| Antimicrobial theta defensins and methods of using same -> Monitor Keywords |
|
|
  Antimicrobial theta defensins and methods of using sameUSPTO Application #: 20050261193Title: Antimicrobial theta defensins and methods of using same Abstract: The present invention relates to an isolated cyclic peptide, theta defensin, having antimicrobial activity, and to theta defensin analogs. A theta defensin can have the amino acid sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa1-Xaa6-Xaa4-Xaa4-Xaa1-Xaa1-Xaa6-Xaa4-Xaa5-Xaa1-Xaa3-Xaa7-Xaa5, wherein Xaa1 to Xaa8 are defined; wherein Xaa1 can be linked through a peptide bond to Xaa8; and wherein crosslinks can be formed between Xaa3 and Xaa3, between Xaa5 and Xaa5, and between Xaa7 and Xaa7. For example, the invention provides a theta defensin having the amino acid sequence Gly-Phe-Cy-Arg-Cys-Leu-Cys-Arg-Arg-Gly-Val-Cys-Arg-Cys-Ile-Cys-Thr-Arg (SEQ ID NO:1), wherein the Gly at position 1 (Gly-1) is linked through a peptide bond to Arg-18, and wherein disulfide bonds are present between Cys-3 and Cys-16, between Cys-5 and Cys-14, and between Cys-7 and Cys-12. The invention also relates to antibodies that specifically bind a theta defensin and to isolated nucleic acid molecules encoding a theta defensin. In addition, the invention relates to methods of using theta defensin or a theta defensin analog to reduce or inhibit microbial growth or survival in an environment capable of sustaining microbial growth or survival by contacting the environment with the theta defensin. (end of abstract) Agent: Mcdermott, Will & Emery - San Diego, CA, US Inventors: Michael E. Selsted, Yi-Quan Tang, Jun Yuan, Andre J. Ouellette USPTO Applicaton #: 20050261193 - Class: 514013000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 16 To 24 Peptide Repeating Units In Known Peptide Chain The Patent Description & Claims data below is from USPTO Patent Application 20050261193. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to antimicrobial agents and, more specifically, to cyclic theta defensin peptides and methods of using a theta defensin to reduce or inhibit microbial growth or survival. [0004] 2. Background Information [0005] Infections by microorganisms, including bacteria, viruses and fungi, are a major cause of human morbidity and mortality. Although anyone can be a victim of such infection, the sick and elderly are particularly susceptible. For example, hospitalized patients frequently acquire secondary infections due to a combination of their weakened condition and the prevalence of microorganisms in a hospital setting. Such opportunistic infections result in increased suffering of the patient, increased length of hospitalization and, consequently, increased costs to the patient and the health care system. Similarly, the elderly, particularly those living in nursing homes or retirement communities, are susceptible to infections because of their close living arrangement and the impaired responsiveness of their immune systems. [0006] Numerous drugs are available for treating infections by certain microorganisms. In particular, various bacterial infections have been amenable to treatment by antibiotics. However, the prolonged use of antibiotics since their discovery has resulted in the selection of bacteria that are relatively resistant to these drugs. Furthermore, few if any drugs are effective against microorganisms such as viruses. As a result, continuing efforts are being made to identify new and effective agents for treating infections by a variety of microorganisms. [0007] The identification of naturally occurring compounds that act as antimicrobial agents has provided novel and effective drugs. Many organisms protect themselves by producing natural products that are toxic to other organisms. Frogs, for example, produce a class of peptides, magainins, which provide a defense mechanism for the frog against potential predators. Magainins have been purified and shown to have antimicrobial activity, thus providing a natural product useful for reducing or inhibiting microbial infections. [0008] Natural products useful as antimicrobial agents also have been purified from mammalian organisms, including humans. For example, the defensins are a class of peptides that have been purified from mammalian neutrophils and demonstrated to have antimicrobial activity. Similarly, indolicidin is a peptide that has been isolated from bovine neutrophils and has antimicrobial activity, including activity against viruses, bacteria, fungi and protozoan parasites. Thus, naturally occurring compounds provide a source of drugs that are potentially useful for treating microbial infections. [0009] Upon identifying naturally occurring peptides useful as antimicrobial agents, efforts began to chemically modify the peptides to obtain analogs having improved properties. Such efforts have resulted, for example, in the identification of indolicidin analogs which, when administered to an individual, have increased selectivity against the infecting microorganisms as compared to the individual's own cells. Thus, the availability of naturally occurring antimicrobial agents has provided new drugs for treating microbial infections and has provided a starting material to identify analogs of the naturally occurring molecule that have desirable characteristics. [0010] Although such natural products and their analogs have provided new agents for treating microbial infections, it is well known that microorganisms can become resistant to drugs. Thus, a need exists to identify agents that effectively reduce or inhibit the growth or survival of microorganisms. The present invention satisfies this need and provides additional advantages. SUMMARY OF THE INVENTION [0011] The present invention relates to an isolated cyclic theta defensin peptide, which exhibits broad spectrum antimicrobial activity, and to theta defensin analogs. In general, a theta defensin or theta defensin analog has the amino acid sequence Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa1-Xaa6-Xaa- 4-Xaa4-Xaa1-Xaa1-Xaa6-Xaa4-Xaa5-Xaa1-Xaa3-Xaa7-Xaa8, wherein Xaa1 independently is Gly, Ile, Leu, Val or Ala; Xaa2 is Phe, Trp or Tyr; Xaa3 is Cys or Trp; Xaa4 independently is Arg or Lys; Xaa5 is Cys or Trp; Xaa6 is Cys or Trp; Xaa7 is Thr or Ser; and Xaa8 is Arg or Lys. Xaa1 can be linked through a peptide bond to Xaa8. Furthermore, crosslinks can be formed between Xaa3 and Xaa3, between Xaa5 and Xaa5, and between Xaa7 and Xaa7. For example, the invention provides theta defensin having the amino acid sequence Gly-Phe-Cys-Arg-Cys-Leu-Cys-Arg-Arg-Gly-Val-Cys-Arg-Cys-Ile- -Cys-Thr-Arg (SEQ ID NO:1), wherein the Gly at position 1 (Gly-1) is linked through a peptide bond to Arg-18, and wherein disulfide bonds are present between Cys-3 and Cys-16, Cys-5 and Cys-14, and Cys-7 and Cys-12. [0012] The invention also relates to methods of using a theta defensin or an analog thereof to reduce or inhibit microbial growth or survival in an environment capable of sustaining microbial growth or survival by contacting the environment with theta defensin. As such, the invention provides methods of reducing or inhibiting microbial growth or survival on a solid surface, for example, surgical instruments, hospital surfaces, and the like. [0013] The invention further relates to methods for reducing or inhibiting microbial growth or survival in an individual, particularly a mammal such as a human. Thus, the invention provides methods of treating an individual suffering from a pathology characterized, at least in part, by microbial infection, by administering theta defensin or an analog thereof to the individual, thereby reducing the severity of the pathologic condition. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIG. 1 shows purification of RTD-1. Panel A shows reverse phase HPLC (RP-HPLC) of peripheral blood leukocyte extracts. An .alpha.-defensin-enriched extract of 6.times.10.sup.6 leukocytes (91% PMNs) was fractionated by RP-HPLC on a 0.46.times.25 cm C-18 column equilibrated in 0.1% aqueous TFA and developed with a linear acetonitrile gradient (dotted line). RTD-1 eluted in the peak marked with an arrow. Panel B shows analytical RP-HPLC of purified RTD-1. The purity of RTD-1 was assessed by RP-HPLC of RTD-1 obtained from the peak marked by an arrow in panel A on an analytical C-18 column developed with acetonitrile at 0.5% per min. Panel C shows acid-urea polyacrylamide gel electrophoresis (PAGE). Samples analyzed were 30% acetic acid extracts, 2.times.10.sup.6 cell equivalents, lane 1; methanol/water extracted phase, 1.times.10.sup.7 cell equivalents lane 2; and 1 .mu.g of RTD-1, lane 3. Samples were resolved on a 12.5% acid-urea polyacrylamide gel and stained with formalin-Coomassie blue. [0015] FIG. 2 shows the peptide backbone structure of RTD-1. Panel A shows the amino acid sequence of the peptide chain, determined by Edman sequencing. The corresponding MALDI-TOF MS analysis of purified proteolytic fragments is also shown. Residues in parentheses were assigned based on MALDI-TOF MS data. Calculated MALDI-TOF MS values are in parentheses. The peptides shown in Panel A (top to bottom) correspond to SEQ ID NOS:2-9, respectively. Panel B shows a schematic of RTD-1 cyclized peptide backbone. [0016] FIG. 3 shows disulfide analysis of RTD-1. A tridisulfide-containing 17-residue oligopeptide generated by trypsin digestion was purified by RP-HPLC and further digested with thermolysin. MS analysis (calculated values in parentheses) of the digest or of HPLC-purified fragments disclosed thermolytic cleavage at Cys-14/Ile-15 and at Cys-5/Leu-6 (arrows), producing four major thermolytic fragments (Th-1 to Th-4). The masses of all fragments were consistent with the disulfide assignments shown. [0017] FIG. 4 shows the structure of RTD-1. Panel A shows a schematic of the covalent structure of RTD-1 compared with that of circulin A, an antiviral peptide isolated from the plant Chassalia parvifolia. Panel B shows a theoretical model of RTD-1 obtained by molecular dynamics and energy minimization in water. The model shows a high degree of structural similarity to porcine protegrin 1 (PG-1) for those residues defined in the PG-1 solution structure. Panel C shows the alignment of the PG-1 and RTD-1 sequences and disulfide motifs. [0018] FIG. 5 shows the coordinates used to generate the molecular model shown in FIG. 4. [0019] FIG. 6 shows synthesis and characterization of RTD-1. Panel A shows the scheme for solid phase peptide synthesis and cyclization entailed chain assembly, cleavage/deprotection, purification of the reduced linear chain, oxidation and cyclization. Panel B shows co-elution of synthetic and natural RTD-1 on RP-HPLC and comigration on acid-urea PAGE (inset). Panel C shows circular dichroic spectra of synthetic and natural RTD-1 determined in water, 10 mM sodium phosphate buffer, and methanol at a peptide concentration of 111 .mu.g/ml (53.3 .mu.M). [0020] FIG. 7 shows the zone of inhibition (mm inhibition) of growth of Staphylococcus aureus 502A (closed circles), Escherichia coli ML35 (open circles); Listeria monocytogenes EGD (open triangles), and Cryptococcus neoformans 271A (closed triangles) at various concentrations of theta defensin. [0021] FIG. 8 shows a comparison of staphylocidal activity of natural and synthetic RTD-1. S. aureus 502a was incubated with increasing-concentrations of natural or synthetic theta defensin peptide. Killing was quantified by colony counts. [0022] FIG. 9 shows microbicidal activity of RTD-1. Panel A shows incubation of S. aureus 502a with increasing concentrations of natural or synthetic peptide. Killing was quantified by colony counts. Panels B-D show incubation of the indicated organisms with RTD-1 peptide: Listeria monocytogenes and Staphylococcus aureus (Panel B); Salmonella typhimurium and Escherichia coli (Panel C); and Cryptococcus neoformans and Candida albicans (Panel D). The limit of detection (1 colony per plate) was equal to 1.times.10.sup.3 colony forming units in the incubation mixture. Panel E shows killing of S. aureus 502a with natural or synthetic RTD-1 supplemented with increasing concentrations of NaCl. Continue reading... Full patent description for Antimicrobial theta defensins and methods of using same Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Antimicrobial theta defensins and methods of using same patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Antimicrobial theta defensins and methods of using same or other areas of interest. ### Previous Patent Application: Vasoregulating compounds and methods of their use Next Patent Application: Myosin light chain kinase inhibitors and methods of use Industry Class: Drug, bio-affecting and body treating compositions ### FreshPatents.com Support Thank you for viewing the Antimicrobial theta defensins and methods of using same patent info. IP-related news and info Results in 1.47336 seconds Other interesting Feshpatents.com categories: Canon USA , Celera Genomics , Cephalon, Inc. , Cingular Wireless , Clorox , Colgate-Palmolive , Corning , Cymer , |
||
