Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1999-09-15
2001-10-09
Low, Christopher S. F. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S328000, C530S329000, C530S330000, C530S333000, C530S334000, C530S335000, C530S387100, C514S002600, C514S012200, C514S015800, C514S016700, C514S017400, C514S018700, C514S021800, C435S007100, C536S023100, C436S503000, C436S547000
Reexamination Certificate
active
06300470
ABSTRACT:
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates generally to antimicrobial peptides and more specifically to cryptdin peptides, nucleic acid molecules encoding cryptdins, and their uses.
BACKGROUND INFORMATION
Survival in a world teaming with microorganisms depends on a network of host defense mechanisms. Among these mechanisms are phagocytosis by cells are resident in tissues or that circulate in the blood system and ingest, kill and digest potentially harmful microbes. Although pathogenic microbes may vary considerably, phagocytes are able to destroy the vast majority by sequestering them in intracytoplasmic vacuoles and exposing them to a lethal mixture of organic and inorganic toxins.
Perhaps the most remarkable ultrastructural feature of phagocytes are their several thousand cytoplasmic granules, which are membrane-bound organelles typically about 0.3 &mgr;m in diameter. During phagocytosis, some of these granules fuse to phagocytic vesicles thus enabling the contents of the granule to enter the lumen of the vesicle. Early observers surmised correctly that the granules contained factors which were responsible for intraphagosomal killing in digestion of microbes. These granules contain a mixture of antimicrobial molecules including various peptides such as the so-called defensins.
Defensins are abundant antimicrobial peptide components of vertebrate neutrophil and macrophage granules. Members of the defensin family have been identified previously in human, rabbit, guinea pig and rat phaodyes,primarily those phagocytes termed phagocytic granulocytes. Defensins are cationic peptides that have molecular weights between about 3 and 4 kiloDaltons (kDa) and that exhibit broad-range antimicrobial activities against gram negative and gram positive bacteria, many fungi and some enveloped viruses. The peptides are characterized by eight invariant amino acids, including six invariant cysteine residues that constitute a unique disulfide motif. The three disulfide bonds stabilize a tertiary conformation consisting predominantly of &bgr;-sheet. The highly ordered structure and the absence of a helix make defensins unique among known antimicrobial peptides. It appears that defensins exert their antibacterial effect by permeabilizing the cytoplasmic membrane of the target microorganism by a Mechanism that may involve the formation of ion channels or transmembrane pores.
Until recently, defensins had been identified only in circulating or tissue phagocytes of myeloid origin. However, based on the presence of a particular mRNA, it has been surmised that similar peptides might be present in the epithelial cells of the small intestine. Such intestinal peptides may prevent access of microorganisms through the small intestine into the systemic circulation and, therefore, can be useful as a therapeutic or prophylactic agent. Thus, a need exists to identify peptides that have antimicrobial activity within the uiucosal epithelium or in the intestinal lumen. The present invention satisfies this need and provides additional benefits as well.
SUMMARY OF THE INVENTION
The present invention provides a substantially purified cryptdin peptide having a consensus amino acid sequence:
X
1
-C-X
2
-C-R-X
3
-C-X
4
-E-X
5
-C-X
6
-C-C-X
7
wherein X
1
is 3 to 9 amino acids; X
2
is 1 amino acid, preferably Y, H or R; X
3
io 2 or 3 amino acids X
4
is 3 amino acids; X
5
is 5 amino acids; X
6
is 6 to 10 amino acids; and X
7
is 0 to 9 amino acids.
The invention also provides a substantially purified mouse cryptdin having a consensus amino acid sequence:
X
1
-L-X
2
-C-Y-C-R-X
3
-C-K-X
4
-E-X
5
-G-T-C-X
6
-C-C-X
7
wherein X
1
is 3 or 4 amino acids, preferably LRD, LSKK (SEQ ID No: 1) or LRG;
X
2
is 1 amino acid, preferably V, L or I;
X
3
is 3 amino acids, preferably KGH or *RG, where * is S, T, K, I or A;
X
4
is 2 amino acids, preferably GR, RR or RG;
X
5
is 3 amino acids, preferably RMN, RVR, RVF HMN or HIN;
X
6
is 6 to 9 amino acids, preferably GIRFLY (SEQ ID NO; 2) or RNLFLTFVF (SEQ ID NO: 3), RRGHLMYTL (SEQ ID NO: 4) or RKGHL*YT* (SEQ ID NO: 5), where * independently is L or M; and
X
7
is 0 to 3 amino acids, preferably R, S or PRR.
For example, the invention provides various mouse, rat or human cryptdins having the sequence:
1) LRDLVCYCRSRGCKGRERMNGTCRKGHLLYTLCCR (SEQ ID NO: 6);
2) LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID NO: 7);
3) LRDLVCYCRKRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID NO: 8);
4) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPR (SEQ ID NO: 9);
5) LSKKLICYCRIRGCKRRERVFGTCRNLFLTFVFCC (SEQ ID NO: 10);
6) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPG (SEQ ID NO: 11);
7) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPRR (SEQ ID NO: 12);
8) LSKKLICYCRIRGCKRRERVFGTCRNLFLTFVFCCS (SEQ ID NO: 13);
9) LRDLVCYCRARGCKGRERMNGTCRKGHLLYMLCCR (SEQ ID NO: 14);
10) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPGRT (SEQ ID NO: 15);
11) IGRPVRRCRCRANCGPKEYATAFCAQGPFKQFKFCCT (SEQ ID NO: 16);
12) IRWPWKRCHCRSFCRPYENATSFCAQGLFKQHKFCCLDTWPPRMK (SEQ ID NO: 17);
13) TSGSQARATCYCRTGRCATRESLSGVCEISGRLYRLCCR (SEQ ID NO: 18); and
14) AFTCHCRRSCYSTEYSYGTCTNMGINHRFCCL (SEQ ID NO: 19).
Cryptdins are typically characterized by being naturally found in the epithelial cells of the small intestine, being cationic, being about 30 to about 45 amino acids in length, having at least three and, preferably, three to nine, amino acids to the N-terminal of the first cysteine residue, exhibiting specific antimicrobial activity against intestinal pathogens and opportunistic pathogens and being relatively non-toxic to cells of the host organism. However, there may be diversity in these structural and functional characteristics. The invention also provides cryptdin analogs, which are devoid of one or more amino acids N-terminal to the first cysteine. In addition, the invention also provides nucleic acid molecules encoding cryptdin peptides. For example, the invention provides genomic DNA sequences and cDNA sequences encoding mouse and rat cryptdins.
The invention further provides a method for detecting an inflammatory pathology in a subject by determining the amount of cryptdin in a biological sample from the subject and comparing that amount to the amount present in a normal subject. such a method can be used to determine the presence of an inflammatory pathology such as inflammatory bowel disease, pancreatitis, malignancy, infection or ileitis.
The invention also provides a method for treating an inflammatory pathology in a subject by administering a cryptdin to the subject Such treatment is particularly advantageous in patients who are immunocompromised due, for example, to malnutrition, radiation burns, immunosuppressive infections, autoimmune disease, neonatality, bone marrow transplantation or chemotherapy. A cryptdin can be administered orally, by nasogastric intubation, by transabdominal catheter, intravenously or by aerosol inhalation. When administered orally, it is preferably in a delayed release formulation designed to permit release in the small intestine The cryptdin can be administered as a composition with a physiologically acceptable medium, and more than one cryptdin can be administered simultaneously or sequentially.
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Bowie et al.,Science,vol. 247, pp. 1306-1310, 1990.*
Houghten et al.,
Ouellette Andre J.
Selsted Michael E.
Campbell & Flores LLP
Low Christopher S. F.
Mohamed Abdel A.
The Regents of the University of California
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