Delivery methods for peptide and protein toxins in insect control.

Since the introduction of DDT in the 1940s, arthropod pest control has relied heavily upon chemical insecticides. However, the development of insect resistance, an increased awareness of the real and perceived environmental and health impacts of these chemicals, and the need for systems with a smaller environmental footprint has stimulated the search for new insecticidal compounds, novel molecular targets, and alternative control methods. In recent decades a variety of biocontrol methods employing peptidic or proteinaceous insect-specific toxins derived from microbes, plants and animals have been examined in the laboratory and field with varying results. Among the many interdependent factors involved with the production of a cost-effective pesticide--production expense, kill efficiency, environmental persistence, pest-specificity, pest resistance-development, public perception and ease of delivery--sprayable biopesticides have not yet found equal competitive footing with chemical counterparts. However, while protein/peptide-based biopesticides continue to have limitations, advances in the technology, particularly of genetically modified organisms as biopesticidal delivery systems, has continually progressed. This review highlights the varieties of delivery methods currently practiced, examining the strengths and weaknesses of each method.

Method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry.

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta.

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting.

Antibodies against metal chelates may potentially be used in biomedical applications such as targeted imaging and therapy of cancer. Highly specific monoclonal antibodies can be developed, but their binding strength needs to be maximized for them to be of practical use. In general, the half-life for dissociation of an antibody-ligand complex is more than an order of magnitude lower than the half-lifetimes for decay of medically useful radiometal ions. Practically speaking, the metal chelate-based ligand will not be bound to its receptor long enough for all of the bound radiometal to decay. A novel approach to this problem is a combination of synthetic chemistry and site-directed mutagenesis, to position a mildly reactive group on the metal chelate adjacent to a complementary reactive group on the antibody when the complex is formed. The partners are chosen to be sufficiently unreactive so that they coexist with other molecules in living systems without undergoing reaction. When the antibody-chelate complex is formed the effective local concentrations of the two groups can be non-physically large, so that a permanent link is formed in the complex even though no reaction occurs when the partners are free in solution.

Irreversible engineering of the multielement-binding antibody 2D12.5 and its complementary ligands.

Engineering the permanent formation of a receptor-ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) J. Am. Chem. Soc. 125, 15039-15048), we used the site-directed incorporation of cysteine nucleophiles at the periphery of the antibody's binding site, paired with the chemical design of a weakly electrophilic ligand, to produce a receptor-ligand pair that associates efficiently and permanently. Protein residues proximal to the ligand's side chain were identified for engineering cysteine mutants. Fab fragments incorporating a cysteine at position 54, 55, or 56 of the heavy chain (complementarity determining region 2) were designed from the structure and then cloned, expressed in Drosophila S2 cells, and tested for reactivity with mildly electrophilic DOTA-yttrium ligands. All showed permanent binding activity, indicating that there is some tolerance for the location of the reactive mutant on the protein surface near the binding site. The G54C Fab mutant displayed the highest expression levels and permanent binding activity in initial experiments and was produced in high yield for further study. Upon examining the behavior of the G54C mutant with a small set of electrophilic ligands, differences in reactivity were observed which indicated that the substituents near the electrophilic atom can be important determinants of permanent binding. The G54C mutant permanently attaches to Y(3+) complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of approximately 13 min at 37 degrees C, making it potentially useful for in vivo pretargeting applications.

Converting weak binders into infinite binders.

Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with K(d) approximately 10(-)(8) M, making it useful for the capture of probe molecules with a variety of properties. To make 2D12.5 even more useful for biological applications, we have engineered a single cysteine residue at position 54 of the heavy chain, a site proximal to the protein's binding site, so that weakly electrophilic metal complexes of (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) may bind and form permanent linkages. At 37 degrees C, pH 7.5, all of the rare earth-AABD complexes bind permanently to the 2D12.5 G54C mutant within 5 min, in yields that correlate with their relative binding affinities. Surprisingly, indium-AABD also binds permanently in >50% yield within 5 min, despite the fact that changing the metal to indium reduces the affinity approximately 100x; even copper-AABD, which has approximately 10 000x lower binding affinity than the rare earths, binds permanently in >70% yield within 2 h. However, acrylamido compounds with no measurable affinity do not bind permanently. The important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only the rare earths, but also an unexpected range of other elements as well. This infinite binding system can exhibit selective and permanent attachment with a remarkable range of structurally related ligands, albeit at slower rates as affinities decrease.

Evaluation of cleavable (Tyr3)-octreotate derivatives for longer intracellular probe residence.

Radioligand targeting of somatostatin receptor subtype 2 (sstr2)-positive tumors with synthetic somatostatin analogues such as octreotide is subject to improvement in tumor to nontumor biodistribution, in part because internalization of such somatostatin analogues is limited by sstr2 recycling to the cell surface. We reasoned that it might be possible to prepare probe-carrying somatostatin analogues that would escape recycling, efficiently depositing probe molecules inside cells and ultimately increasing their intracellular concentration. We have incorporated cathepsin-B-cleavable linkers into (Tyr3)-octreotate chelate conjugates and examined these constructs as to cellular uptake, externalization, subcellular localization, and cleavage in the rat pancreatic tumor cell line AR42J in culture. Comparison of the cleavable radioligands with a noncleavable control indicates that scission of the constituent cathepsin B substrate occurs at a rate faster than ligand externalization, depositing virtually all internalized cleaved radiochelates within lysosomal compartments.

Element-coded affinity tags for peptides and proteins.

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.

Molecular tools for targeted imaging and therapy of cancer.

Here we review an approach to the design and production of antibody/ligand pairs for use in cell targeting procedures, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This methodology is applicable to other biological binding pairs.

A rare earth-DOTA-binding antibody: probe properties and binding affinity across the lanthanide series.

An antibody that binds rare earth complexes selectively could be used as a docking station for a set of probe molecules, of particular interest for medical imaging and therapy. The rare earths are rich in probe properties, such as the paramagnetism of Gd, the luminescence of Tb and Eu, and the nuclear properties of Lu and Y. We find that antibody 2D12.5, initially developed to bind analogues of Y-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' ''-tetraacetic acid) for radiotherapy, binds not only Y-DOTA analogues but also analogous DOTA complexes of all of the lanthanides. Surprisingly, chelates of some metals such as Gd3+ bind more tightly than the original Y3+ complex. When the shape of the complex is perturbed by either increasing or decreasing the radius of the lanthanide ion, the thermodynamic stability of the protein-ligand complex changes in a regular fashion. The behavior of DeltaDeltaG as a function of ionic radius fits a parabola, as might be expected for a system that behaves in a thermodynamically elastic way. The broad specificity and high affinity of this antibody for all rare earth-DOTA complexes make it particularly interesting for applications that take advantage of the unique characteristics of lanthanides. For example, UV excitation of the Tb-DOTA-2D12.5 complex leads to energy transfer from aromatic side chains of the antibody to bound Tb-DOTA, enhancing green terbium luminescence >104 relative to unbound Tb-DOTA.