Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
  • G16C20/50—Molecular design, e.g. of drugs
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

• omega-atracotoxins A class of peptide toxins known as the omega-atracotoxins are disclosed in U.S. Pat. No. 5,763,568 as being isolated from Australian funnel-web spiders by screening the venom for "anti-cotton bollworm" activity.
• omega-ACTX-Hvla One of these compounds, designated omega-ACTX-Hvla, has been shown to selectively inhibit insect, as opposed to mammalian, voltage-gated calcium channel currents.
• a second, unrelated family of insect-specific peptidic calcium channel blockers are disclosed as being isolated from the same family of spiders in U.S. Pat. No. 6,583,264.
• a method of identifying a candidate molecule that mimics at least a portion of a three-dimensional structure of a U-ACTX insecticidal toxin comprises providing a molecular model made from the atomic coordinates for the rU-ACTX-Hvla insecticidal toxin having PDB ID 2HlZ and RCSB ID RCSB037828; using the molecular model to identify a candidate molecule that mimics the structure of the rU-ACTX-Hvla molecular model; and providing the candidate molecule that is identified.
• a method for selecting a candidate molecule that mimics at least a portion of a three-dimensional structure of rU-ACTX-Hvla comprises providing a computer having a memory means, a data input means, and a visual display means, the memory means containing three-dimensional molecular simulation software operable to retrieve coordinate data from the memory means and to display a three-dimensional representation of rU- ACTX-HvIa on the visual display means; inputting three-dimensional coordinate data of atoms of rU-ACTX-Hvl having PDB ID 2HlZ and RCSB ID RCSB037828 into the computer and storing the data in the memory means; displaying the three-dimensional representation of the candidate molecule on the visual display means; comparing the three-dimensional structure of rU-ACTX-Hvla and the candidate molecule; and providing the candidate molecule.
• a method of identifying a molecule that mimics at least a portion of a three-dimensional structure of a U-ACTX insecticidal toxin comprising: generating a three-dimensional model of the U-ACTX polypeptide, identifying pharmacophore residues Q 8 , P 9 , N 28 , and V 34 in the three-dimensional model, and performing a computer analysis to identify a candidate molecule that mimics the pharmacophoric residues of the U- ACTX polypeptide.
• Figure 1 shows a comparison of the primary structures of various members of the U-ACTX family of insecticidal peptide toxins (SEQ ID NOs. 1-7).
• rU-ACTX-Hvla (SEQ ID NO:1) is a recombinant version of one of the native peptide toxins (SEQ ID NO:2) in which the two N-terminal residues (Gln-Tyr) have been replaced with Gly-Ser for cloning purposes.
• Figure 2 shows a wall-eyed stereo view of the ensemble of 25 rU-ACTX-Hvla structures (PDB file 2Hl Z) overlaid for optimal superposition over the backbone atoms (C ⁇ , C, and N) of residues 3-39.
• the N- and C-termini of the peptide toxin are labeled "N" and "C", respectively.
• the three disulfide bonds are shown as light grey tubes, and each disulfide bond is labeled with the residue numbers of the two cysteine residues that form the disulfide bond.
• Figure 3 shows a Ramachandran plot for the ensemble of 25 rU-ACTX-Hvla structures as determined using the computer program PROCHECK. The statistics calculated by the PROCHECK program are shown below the Ramachandran plot.
• Figure 4 shows a Richardson schematic of the three-dimensional structure of rU- ACTX-HvIa based on the coordinates of the model from the ensemble with the lowest molecular energy (Model 1 in PDB file 2H1Z).
• the schematic is shown as a wall-eyed stereo image.
• the N- and C-termini of the peptide toxin are labeled "N" and "C", respectively.
• N N and C-termini of the peptide toxin
• Figure 5 shows a Richardson schematic of the three-dimensional structure of rU- ACTX-HvIa based on the coordinates of the model from the ensemble with the lowest molecular energy (Model 1 in PDB file 2Hl Z).
• the sidechains of key functional residues Gln8, Pro9, Asn28, and Val34, as determined from alanine scanning mutagenesis experiments, are shown as black tubes.
• the orientation of the molecule is similar to that shown in Figure 4.
• the N-terminus of the peptide toxin is labeled "N".
• Figure 6 shows a representation of the molecular surface of the three-dimensional structure of rU-ACTX-Hvla based on the coordinates of the model from the ensemble with the lowest molecular energy (Model 1 in PDB file 2Hl Z).
• the surface of the key pharmacophore elements of rU- ACTX-Hv Ia (Gln8, Pro9, Asn28, and Val34) are highlighted in black.
• the present invention is based, at least in part, upon the determination of the three-dimensional structure of an insecticidal peptide toxin known as U-ACTX-HvIa.
• the present invention is also based, at least in part, upon the determination of the pharmacophore of this toxin. It has been unexpectedly discovered by the inventors herein that four residues, Q , P , N 28 , and V 34 , of the U-ACTX polypeptides provide the insecticidal activity of the polypeptides.
• U-ACTX-HvIa is the prototypic member of a family of insecticidal peptide toxins described in US 2006/242734, which is incorporated herein by reference in its entirety. These insecticidal toxins comprise 38-39 residues, including six conserved cysteine residues that are paired to form three disulfide bonds. U-ACTX polypeptides cause irreversible toxicity when injected into insects such as the house fly Musca domestica, the house cricket Acheta domestica, and other insect species. These toxins have the unique ability to block both insect voltage-gated calcium channels and insect calcium-activated potassium channels.
• rU-ACTX-Hvla (SEQ ID NO: 1) is a recombinant polypeptide in which the first two residues of the native sequence of U-ACTX-HvIa (Gln-Tyr) (SEQ ID. NO: 2) have been replaced with Gly-Ser to give the following sequence:
• SEQ ID NO:1 Gly-Ser-Cys-Val-Pro-Val-Asp-Gln-Pro-Cys-Ser-Leu-Asn-Thr- Gln-Pro-Cys-Cys-Asp-Asp-Ala-Thr-Cys-Thr-Gln-Glu-Arg-Asn-Glu-Asn-Gly-His-Thr-Val-Tyr- Tyr-Cys-Arg-Ala [002O] (GSCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA)
• U-ACTX SEQ ID NOs: 3-7 are shown in Figure 1.
• Other homologs of U-ACTX-HvIa may be employed, for example, homologs that are greater than or equal to about 70%, 85%, 90%, or 95% identical to SEQ ID NO: 1, wherein the homologous polypeptide has insecticidal activity.
• "Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the sequences being compared.
• percent homology of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. ScI, U.S.A. 87, 2264-2268. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. MoI. Biol. 215, 403-410.
• bind when used in reference to the association of atoms, molecules, or chemical groups, refer to physical contact or association of two or more atoms, molecules, or chemical groups. Such contacts and associations include covalent and non-covalent types of interactions.
• hydrogen bond refers to two electronegative atoms (either O or N) which share a hydrogen that is covalently bonded to only one atom, while interacting with the other.
• hydrophobic interaction refers to interactions made by two hydrophobic residues.
• noncovalent bond refers to an interaction between atoms and/or molecules that does not involve the formation of a covalent bond between them.
• molecular graphics refers to three-dimensional representations of atoms, preferably on a computer screen.
• molecular model or “molecular structure” refer to the three-dimensional arrangement of atoms within a particular object (e.g., the three-dimensional structure of the atoms that comprise a toxin).
• molecular modeling refers to a method or procedure that can be performed with or without a computer to make one or more models, and, optionally, to make predictions about structure-activity relationships of ligands.
• the methods used in molecular modeling range from molecular graphics to computational chemistry.
• the term "pharmacophore” refers to an ensemble of interactive functional groups with a defined geometry that are responsible for the biological activity of a U- ACTX polypeptide.
• a pharmacophore is specified by the precise electronic properties on the surface of the active residues that cause binding to the surface of the target molecule (i.e., an insect ion channel). Typically, these properties are specified by the underlying chemical structures (e.g., aromatic groups, functional groups such as -COOH, etc.) and their geometric relationships. In a nonlimiting aspect, the geometric relations are precise to at least 2 Angstroms, more specifically, at least 1 Angstrom.
• a pharmacophore may include the identification of 2 to 4 of such groups (i.e., pharmacophoric elements or features). However, for complex protein recognition targets, a pharmacophore may include a greater number of groups.
• the core pharmacophore residues of the U-ACTX toxin include the amino acid residues Q 8 , P 9 , N 28 , and V 34 , as shown in Figures 5-6.
• “Fundamental pharmacophore specification” refers to both the chemical groups making up the pharmacophore and the geometric relationships of these groups. Several chemical arrangements may have similar electronic properties. For example, if a pharmacophore specification includes an -OH group at a particular position, a substantially equivalent specification includes an -SH group at the same position. Equivalent chemical groups that may be substituted in a pharmacophore specification without substantially changing its nature are homologous.
• U-ACTX mimic refers to a molecule that interacts with an insect voltage-gated calcium channel, an insect calcium-activated potassium channel, or both of these channels, and thus functions as a U-ACTX toxin.
• the U-ACTX mimic interacts with both an insect voltage-gated calcium channel and an insect calcium-activated potassium channel.
• the term mimic encompasses molecules having portions similar to corresponding portions of the U-ACTX pharmacophore in terms of structure and/or functional groups.
• the methods described herein include the use of molecular and computer modeling techniques to design and/or select novel molecules that mimic the U- ACTX family of toxins.
• a method of identifying a candidate molecule that mimics at least a portion of a three-dimensional structure of a U-ACTX insecticidal toxin comprises providing a molecular model made from the atomic coordinates for the rU-ACTX-Hvla insecticidal toxin having PDB ID 2HlZ and RCSB ID RCSB037828 (Table 3); using the molecular model to identify a candidate molecule that mimics the structure of the rU- ACTX- HvIa molecular model; and providing the candidate molecule that is identified.
• the method further comprises identifying the pharmacophore residues Q 8 , P , N 28 , and V 34 in the molecular model while using the molecular model.
• the atomic coordinates of rU- ACTX-Hv Ia may be used in rational drug design (RDD) to design a novel molecule of interest, for example, novel ion channel modulators (for example, rational design of insecticides that behave as structural and functional mimics of rU-ACTX-Hvla).
• the skilled artisan can design, make, test, refine and use novel insecticides specifically engineered to kill or paralyze insects, or to inhibit insect development or growth in such a manner that, for example in the case of agricultural applications, the insects provide less damage to a plant, and plant yield is not significantly adversely affected.
• the skilled artisan can engineer new molecules that functionally mimic rU-ACTX-Hvla.
• the molecular structure and optionally the fundamental pharmacophoric specification provided and discussed herein permit the skilled artisan to design new insecticidal toxins, including small molecule toxins as well as polypeptide toxins.
• RDD using the atomic coordinates of a U-ACTX-HvIa can be facilitated most readily via computer-assisted drug design (CADD) using computer hardware and software known and used in the art.
• the candidate molecules may be designed de novo or may be designed as a modified version of an already existing molecule, for example, a pre-existing toxin. Once designed, candidate molecules can be synthesized using methodologies known and used in the art, or obtained from a library of compounds. Once they have been obtained, the candidate molecules are optionally screened for bioactivity, for example, for their ability to inhibit insect ion channels.
• the structure of the candidate molecule is elucidated to determine how closely the structure mimics the pharmacophoric elements of rU- ACTX-Hv Ia. Based in part upon these results, the candidate molecules may be refined iteratively using one or more of the foregoing steps to produce a more desirable molecule with a desired biological activity.
• the tools and methodologies provided herein may be used to identify and/or design molecules that have insecticidal activity. Essentially, the procedures utilize an iterative process whereby the candidate molecules are synthesized, tested, and characterized. New molecules are designed based on the information gained in the testing and characterization of the initial molecules and then such newly identified molecules are themselves tested and characterized. This series of processes may be repeated as many times as necessary to obtain molecules with desirable binding properties and/or biological activities. Methods for identifying candidate molecules are discussed in more detail below.
• candidate molecules of interest can be facilitated by ball and stick- type physical modeling procedures.
• the ability to design candidate molecules may be enhanced significantly using computer-based modeling and design protocols.
• selection of a candidate molecule also includes providing a computer having a memory means, a data input means and a visual display means in operable communication.
• the memory means contains three-dimensional molecular simulation software operable to retrieve coordinate data from the memory means and operable to display a three- dimensional representation of the molecule or a portion thereof on the visual display means.
• This software is operable to produce a modified three-dimensional analog representation responsive to operator-selected changes to the chemical structure of the domain and is operable to display the three-dimensional representation of the modified analog.
• the date input means includes a central processing unit for processing computer readable data.
• This method optionally also includes inputting three-dimensional coordinate data of atoms of rU-ACTX-Hvla into the computer and storing the data in the memory means; inputting into the data input means of the computer at least one operator- selected change in chemical structure of rU- ACTX-Hv Ia; executing the molecular simulation software to produce a modified three-dimensional molecular representation of the analog structure; displaying the three-dimensional representation of the analog on the visual display means; whereby changes in three-dimensional structure of rU- ACTX-HvIa consequent on changes in chemical structure can be visually monitored.
• the method also optionally includes inputting operator-selected changes in the chemical structure of rU-ACTX-Hvla; executing the software to produce a modified three-dimensional molecular representation of the analog structure; and displaying the three-dimensional representation of the analog on the visual display means.
• the method also includes selecting a candidate compound structure represented by a three-dimensional representation and comparing the three-dimensional representation to the three-dimensional configuration and spatial arrangement of pharmacophore regions involved in function of rU- ACTX-Hv Ia.
• candidate molecules is optionally facilitated using computers or workstations, available commercially from, for example, Silicon Graphics Inc., Apple Computer Inc., and Sun Microsystems, running, for example, UNIX based, or Windows operating systems, and capable of running suitable computer programs for molecular modeling and rational drug design.
• computers or workstations available commercially from, for example, Silicon Graphics Inc., Apple Computer Inc., and Sun Microsystems, running, for example, UNIX based, or Windows operating systems, and capable of running suitable computer programs for molecular modeling and rational drug design.
• the computer-based systems comprise a data storage means having stored therein the atomic coordinates and optionally the fundamental pharmacophoric specification of rU- ACTX-Hv Ia as described herein, and the necessary hardware means and software means for supporting and implementing an analysis means.
• a computer system or “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the sequence, molecular structure and optionally the fundamental pharmacophoric specification as described herein.
• data storage means is understood to refer to a memory which can store sequence data, or a memory access means which can access manufactures having recorded thereon the molecular structure of the present invention.
• the atomic coordinates of rU- ACTX-Hv Ia and optionally the fundamental pharmacophoric specification of this polypeptide toxin are recorded on a computer readable medium.
• computer readable medium is understood to mean a medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
• magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
• optical storage media such as optical discs or CD-ROM
• electrical storage media such as RAM and ROM
• hybrids of these categories such as magnetic/optical storage media.
• the term "recorded” refers to a process for storing information on a computer readable medium.
• a skilled artisan can readily adopt the presently known methods for recording information on a computer readable medium to generate manufactures comprising an amino acid or nucleotide sequence, atomic coordinates and/or NMR data.
• a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon amino acid and/or nucleotide sequences, atomic coordinates and/or NMR data.
• the choice of the data storage structure will generally be based on the means chosen to access the stored information.
• a variety of data processor programs and formats can be used to store the sequence information, NMR data, and/or atomic coordinates on computer readable medium.
• the foregoing information, data and coordinates can be represented in a word processing text file, formatted in commercially- available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
• a skilled artisan can readily adapt a number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the information.
• molecular modeling can be most readily facilitated by using computers to build realistic models of rU- ACTX-Hv Ia, or portions thereof, such as the fundamental pharmacophoric specification of rU-ACTX-Hvla.
• Molecular modeling also permits the modeling of smaller molecules that structurally mimic the toxin.
• the methods utilized in molecular modeling range from molecular graphics (i.e., three-dimensional representations) to computational chemistry (i.e., calculations of the physical and chemical properties) to make predictions about the structure and activity of the smaller molecules, and to design new molecules.
• molecular graphics i.e., three-dimensional representations
• computational chemistry i.e., calculations of the physical and chemical properties
• Three-dimensional modeling can include, but is not limited to, making three- dimensional representations of structures, drawing pictures of structures, building physical models of structures, and determining the structures of related toxins and toxin/ligand complexes using the known coordinates. The appropriate coordinates are entered into one or more computer programs for molecular modeling.
• a list of computer programs useful for viewing or manipulating three-dimensional structures include: Midas (University of California, San Francisco); MidasPlus (University of California, San Francisco); MOIL (University of Illinois); Yummie (Yale University); Sybyl (Tripos, Inc.); Insight/Discover (Biosym Technologies); MacroModel (Columbia University); Quanta (Molecular Simulations, Inc.); Cerius (Molecular Simulations, Inc.); Alchemy (Tripos, Inc.); Lab Vision (Tripos, Inc.); Rasmol (Glaxo Research and Development); Ribbon (University of Alabama); NAOMI (Oxford University); Explorer Eyechem (Silicon Graphics, Inc.); Univision (Cray Research); Molscript (Uppsala University); Chem-3D (Cambridge Scientific); Chain (Baylor College of Medicine); 0 (Uppsala University); GRASP (Columbia University); X-Plor (Molecular Simulations, Inc.; Yale University); Spartan (Wa
• RDD One approach to RDD is to search for known molecular structures that mimic a site of interest.
• RDD programs can look at a range of different molecular structures of molecules that mimic a site of interest, and by moving them on the computer screen or via computation it can be decided which compounds are the best structural mimics of the site of interest.
• molecular modeling programs could be used to determine which one of a given set of compounds was the best structural mimic of the pharmacophoric regions of rU-ACTX-Hvla.
• the atomic coordinates provided herein are also useful in designing improved analogues of known insecticidal toxins.
• the atomic coordinates presented herein also permit comparing the three- dimensional structure of a U-ACTX toxin or a portion thereof with molecules composed of a variety of different chemical features to determine optimal sites to mimic the U-ACTX toxin structure.
• U-ACTX toxin The atomic coordinates of a U-ACTX toxin permit the skilled artisan to identify target locations in a toxin that can serve as a starting point in rational drug design.
• identification of the fundamental pharmacophoric specification of the U-ACTX toxins allows one to identify residues and functional groups that are key for toxin function.
• a candidate molecule comprises, but is not limited to, at least one of a lipid, nucleic acid, peptide, small organic or inorganic molecule, chemical compound, element, saccharide, isotope, carbohydrate, imaging agent, lipoprotein, glycoprotein, enzyme, analytical probe, and an antibody or fragment thereof, any combination of any of the foregoing, and any chemical modification or variant of any of the foregoing.
• a candidate molecule may optionally comprise a detectable label.
• labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
• Methods useful for synthesizing candidate molecules such as lipids, nucleic acids, peptides, small organic or inorganic molecules, chemical compounds, saccharides, isotopes, carbohydrates, imaging agents, lipoproteins, glycoproteins, enzymes, analytical probes, antibodies, and antibody fragments are well known in the art.
• Such methods include the approach of synthesizing one such candidate molecule, such as a single defined peptide, one at a time, as well as combined synthesis of multiple candidate molecules in one or more containers.
• Such multiple candidate molecules may include one or more variants of a previously identified candidate molecule.
• Methods for combined synthesis of multiple candidate molecules are particularly useful in preparing combinatorial libraries, which may be used in screening techniques known in the art.
• peptides and oligonucleotides may be simultaneously synthesized.
• Candidate molecules that are small peptides, up to about 50 amino acids in length, may be synthesized using standard solid-phase peptide synthesis procedures. For example, during synthesis, N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal end to an insoluble polymeric support, e.g., polystyrene beads.
• the peptides are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxy group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
• a reagent such as dicyclohexylcarbodiimide.
• the attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
• the most commonly used N- ⁇ -protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
• the C-terminal N- ⁇ -protected amino acid is first attached to the polystyrene beads. Then, the N- ⁇ -protecting group is removed. The deprotected ⁇ -amino group is coupled to the activated ⁇ -carboxylate group of the next N- ⁇ -protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are cleaved from the insoluble polymer support and the amino acid side chains are deprotected. Longer peptides, for example greater than about 50 amino acids in length, are derived by condensation of protected peptide fragments.
• a synthetic peptide comprises naturally occurring amino acids, unnatural amino acids, and/or amino acids having specific characteristics, such as, for example, amino acids that are positively charged, negatively charged, hydrophobic, hydrophilic, or aromatic.
• Amino acids used in peptide synthesis include L- or D-stereoisomers.
• Candidate molecules can be designed entirely de novo or may be based upon a pre-existing insecticidal toxin. Either of these approaches can be facilitated by computationally screening databases and libraries of small molecules for chemical entities, agents, ligands, or compounds that can mimic an insecticidal toxin.
• the mimic should mimic at least a portion of the pharmacophoric specification of rU- ACTX-Hv Ia.
• the functional groups on the mimic should be assessed for their ability to participate in hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
• the mimic should be able to assume a conformation that allows it to mimic at least a portion of the structure of rU-ACTX-Hvla.
• Such conformational factors include the overall three-dimensional structure and orientation of the mimic in relation to all or a portion of the structure of rU-ACTX-Hvla, or the spacing between functional groups of a mimic comprising several chemical entities that directly interact with the molecular targets of U-ACTX-HvIa and similar toxins.
• One skilled in the art may use one or more of several methods to identify chemical moieties or entities, compounds, or other agents for their ability to mimic the three- dimensional structure of rU- ACTX-Hv Ia, or a portion thereof, such as the fundamental pharmacophoric specification as identified herein.
• This process may begin by visual inspection or computer assisted modeling of, for example, the pharmacophore of rU- ACTX-Hv Ia, using the atomic coordinates deposited in the RCSB Protein Data Bank (PDB) with Accession Number PDB ID 2HlZ and RCSB ID RCSB037828.
• PDB RCSB Protein Data Bank
• compound design uses computer modeling programs that calculate how well a particular molecule mimics the structure of rU- ACTX-Hv Ia.
• Selected chemical moieties or entities, compounds, or agents are positioned in a variety of orientations.
• Databases of chemical structures are available from, for example, Cambridge Crystallographic Data Center (Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).
• Specialized computer programs also assist in the process of selecting chemical entities. Once suitable chemical moieties or entities, compounds, or agents have been selected, they can be assembled into a single molecule. Assembly may proceed by visual inspection and/or computer modeling and computational analysis of the spatial relationship of the chemical moieties or entities, compounds or agents with respect to one another in three-dimensional space. This could then be followed by model building and energy minimization using software such as Quanta or Sybyl optionally followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.
• Useful programs to aid in choosing and connecting the individual chemical entities, compounds, or agents include but are not limited to: GRID (University of Oxford); CATALYST (Accelrys, San Diego, CA); AUTODOCK (Scripps Research Institute, La Jolla, CA); DOCK (University of California, San Francisco, CA); ALADDIN; CLIX; GROUPBUILD; GROW; and MOE (Chemical Computing Group).
• the test molecule mimics one or more key chemical features of the rU-ACTX-Hvla pharmacophoric specification, such as the hydrogen-bonding capacity.
• a test compound mimics the hydrogen-bonding capacity of the sidechain amide moiety of GIn .
• the molecule of interest are designed as a complete entity using either the complete fundamental pharmacophoric specification of rU- ACTX-Hv Ia, or a portion thereof.
• chemical moieties that are beneficial for a molecule that is to be administered as an insecticide.
• Knowledge of the structure of an insecticidal toxin relative to the structure of rU- ACTX-HvIa may allow for the design of a new toxin that has better insecticidal activity relative to the molecule from which it was derived.
• a variety of modified molecules are designed using the atomic coordinates provided herein. For example, by knowing the spatial relationship of one or more insecticidal peptide toxins relative to the structure of rU-ACTX-Hvla, it is possible to generate new polypeptide toxins with improved insecticidal properties.
• a candidate molecule has been designed or selected by the above methods, its similarity to a rU- ACTX-Hv Ia toxin is determined by computational evaluation and/or by testing its biological activity after the compound has been synthesized.
• substitutions may then be made in some of the atoms or side groups of the candidate molecule in order to improve or modify its properties.
• initial substitutions are conservative, i.e., the replacement group will approximate the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known to alter conformation should be avoided.
• such substituted chemical compounds are analyzed for structural similarity with U-ACTX by the same computer methods described in detail, above.
• the method further comprises using the molecular model, or a portion thereof, to identify a modified candidate molecule and produce a modified candidate molecule having a higher lethality to insects, enhanced inhibition of insect calcium channels, enhanced inhibition of insect calcium-activated potassium channels, enhanced binding to insect calcium channels, enhanced binding to insect calcium-activated potassium channels, or an enhancement of one or more of the foregoing functionalities relative to the candidate molecule.
• molecules designed, selected and/or optimized by methods described above, once produced are characterized using a variety of assays to determine whether the compounds have biological activity.
• the molecules are characterized by assays, including but not limited to those assays described below, to determine whether they have a predicted activity, binding activity and/or binding specificity.
• Suitable assays measure, for example, the ability of the chosen molecule to kill or paralyze insects, inhibit insect calcium channels, inhibit insect calcium-activated potassium channels, bind insect calcium channels, bind insect calcium-activated potassium channels, and combinations comprising one or more of the foregoing functions.
• the activity of a candidate molecule can be determined quantitatively by direct injection of the candidate molecule into an insect such as Musca domestica (house flies).
• house flies body weight 10 to 25 mg
• Control flies are injected with 2 ⁇ l of insect saline.
• An Arnold microapplicator (Burkard Scientific Supply, Rickmansworth, England) equipped with a 29-gauge needle, for example, is employed to administer the injections.
• Specimens can be temporarily immobilized at 4°C for the injections and then immediately returned to room temperature (24°C).
• the LD 50 value i.e., the dose of candidate molecule that kills 50% of flies at 24 hours post-injection
• y is the percentage deaths in the sample population at 24 hours post-injection
• x is the toxin dose in pmol g ⁇ '
• n is a variable slope factor
• a is the maximum response
• b is the minimum response.
• Electrophysiological assays Inhibition of insect ion channels may be studied using isolated insect neurons, in recombinant cells or oocytes expressing a specific channel, or a combination comprising one or more of the foregoing.
• the ion channels to be tested are voltage-gated calcium channels and/or calcium-activated potassium channels naturally found in an insect neuronal system.
• the activity of a test compound is assessed by its ability to inhibit the activity of an isolated insect neuron.
• DUM dorsal unpaired median (DUM) neurons isolated from the terminal abdominal ganglion (TAG) of cockroach Periplaneta americana are employed.
• DUM neurons contain voltage-gated calcium channels (Cav channels) from which Ca v channel currents (/c a ) can be recorded using whole-cell patch-clamp recording techniques.
• DUM neuron cell bodies are isolated from the midline of the TAG of the nerve cord of P. Americana.
• cockroaches are anaesthetized by cooling at -20°C for approximately 5 minutes.
• NES normal insect saline
• HEPES normal insect saline
• 50 mM sucrose with 5% volume/volume bovine calf serum and 50 IU mP 1 penicillin and 50 ⁇ g ml "1 streptomycin added, and the pH adjusted to 7.4 using NaOH.
• the TAG is carefully dissected and placed in sterile Ca 2+ /Mg 2+ -free insect saline containing 200 mM NaCl, 3.1 mM KCl, 10 mM HEPES, 60 mM sucrose, 50 IU/mL penicillin, and 50 IU/ml streptomycin, with the pH adjusted to 7.4 using NaOH.
• the ganglia are then desheathed and incubated for 20 minutes in Ca 2+ /Mg 2+ -free insect saline containing 1.5 mg/ml collagenase.
• the ganglia are rinsed three times in normal insect saline.
• the resulting suspension is distributed into eight wells of a 24- well cluster plate. Each well contains a 12-mm diameter glass coverslip that had been previously coated with concanavalin A (2 mg/ml). Isolated cells attach to coverslips overnight in an incubator (100% relative humidity, 37°C).
• electrophysiological experiments employ the patch-clamp recording technique in whole-cell configuration to measure voltage-gated sodium, potassium, and calcium currents from cockroach DUM neurons.
• Coverslips with isolated cells are transferred to a 1 -ml glass-bottom perfusion chamber mounted on the stage of a phase-contrast microscope.
• Whole-cell recordings of sodium, potassium, and calcium currents are made using an Axopatch 200A-integrating amplifier (Axon Instruments, Foster City, CA). Borosilicate glass-capillary tubing is used to pull single-use recording micropipettes.
• the contents of the external and internal solutions are varied according to the type of recording procedure undertaken and also the particular ionic current being studied.
• the holding potential can be, for example, -80 mV.
• Electrode tip resistances can be in the range 0.8- 4.0 M ⁇ .
• the osmolality of both external and internal solutions may be adjusted to 310 mosmol/liter with sucrose to reduce osmotic stress.
• the liquid junction potential between internal and external solutions may be determined using the program JPCaIc.
• large tear-shaped DUM neurons with diameters greater than 45 ⁇ m are selected for experiments.
• Inverted voltage-clamp command pulses are applied to the bath through an Ag/ AgCl pellet/3 M KCl-agar bridge. After formation of a gigaohm seal, suction is applied to break through the membrane. Experiments should not commence for a period of 5 to 10 minutes to allow for complete block of unwanted currents.
• Stimulation and recording may both be controlled by an AxoData data acquisition system (Axon Instruments) running on an Apple Macintosh computer. Data is filtered at 5 kHz (low-pass Bessel filter) and digital sampling rates are between 15 and 25 kHz depending on the length of the voltage protocol. Leakage and capacitive currents are digitally subtracted with P- P/4 procedures. Data analysis is performed off-line following completion of the experiment. I/V data are fitted by nonlinear regression of the following equation onto the data:
• V is the amplitude of the peak current at a given potential
• V is the amplitude of the peak current at a given potential
• g max is the maximal conductance
• Vi/ 2 is the voltage at half-maximal activation
• s is the slope factor
• V rev is the reversal potential
• the insect ion channel comprises a heterologously expressed insect calcium-activated potassium channel (also known as BKc a , Kc 8 Ll, Maxi-K, or SIo 1), such as the pore-forming ⁇ subunit of the pSlo channel from the cockroach Periplaneta americana.
• a heterologously expressed insect calcium-activated potassium channel also known as BKc a , Kc 8 Ll, Maxi-K, or SIo 1
• BKc a , Kc 8 Ll, Maxi-K, or SIo 1 such as the pore-forming ⁇ subunit of the pSlo channel from the cockroach Periplaneta americana.
• HEK293 monolayers in 35 mm dishes are transfected using 9 ⁇ l Lipofectamine Reagent (Gibco, BRL) and 5 ⁇ g DNA.
• Stably transfected cells are then selected with 1000 ⁇ g mF 1 G418 (Gibco, Grand Island, NY, USA). These cells are maintained in the normal growth media described above and cultured on sterile glass coverslips to be used for the patch clamp experiments.
• pipettes are filled with a solution containing 4 mM NaCl, 140 mM KCl, 2 mM ATP-Mg 2 , 0.6 mM CaCl 2 , and 10 mM N-(2-hydroxyethyl)piperazine-N > -[2-ethanesulfonic acid] (HEPES), with the pH adjusted to 7.25 with 2 M KOH.
• the external solution contains 135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.33 mM NaH 2 PO 4 , 10 mM glucose, and 10 mM HEPES, with the pH adjusted to 7.4 with 2 M NaOH.
• the osmolality is approximately 290 mosmol/L.
• the efficacy of a test compound can be expressed as the IC 50 (the dose that inhibits 50% of the activity) of an insect calcium channel or insect calcium-activated potassium channel.
• IC 50 the dose that inhibits 50% of the activity
• the effective insecticidal amount of such compounds lies preferably within a range of concentrations that include the IC 5 0.
• the human ether-a go-go-related gene that encodes the pore-forming subunit of cardiac Ikr potassium (K + ) channels
• HERG human ether-a go-go-related gene
• CHO Chinese hamster ovary
• Cells are seeded into 96-well plates at a density of about 200 cells/ ⁇ L of media. After seeding, the cells are incubated at 37°C and 5% CO 2 overnight prior to use.
• the cell culture medium is changed to rubidium ( 86 Rb). Cells are incubated with 86 Rb for four hours, the medium is removed and the cells are washed.
• the cells are washed with a 40 mM K + test buffer containing: 105 mM NaCl, 2 mM CaCl 2 , 40 mM KCl, 2 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose.
• the pH is adjusted to 7.4 with NaOH.
• Test compounds are carried in the high K + test buffer.
• the effect of the test compounds on the channels is measured using electrophysiology recordings. Electrodes have resistances between 2 and 3 M ⁇ when filled with internal solution.
• the internal solution contains: 100 mM KF, 40 mM KCl, 5 mM NaCl, 10 mM EGTA, and 10 mM HEPES, adjusted to pH 7.4 with KOH.
• the cells are perfused with external solution containing: 140 mM NaCl, 2 mM CaCl 2 , 5 mM KCl, 2 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH.
• the junction potential may be calculated using pClamp 8 software. Currents may be filtered at 5 kHz on an Axopatch-1D amplifier (Axon Instruments) and recorded onto a PC with sample rate of 1 kHz using pClamp 8 (Axon Instruments). Data may be analyzed using Clamp fit (Axon Instruments) and Origin software (Microcal).
• membrane-potential-sensitive fluorescent dyes are used to indirectly monitor channel activity by monitoring changes in membrane potential.
• Membrane potential sensors based on FRET are useful for high throughput screening of ion channels.
• the sensor is a two-component sensor comprising a fist component that is a highly fluorescent hydrophobic ion that binds to the plasma membrane and senses the membrane potential and a second component that is a fluorescent molecule that binds to one face of the plasma membrane and functions as a FRET partner to the mobile voltage sensing ion.
• the first component is a coumarin-labeled phospholipid (CC2- DMPE) and the second component is a bis-(l,3- dialkylthiobarbituric acid) trimethine oxonol, DiSBACn(3), where n corresponds to the number of carbon atoms in the n alkyl group.
• Vertex for example, has developed a kinetic plate reader that is compatible with such FRET-based voltage sensors.
• the VIPRTM is a 96- or 384-well integrated liquid handler and fluorescent reader, The reader uses a scanning fiber optic illumination and detection system. Other similar systems and probes may also be employed.
• SPR surface plasmon resonance
• SPR methodologies measure the interaction between two or more macromolecules in real-time through the generation of a quantum mechanical surface plasmon.
• One device the BIAcore Biosensor RTM (Pharmacia, Piscataway, N.J.), provides a focused beam of polychromatic light to the interface between a gold film (provided as a disposable biosensor "chip") and a buffer compartment that can be regulated by the user.
• a 100 nm thick "hydrogel” composed of carboxylated dextran which provides a matrix for the covalent immobilization of analytes of interest is attached to the gold film. When the focused light interacts with the free electron cloud of the gold film, plasmon resonance is enhanced.
• the resulting reflected light is spectrally depleted in wavelengths that optimally evolved the resonance.
• the BIAcore establishes an optical interface which accurately reports the behavior of the generated surface plasmon resonance.
• Fluorescence polarization is a measurement technique is readily applied to protein-protein and protein-ligand interactions in order to derive IC 50 and K 1 ⁇ values for the association reaction between two molecules.
• one of the molecules of interest is conjugated with a fluorophore.
• This is generally the smaller molecule, such as a small-molecule mimic of rU- ACTX-Hv Ia.
• the sample mixture containing both the conjugated small molecule and either an insect voltage-gated calcium channel or an insect calcium-activated potassium channel, is excited with vertically polarized light. Light is absorbed by the probe fluorophores, and re-emitted a short time later.
• the degree of polarization of the emitted light is measured. Polarization of the emitted light is dependent on several factors, such as on viscosity of the solution and on the apparent molecular weight of the fluorophore. With proper controls, changes in the degree of polarization of the emitted light depends only on changes in the apparent molecular weight of the fluorophore, which in-turn depends on whether the probe-ligand conjugate is free in solution, or is bound to a receptor. Binding assays based on FP have a number of important advantages, including the measurement of IC 50 and K d values under true homogenous equilibrium conditions, speed of analysis, amenity to automation, and ability to screen in cloudy suspensions and colored solutions.
• the ability of a molecule to mimic the binding of rU- ACTX- Hv Ia to a particular insect ion channel is measured from its ability to competitively displace rU-ACTX-Hvla from that channel.
• fluorescently or radioactively labeled rU-ACTX-Hvla are first bound to neuronal membranes or cell lines containing the ion channel of interest.
• Repetition of the displacement assay with varying concentrations of the molecule of interest permit its IC 50 value to be calculated and compared with that of other putative mimics, enabling the molecules to be ranked in order of binding affinity.
• high-throughput screening may be used to speed up analysis using such assays. As a result, it may be possible to rapidly screen new molecules for their ability to interact with an insect ion channel using the tools and methods disclosed herein.
• General methodologies for performing high-throughput screening are described, for example, in U.S. Pat. No. 5,763,263, incorporated herein by reference.
• High-throughput assays can use one or more different assay techniques including, but not limited to, those described above.
• the active molecules are optionally incorporated into a suitable carrier prior to use. More specifically, the dose of active molecule, mode of administration, and use of suitable carrier will depend upon the target and non-target organism(s) of interest.
• a method of controlling an insect comprises contacting the insect or an insect larva with an insecticidally effective amount of a U-ACTX mimic.
• the U-ACTX mimic may be, for example, in the form of a small organic molecule, a chemical compound, a purified polypeptide, a polynucleotide encoding the U-ACTX mimic optionally in an expression vector, an insect virus expressing the U-ACTX mimic, a cell such as a plant cell or a bacterial cell expressing the U-ACTX mimic, or a transgenic plant expressing the U-ACTX mimic.
• the U- ACTX mimic is optionally fused to, or delivered in conjunction with, an agent that enhances the activity of the compound when ingested by insects, such as snowdrop lectin or one of the Bacillus thuringiensis ⁇ -endotoxins.
• Contacting includes, for example, injection of the U-ACTX mimic, external contact, ingestion of the U-ACTX mimic, or ingestion of a polynucleotide, virus, or bacterium expressing the U-ACTX mimic.
• a method of treating a plant comprises contacting the plant with an insecticidally effective amount of a U-ACTX mimic.
• the U-ACTX mimic is, for example, in the form of a small organic molecule, a chemical compound, a purified polypeptide, a polynucleotide encoding the U-ACTX mimic optionally in an expression vector, a virus expressing the U-ACTX mimic, or a cell such as a plant cell or a bacterial cell expressing the U-ACTX mimic.
• an insecticidal composition comprising a U- ACTX mimic and an agriculturally acceptable carrier, diluent and/or excipient.
• an insecticidal composition comprises a virus expressing a U-ACTX mimic. Insect viruses can be replicated and expressed inside a host insect once the virus infects the host insect. Infecting an insect with an insect virus can be achieved via methods, including, for example, ingestion, inhalation, direct contact of the insect or insect larvae with the insect virus, and the like.
• the insecticidal composition is, for example, in the form of a flowable solution or suspension such as an aqueous solution or suspension.
• aqueous solutions or suspensions are provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.
• an insecticide composition comprises a water dispersible granule.
• an insecticide composition comprises a wettable powder, dust, pellet, or colloidal concentrate.
• Such dry forms of the insecticidal compositions are formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner.
• the virus expressing the U-ACTX mimic can be applied to the crop to be protected.
• the virus may be engineered to express a U-ACTX mimic, either alone or in combination with one or several other U-ACTX polypeptides or mimics, or in combination with other insecticides such as other insecticidal polypeptide toxins that may result in enhanced or synergistic insecticidal activity.
• Suitable viruses include, but are not limited to, baculoviruses.
• the insecticidal compositions comprise intact cells (e.g., bacterial cells) expressing a U-ACTX mimic, such cells are formulated in a variety of ways.
• the formulations may include spreader- sticker adjuvants, stabilizing agents, other pesticidal additives, surfactants, and combinations comprising one or more of the foregoing additives.
• Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, and the like.
• the ingredients may include rheological agents, surfactants, emulsif ⁇ ers, dispersants, polymers, liposomes, and combinations comprising one or more of the foregoing ingredients.
• the U-ACTX mimics are expressed in vitro and isolated for subsequent field application.
• Such mimics are, for example, in the form of crude cell lysates, suspensions, colloids, etc., or may be purified, refined, buffered, and/or further processed, before formulating in an active insecticidal formulation.
• the amount of the active component(s) is applied at an insecticidally-effective amount, which will vary depending on such factors as, for example, the specific insects to be controlled, the specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the insecticidally- active composition.
• Insecticidal compositions comprising the U-ACTX mimics are, for example, formulated with an agriculturally-acceptable carrier.
• the compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer.
• the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination another other carrier material suitable for agricultural application.
• Suitable agricultural carriers can be solid or liquid and are well known in the art.
• agriculturally-acceptable carrier covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation.
• the formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the insecticidal composition with suitable adjuvants using conventional formulation techniques.
• the insecticidal compositions are, for example, applied to the environment of the target insect, for example onto the foliage of the plant or crop to be protected, by methods, preferably by spraying.
• the strength and duration of insecticidal application may be set with regard to conditions specific to the particular pest(s), crop(s) to be treated, and particular environmental conditions.
• the proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the insecticidal composition, as well as the particular formulation contemplated.
• the insecticidal compositions are employed singly or in combination with other compounds, including but not limited to other pesticides. They may be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations.
• the insecticidal compositions optionally comprise an insect attractant.
• the insecticidal compositions are formulated for either systemic or topical use. Such agents may are optionally applied to insects directly.
• concentration of the insecticidal composition that is used for environmental, systemic, or foliar application varies depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity.
• a crop is engineered to express a U-ACTX mimic, either alone, or in combination with insecticidal polypeptide toxins that may result in enhanced or synergistic insecticidal activity.
• Crops for which this approach would be useful include, but are not limited to, cotton, tomato, sweet corn, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, sunflower, and field lupins.
• Arthopods of suitable agricultural, household and/or medical/veterinary importance for treatment with the insecticidal polypeptides include, for example, members of the classes and orders: Coleoptera such as the American bean weevil Acanthoscelides obtectus, the leaf beetle Agelastica alni, click beetles (Agriotes Meatus, Agriotes obscurus, Ag ⁇ otes bicolor), the grain beetle Ahasverus advena, the summer schafer Amphimallon solstitialis, the furniture beetle Anobium punctatum, Anthonomus spp.
• Coleoptera such as the American bean weevil Acanthoscelides obtectus
• the leaf beetle Agelastica alni click beetles (Agriotes Meatus, Agriotes obscurus, Ag ⁇ otes bicolor)
• the insecticidal compositions comprising the U-ACTX mimics are employed to treat ectoparasites.
• Ectoparasites include, for example, fleas, ticks, mange, mites, mosquitoes, nuisance and biting flies, lice, and combinations comprising one or more of the foregoing ectoparasites.
• the term fleas includes the usual or accidental species of parasitic flea of the order Siphonaptera, and in particular the species Ctenocephalides, in particular C. felis and C. canis, rat fleas ⁇ Xenopsylla cheopis) and human fleas (Pulex irritans).
• Ectoparasites on farm animals e.g., cattle
• companion animals e.g., cats and dogs
• treatment may include impregnation in a collar or topical application to a localized region followed by diffusion through the animal's dermis and/or accumulation in sebaceous glands.
• treatment may include a composition suitable for the treatment of lice in humans. Such a composition may be suitable for application to a human scalp such as a shampoo or a conditioner.
• a derivative of the prototypic U-ACTX family member, rU-ACTX-Hvla (SEQ ID NO: 1) ( Figure 1), was chosen for structure-function analyses.
• the toxin is produced from this plasmid as a fusion to the C-terminus of glutathione 5-transferase (GST), with a thrombin cleavage site between the GST and toxin coding regions.
• GST glutathione 5-transferase
• the cells were grown in LB medium at 37°C to an ⁇ 600 of 0.6-0.8 before induction of the fusion protein with 300 ⁇ M isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG). The cells were harvested by centrifugation at an ⁇ 600 of 1.9-2.1 and frozen until further use. Cell pellets were defrosted and then resuspended in lysis buffer (50 mM NaCl, 50 mM Tris, 1 mM EDTA, pH 8.0). Cells were then lysed by sonication. The recombinant fusion protein was purified from the soluble cell fraction using affinity chromatography on GSH-Sepharose columns (Amersham Biosciences).
• the column beads were equilibrated and resuspended in thrombin buffer (150 mM NaCl, 20 mM Tris, 1 mM CaCl 2 , pH 8.0) before addition of 50 U bovine thrombin (Sigma).
• thrombin buffer 150 mM NaCl, 20 mM Tris, 1 mM CaCl 2 , pH 8.0
• the column was placed in a 37 0 C incubator overnight to allow proteolytic cleavage of the fusion protein.
• the liberated toxin was eluted from the column with Tris-buffered saline (150 mM NaCl, 50 mM Tris, pH 8.0).
• the toxin was purified immediately using reverse-phase (rp) HPLC before being lyophilized. Lyophilized toxin was then resuspended in the appropriate buffer.
• the correctly folded recombinant toxin was separated from non-native disulfide bond isomers and other contaminants by rpHPLC using a Vydac Ci 8 analytical column (4.6 x 250 mm, 5- ⁇ m pore size).
• the toxin was eluted from the column at a flow rate of 1 ml min "1 using a linear gradient of 10-18% acetonitrile over 20 minutes.
• Correctly folded toxin eluted as the major peak with a retention time of 9-10 minutes.
• the toxin molecular weight was verified using electrospray mass spectrometry.
• the yield of the correctly folded recombinant toxin was estimated from integration of the relevant HPLC peaks and found to be ⁇ 70-80%.
• NMR experiments were performed using a four-channel Varian INOVA 600 NMR spectrometer equipped with pulse-field gradients. All experiments were performed at 25°C. All data were processed using NMRPipe. Processed spectra were analyzed and peaks were integrated using the program XEASY.
• Interproton distance restraints were obtained from integration of peak intensities in 3D 15 N-edited NOESY and 13 C-edited NOESY spectra. NOE assignments were initially made using the CANDID macro in CYANA, then refined manually. Crosspeak intensities from NOESY spectra were converted into distance restraints using the CALIBRA macro in the program CYANA.
• Dihedral-angle restraints were obtained from TALOS analysis of H ⁇ , C ⁇ , Cp, and H N chemical shifts; for structure calculations, the range of each restraint was set to twice the standard deviation of the TALOS prediction.
• Disulfide bonds were assigned from the experimentally determined disulfide-bond pattern in the ⁇ -ACTX-Hvla and J-ACTX-HvIc toxins, which are part of the same toxin superfamily.
• the disulfide bonds in rU-ACTX-Hvla are thus Cys3-Cysl8, CyslO-Cys23, and Cysl7-Cys37.
• Hydrogen bonds were determined in two ways: (i) from direct observation of hydrogen-bond scalar couplings in a 2D HNCO experiment; (ii) from analysis of a hydrogen- deuterium exchange experiment in which a sample of lyophilized rU-ACTX-Hvla was dissolved in 100% D 2 O and the exchange of H N protons with solvent deuterons was monitored from the change in peak intensities in a time course of 2D HSQC spectra. These analyses led to the assignment of 14 hydrogen bonds, as summarized in Table 1. For structure calculations, the O- N and O-H N distance for each hydrogen bond was restrained to range of 2.7-3.1 A and 1.7- 2.1 A, respectively.
• Figure 4 shows a Richardson schematic of the three-dimensional structure of rU- ACTX-HvIa generated using the computer program MOLMOL.
• the major secondary structure element is a C-terminal ⁇ -hairpin comprising ⁇ -strand 1 ( ⁇ l, residues 22-27) and ⁇ -strand 2 ( ⁇ 2, residues 33-38).
• Residues that are critical for the function of rU- ACTX-Hv Ia were determined using alanine scanning mutagenesis. In this approach, individual residues were mutated to alanine, then the activity of the mutant toxin was compared to that of wild-type rU-ACTX-Hvla (SEQ ID NO:1).
• the six cysteine residues comprising the inhibitory cysteine knot motif of rU- ACTX-HvIa were excluded from the alanine scan because they are presumed to be important for defining the three-dimensional structure of the toxin; these buried cysteine residues are not expected to interact with the insect ion channels targeted by rU- ACTX-Hv Ia. Ala21 and Ala39 were also not mutated. The remaining 28 non-alanine residues present in the primary structure of rU- ACTX-Hv Ia were mutated individually to alanine.
• the alanine side chain can be accommodated in most types of polypeptide secondary structure (i.e., ⁇ -helix, ⁇ -sheet, and ⁇ -turn) and therefore it is commonly utilized in scanning mutagenesis in order to minimize the possibility of introducing major structural perturbations.
• polypeptide secondary structure i.e., ⁇ -helix, ⁇ -sheet, and ⁇ -turn
• scanning mutagenesis i.e., ⁇ -helix, ⁇ -sheet, and ⁇ -turn
• each rU-ACTX-Hvla mutant was analyzed for structural perturbations relative to the wild-type toxin.
• rU- ACTX-Hv Ia SEQ ID NO:1
• mutants thereof were prepared for acquisition of 2D 1 H- 15 N HSQC spectra by overproduction in Escherichia coli BL21 cells grown in minimal media with 15 N as the sole nitrogen source.
• the 2D HSQC spectrum of each uniformly 15 N-labeled mutant toxin was compared with the HSQC spectrum of wild-type rU- ACTX-HvIa acquired using identical experimental conditions. If the HSQC spectrum of the mutant toxin superimposed closely on the HSQC spectrum of rU- ACTX-H via, it was concluded that the introduced alanine substitution does not cause any significant structural perturbations in the mutant toxin.
• HSQC spectra were quantitatively compared by measuring the difference between the chemical shift of each peak in the HSQC spectrum of rU- ACTX-Hv Ia and the chemical shift of the corresponding peak in the spectrum of the mutant toxin.
• the chemical shift difference ( ⁇ ) was calculated using the following equation:
• ⁇ 6 N and ⁇ are the differences in chemical shifts of HSQC peaks in the nitrogen ( 15 N) and proton ( 1 H) dimensions respectively.
• a mutant was considered structurally perturbed relative to rU-ACTX-Hvla if 10% or more of the peaks in the HSQC spectrum had ⁇ values greater than 0.25 ppm. Based on this criterion, only four mutant toxins showed chemical shift differences indicative of a structural perturbation.
• the HSQC spectrum of the E26A mutant was dramatically different to that of rU- ACTX-HvIa, indicating a major structural perturbation. It was therefore excluded from the functional assays.
• the HSQC spectra of three other mutants, namely, T14A, Y35A and R38A revealed that peaks from 4-6 residues has ⁇ > 0.25 ppm, indicative of a minor structural perturbation.
• a LD 50 values were determined by injection into Musca domes tica.
• c Folding was assessed by comparing the HSQC spectrum of the mutant with that of rU- ACTX- HvIa.
• a molecular model made from the atomic coordinates for the rU-ACTX-Hvla insecticidal toxin having PDB ID 2HlZ and RCSB ID RCSB037828 is employed to identify a candidate molecule that mimics the structure of the rU-ACTX-Hvla molecular model. Identifying the pharmacophoric residues Q , P 9 , N 28 , and V 34 in the molecular model while using the molecular model aids in identifying functional U-ACTX mimics. In particular, mimics exhibiting lethality to insects, inhibition of insect calcium channels, inhibition of insect calcium-activated potassium channels, binding to insect calcium channels, binding to insect calcium-activated potassium channels, or a combination of one or more of the foregoing are particularly desirable.

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