Stabilized bioactive peptides and methods of identification, synthesis, and use

#This invention relates to stabilized bioactive peptides, and more particularly to bioactive peptides that contain heterologous stabilizing groups attached to one or both of the bioactive peptide's termini.

#Bioactive peptides are small peptides that elicit a biological activity. Since the discovery of secretin in 1902, over 500 of these peptides which average 20 amino acids in size have been identified and characterized. They have been isolated from a variety of systems, exhibit a wide range of actions, and have been utilized as therapeutic agents in the field of medicine and as diagnostic tools in both basic and applied research. Tables 1 and 2 list some of the best known bioactive peptides. 1 TABLE 1 Bioactive peptides utilized in medicine Size in Amino Name Isolated From Acids Therapeutic Use Angiotensin II Human Plasma 8 Vasoconstrictor Bradykinin Human Plasma 9 Vasodilator Caerulein From Skin 10 Choleretic Agent Calcitonin Human Parathyroid Gland 32 Calcium Regulator Cholecystokinin Porcine Intestine 33 Choleretic Agent Corticotropin Porcine Pituitary Gland 39 Hormone Eledoisin Octopod Venom 11 Hypotensive Agent Gastrin Porcine Stomach 17 Gastric Activator Glucagon Porcine Pancreas 29 Antidiabetic Agent Gramicidin D Bacillus brevis Bacteria 11 Antibacterial Agent Insulin Canine Pancreas Antidiabetic Agent Insulin A 21 Insulin B 30 Kallidin Human Plasma 10 Vasodilator Luteinizing Bovine Hypothalamus 10 Hormone Hormone-Releasing Stimulator Factor Melittin Bee Venom 26 Antirheumatic Agent Oxytocin Bovine Pituitary Gland 9 Oxytocic Agent Secretin Canine Intestine 27 Hormone Sermorelin Human Pancreas 29 Hormone Stimulator Somatostatin Bovine Hypothalamus 14 Hormone Inhibitor Vasopressin Bovine Pituitary Gland 9 Antidiuretic Agent

#2 TABLE 2 Bioactive peptides utilized in applied research Size in Amino Name Isolated From Acids Biological Activity Atrial Natriuretic Rat Atria 28 Natriuretic Agent Peptide Bombesin Frog Skin 14 Gastric Activator Conantokin G Snail Venom 17 Neurotransmitter Conotoxin G1 Snail Venom 13 Neuromuscular Inhibitor Defensin HNP-1 Human Neutrophils 30 Antimicrobial Agent Delta Sleep- Rabbit Brain 9 Neurological Affector Inducing Peptide Dermaseptin Frog Skin 34 Antimicrobial Agent Dynorphin Porcine Brain 17 Neurotransmitter EETI II Ecballium elaterium 29 Protease Inhibitor seeds Endorphin Human Brain 30 Neurotransmitter Enkephalin Human Brain 5 Neurotransmitter Histatin 5 Human Saliva 24 Antibacterial Agent Mastoparan Vespid Wasps 14 Mast Cell Degranulator Magainin 1 Frog Skin 23 Antimicrobial Agent Melanocyte Porcine Pituitary 13 Hormone Stimulator Stimulating Gland Hormone Motilin Canine Intestine 22 Gastric Activator Neurotensin Bovine Brain 13 Neurotransmitter Physalaemin Frog Skin 11 Hypotensive Agent Substance P Horse Intestine 11 Vasodilator Vasoactive Porcine Intestine 28 Hormone Intestinal Peptide

#FIG. 1 shows the control region (SEQ ID NO: 1) of the wild-type lac operon from the auxiliary operator O3 through the translational start of the lacZ gene. DNA binding sites include the operators O3 and O1 (both underlined), CAP (boxed), the −35 site (boxed), and the −10 site (boxed), while important RNA and protein sites include the Lacl translation stop site (TGA), the +1 lacZ transcription start site, the Shine Dalgarno (SD) ribosome binding site for lacZ, and the LacZ translation start site (ATG).

#FIG. 2 is a map of plasmid pLAC11. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Tet (98-1288), Rop (1931-2122), ori (2551-3138), Amp (3309-25 4 169), and lacPO (4424-4536).

#FIG. 3 is a map of plasmid pLAC22. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Tet (98-1288), Rop (1927-2118), ori (2547-3134), Amp (3305-4165), lacIq (4452-5536), and lacPO (5529-5641).

#The present invention represents a significant advance in the art of peptide drug development by allowing concurrent screening for peptide bloactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated inducible expression system that permits essentially complete repression of peptide expression in the host cell. Subsequent induction of peptide expression can then be used to identify peptides that inhibit host cell growth or possess other bioactivities.

#Intracellular screening of randomized peptides has many advantages over existing methods. Bioactivity is readily apparent, many diverse bioactivities can be screened for simultaneously, very large numbers of peptides can be screened using easily generated peptide libraries, and the host cell, if desired, can be genetically manipulated to identify an affected protein target. Advantageously, randomized peptides can be screened in a host cell that is identical to or closely resembles the eventual target cell for antimicrobial, anticancer, and other therapeutic applications. An additional and very important feature of this system is that selection is naturally biased in favor of peptides that are stable in an intracellular environment, i.e., that are resistant to proteases and peptidases. Fortuitously, bacterial peptidases are very similar to eukaryotic peptidases. Peptides that are stable in a bacterial host are thus likely to be stable in a eukaryotic cell as well, allowing bacterial cells to be used in initial screens to identify drugs that may eventually prove useful as human or animal therapeutics.

#The invention is directed to the identification and use of bioactive peptides. A bioactive peptide is a peptide having a biological activity. The term “bioactivity” as used herein includes, but is not limited to, any type of interaction with another biomolecule, such as a protein, glycoprotein, carbohydrate, for example an oligosaccharide or polysaccharide, nucleotide, polynucleotide, fatty acid, hormone, enzyme, cofactor or the like, whether the interactions involve covalent or noncovalent binding. Bioactivity further includes interactions of any type with other cellular components or constituents including salts, ions, metals, nutrients, foreign or exogenous agents present in a cell such as viruses, phage and the like, for example binding, sequestration or transport-related interactions. Bioactivity of a peptide can be detected, for example, by observing phenotypic effects in a host cell in which it is expressed, or by performing an in vitro assay for a particular bioactivity, such as affinity binding to a target molecule, alteration of an enzymatic activity, or the like. Examples of bioactive peptides include antimicrobial peptides and peptide drugs. Antimicrobial peptides are peptides that adversely affect a microbe such as a bacterium, virus, protozoan, or the like. Antimicrobial peptides include, for example, inhibitory peptides that slow the growth of a microbe, microbiocidal peptides that are effective to kill a microbe (e.g., bacteriocidal and virocidal peptide drugs, sterilants, and disinfectants), and peptides effective to interfere with microbial reproduction, host toxicity, or the like. Peptide drugs for therapeutic use in humans or other animals include, for example, antimicrobial peptides that are not prohibitively toxic to the patient, and peptides designed to elicit, speed up, slow down, or prevent various metabolic processes in the host such as insulin, oxytocin, calcitonin, gastrin, somatostatin, anticancer peptides, and the like.