Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-24
2003-10-14
Moran, Marjorie (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C536S023700, C530S350000, C530S388600, C930S210000
Reexamination Certificate
active
06632621
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to G-protein, seven-transmembrane-receptor-like polynucleotides and polypeptides, as well as modulators of the interaction between the polypeptides and their ligand(s) that are useful for inhibiting the neurological activity of organisms, e.g., invertebrates.
BACKGROUND
It has been estimated that as many as one in three humans is infected by one or more species of parasitic helminth (flatworms and roundworms). Helminths also represent a chronic and continuing threat to the health of livestock and companion animals; their abundance and resistance to anthelmintic drugs effectively prevents animal agriculture in certain environments. Despite the prevalence of helminth-caused disease, little is known of helminth physiology. This ignorance, in turn, hampers efforts to identify agents capable of controlling these pathogens.
Flatworms (platyhelminths) are evolutionarily quite distinct from the roundworms (nematodes) and differ markedly in neuromuscular anatomy and physiology. Only a subset of one class of drugs (some of the benzimidazoles) shows activity against both flatworms and roundworms. The diversity among the animal species that threaten the health of man, livestock, crops, and sensitive environmental niches, presents a challenge to efforts to mount broad-based attacks on the pest organisms.
G protein-coupled receptors (i.e., GPCRs) form a vast superfamily of cell surface receptors which are present in virtually all animal cells and are characterized by an amino-terminal extracellular domain, a carboxy-terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times. Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors. These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains. The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream effector molecules.
The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons. Not surprisingly, the GPCRs are important in the normal (and sometimes the aberrant) function of many cell types. [See generally Strosberg, Eur. J. Biochem., 196: 1-10 (1991) and Bohm et al., Biochem J., 322: 1-18 (1997).] When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine nucleotide-binding regulatory protein (G protein) that is coupled to the intracellular portion or region of the receptor. The G protein, in turn, transmits a signal to an effector molecule within the cell by either stimulating or inhibiting the activity of that effector molecule. These effector molecules include adenylate cyclase, phospholipases and ion channels. Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol. It is through this sequence of events that an extracellular ligand stimulus exerts intracellular changes through a G protein-coupled receptor. Each such receptor has its own characteristic primary structure, expression pattern, ligand binding profile, and intracellular effector system.
Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, and drugs that activate or antagonize the activation of such receptors are known. For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically for mimicking, enhancing or inhibiting the action of the ligand. Thus, GPCRs show promise as potential targets of methods for treating infestations and/or infections caused by a variety of invertebrate pests, including both ecto- and endo-parasites. However, such methods must be able to discriminate the GPCRs of the invertebrate pest organisms from the GPCRs found in those species of plants and vertebrate animals upon whom the pests prey.
A large family of peptides (typically 4-15 amino acids in length) that is largely, if not exclusively, found in invertebrate animals such as helminths is a class of neuropeptides known as FMRFamide related peptides (i.e., FaRPs). The prototypical FMRFamide peptides are so named because of the “FMRF” amino acid sequence, including the consensus “RF” sequence, at their C-termini. As neuropeptides, these molecules are involved in vital biological processes requiring controlled neuromuscular activity. Although some neurotransmitters and neuromodulators (including neuropeptides) have been shown to function as ligands for receptors, to date there has been no identification of a FaRP neuropeptide as a ligand of a GPCR.
Because of the toxic potential of broad-spectrum chemical parasiticides, there exists a need in the art for targeted biologicals capable of selectively interfering with the life cycle of harmful invertebrates such as helminths and insects without harming host plant and animal species, as well as the environment.
SUMMARY OF THE INVENTION
The present invention generally relates to materials and methods for the targeted interference with vital biological processes of pest invertebrates. By providing materials and methods for modulating the activity of invertebrate GPCRs involved in neuromuscular activity, the invention provides a biological approach to invertebrate pest control that can minimize the deleterious consequences to non-pest species of animals, including man, as well as plants and the environment in general.
One aspect of the invention is a screening method for identifying candidate anti-invertebrate modulators that affect one or more activities of an invertebrate GPCR-like receptor involved in neuromuscular functioning including, e.g., binding of a GPCR-like receptor to a ligand, typically a peptide ligand, and signal transduction. The method comprises the steps of: (a) contacting a test compound with a composition, wherein the composition contains a GPCR-like receptor encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178, or a polynucleotide hybridizing to the GPCR-like receptor under stringent conditions of hybridizing at 42° C. in a solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and (b) measuring the activity of the GPCR-like receptor in the presence and absence of the test compound. In preferred embodiments, the screening method includes a receptor, or fragment, variant or derivative thereof, having a sequence set forth in SEQ ID NOS: 7, 21, 25, 35, 43, 45, 105, 107, 109, 111, 113, 115, 117, 177 and 179.
As one of ordinary skill in the art would recognize, the above-described method can be practiced with a variety of GPCR-like receptors. For example, the GPCR-like receptor used in the screening method may be encoded by a polynucleotide having a sequence set forth in any one of SEQ ID NOS:43, 21, 45, 35, 7, 106, and 104. As noted above, such GPCR-like receptors may be used in screening assays designed to measure a GPCR-like receptor activity, including binding activity. Expressly contemplated are embodiments of the screening method comprising a GPCR-like receptor encoded by a polynucleotide comprising a sequence set forth in SEQ ID NO:43 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:85, 86, 88, 89, and 118, wherein the peptide binds to the GPCR-like receptor. encoded by a polynucleotide having t
Geary Timothy G.
Kubiak Teresa M.
Larsen Martha J.
Lowery David E.
Moran Marjorie
Pharmacia & Upjohn Company
Pharmacia & Upjohn Company
Rehberg Edward F.
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