin is used widely to inhibit blood clotting, but problems with heparin overdose are widespread. In the operating room, overdoses are corrected by administering cationic molecules that complex with heparin to reduce its effect, but the cationic molecules themselves are anticoagulants when used at too high a concentration. We created viruslike particles with enhanced levels of positive charge on their surfaces and found that these species efficiently inhibit the action of heparin (Fig. 2). Furthermore, the particles are not anticoagulants by themselves or in the presence of heparin and so appear to circumvent the major difficulty with current antiheparin agents in clinical use. Further testing of these particles in animal models is under way.

Fig. 2. Measurement of the time required for clotting of a standard sample of normal human plasma upon the administration of heparin and test antagonist. Control experiments indicated that clotting required approximately 55 seconds in the absence of heparin and 2 minutes in the presence of heparin. The active viruslike particles T18R and D14R and the wild-type particle to which 95 copies of cationic peptide 1 (WT-(1)95) were attached by click chemistry completely inhibited the anticoagulant activity of heparin without themselves inhibiting coagulation at higher concentrations. This finding contrasts sharply with the actions of peptide 1 and the clinical agent protamine. These molecules inhibit heparin at low concentrations but then give rise to strong anticoagulation when added at slightly higher concentrations, making them difficult to use in a clinical setting. The data points for protamine and peptide 1 at >400 seconds represent experiments in which clotting was not observed within that time.

Publications

Bourne, C., Lee, S., Venkataiah, B., Lee, A., Korba, B., Finn, M.G., Zlotnick, A. Small-molecule effectors of hepatitis B virus capsid assembly give insight into virus life cycle. J. Virol. 82:10262, 2008.

Hong, V., Udit, A.K., Evans, R.A., Finn, M.G. Electrochemically protected copper(I)-catalyzed azide-alkyne cycloaddition. ChemBioChem 9:1481, 2008.

Kaltgrad, E., O'Reilly, M.K., Liao, L., Han, S., Paulson, J., Finn, M.G. On-virus construction of polyvalent glycan ligands for cell-surface receptor. J. Am. Chem. Soc. 130:4578, 2008.

Miermont, A., Barnhill, H., Strable, E., Lu, X., Wall, K.A., Wang, Q., Finn, M.G., Huang, X. Cowpea mosaic virus capsid, a promising carrier for the development of carbohydrate based antitumor vaccines. Chem. Eur. J. 14:4939, 2008.

Prasuhn, D.E., Jr., Singh, P., Strable, E., Brown, S., Manchester, M., Finn, M.G. Plasma clearance of bacteriophage Qβ particles as a function of surface charge. J. Am. Chem. Soc. 130:1328, 2008.

Strable, E., Prasuhn, D.E., Jr., Udit, A.K., Brown, S., Link, A.J., Ngo, J.T., Lander, G., Quispe, J., Potter, C.S., Carragher, B., Tirrell, D.A., Finn, M.G. Unnatural amino acid incorporation into virus-like particles. Bioconjug. Chem. 19:866, 2008.

Udit, A.K., Brown, S., Baksh, M.M., Finn, M.G. Immobilization of bacteriophage Qβ on metal-derivatized surfaces via polyvalent display of hexahistidine tags. J. Inorg. Biochem. 102:2142, 2008.

Udit, A.K., Everett, C., Gale, A.J., Kyle, J.R., Ozkan, M., Finn, M.G. Heparin antagonism by polyvalent display of cationic motifs on virus-like particle. ChemBioChem, in press.

Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W.-X., Finn, M.G., Stevens, R.C., Wüthrich, K. Microscale NMR screening of new detergents for membrane protein structural biology. J. Am. Chem. Soc. 130:7357, 2008.