An intracellular selection system allows concurrent screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at either the N-terminus, the C-terminus, or both. The stabilizing group can take the form of a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, or one or more proline residues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention represents a significant advance in the art of peptide drug development by allowing concurrent screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated inducible expression system, preferably derived from the wild-type lac operon, 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 elucidate 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 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, 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.

The term "peptide" as used herein refers to a plurality of amino acids joined together in a linear chain via peptide bonds. Accordingly, the term "peptide" as used herein includes a dipeptide, tripeptide, oligopeptide and polypeptide. A dipeptide contains two amino acids; a tripeptide contains three amino acids; and the term oligopeptide is typically used to describe peptides having between 2 and about 50 or more amino acids. Peptides larger than about 50 are often referred to polypeptides or proteins. For purposes of the present invention, a "peptide" is not limited to any particular number of amino acids. Preferably, however, the peptide contains about 2 to about 50 amino acids, more preferably about 5 to about 40 amino acids, most preferably about 5 to about 20 amino acids.

The library used to transform the host cell is formed by cloning a randomized, peptide-encoding oligonucleotide into a nucleic acid construct having a tightly regulable expression control region. An expression control region can be readily evaluated to determine whether it is "tightly regulable," as the term is used herein, by bioassay in a host cell engineered to contain a mutant nonfunctional gene "X." Transforming the engineered host cell with an expression vector containing a tightly regulable expression control region operably linked to a cloned wild-type gene "X" will preserve the phenotype of the engineered host cell under repressed conditions. Under induced conditions, however, the expression vector containing the tightly regulable expression control region that is operably linked to the cloned wild-type gene "X" will complement the mutant nonfunctional gene X to yield the wild-type phenotype. In other words, a host cell containing a null mutation which is transformed with a tightly regulable expression vector capable of expressing the chromosomally inactivated gene will exhibit the null phenotype under repressed conditions; but when expression is induced the cell will exhibit a phenotype indistinguishable from the wild-type cell. It should be understood that the expression control region in the tightly regulable expression vector of the present invention can be readily modified to produce higher levels of an encoded biopeptide, if desired (see, e.g., Example I, below). Such modification may unavoidably introduce some "leakiness" into expression control, resulting in a low level of peptide expression under repressed conditions.

In a preferred embodiment, the expression control region of the inducible expression vector is derived from the wild-type E. coli lac promoter/operator region. In a particularly preferred form, the expression vector contains a regulatory region that includes the auxiliary operator O3, the CAP binding region, the -35 promoter site, the -10 promoter site, the operator O1, the Shine-Dalgarno sequence for lacZ, and a spacer region between the end of the Shine-Dalgarno sequence and the ATG start of the lacZ coding sequence (see FIG. 1).

It is to be understood that variations in the wild-type nucleic acid sequence of the lac promoter/operator region can be tolerated in the expression control region of the preferred expression vector and are encompassed by the invention, provided that the expression control region remains tightly regulable as defined herein. For example, the -10 site of the wild-type lac operon (TATGTT) is weak compared to the bacterial consensus -10 site sequence TATAAT, sharing four out of six positions. It is contemplated that other comparably weak promoters are equally effective at the -10 site in the expression control region; a strong promoter is to be avoided in order to insure complete repression in the uninduced state. With respect to the -35 region, the sequence of the wild-type lac operon, TTTACA, is one base removed from the consensus -35 sequence TTGACA. It is contemplated that a tightly regulable lac operon-derived expression control region could be constructed using a weaker -35 sequence (i.e., one having less identity with the consensus -35 sequence) and a wild-type -10 sequence (TATAAT), yielding a weak promoter that needs the assistance of the CAP activator protein. Similarly, it is to be understood that the nucleic acid sequence of the CAP binding region can be altered as long as the CAP protein binds to it with essentially the same affinity. The spacer region between the end of the Shine-Dalgarno sequence and the ATG start of the lacZ coding sequence is typically between about 5 and about 10 nucleotides in length, preferably about 5 to about 8 nucleotides in length, more preferably about 7 9 nucleotides in length. The most preferred composition and length of the spacer region depends on the composition and length of Shine-Dalgarno sequence with which it is operably linked as well as the translation start codon employed (i.e., AUG, GUG, or UUG), and can be determined accordingly by one of skill in the art. Preferably the nucleotide composition of the spacer region is "AT rich"; that is, it contains more A's and T's than it does G's and C's.

In a preferred embodiment of the method of the invention, the expression vector has the identifying characteristics of pLAC11 (ATCC No. 207108). More preferably, the expression vector is pLAC11 (ATCC No. 207108).

As used in the present invention, the term "vector" is to be broadly interpreted as including a plasmid, including an episome, a viral vector, a cosmid, or the like. A vector can be circular or linear, single-stranded or double-stranded, and can comprise RNA, DNA, or modifications and combinations thereof. Selection of a vector or plasmid backbone depends upon a variety of characteristics desired in the resulting construct, such as selection marker(s), plasmid copy number, and the like. A nucleic acid sequence is "operably linked" to an expression control sequence in the regulatory region of a vector, such as a promoter, when the expression control sequence controls or regulates the transcription and/or the translation of that nucleic acid sequence. A nucleic acid that is "operably linked" to an expression control sequence includes, for example, an appropriate start signal (e.g., ATG) at the beginning of the nucleic acid sequence to be expressed and a reading frame that permits expression of the nucleic acid sequence under control of the expression control sequence to yield production of the encoded peptide. The regulatory region of the expression vector optionally includes a termination sequence, such as a codon for which there is no corresponding aminoacetyl-tRNA, thus ending peptide synthesis. Typically, when the ribosome reaches a termination sequence or codon during translation of the mRNA, the polypeptide is released and the ribosome-mRNA-tRNA complex dissociates.

An expression vector optionally includes one or more selection or marker sequences, which typically encode an enzyme capable of inactivating a compound in the growth medium. The inclusion of a marker sequence can, for example, render the host cell resistant to an antibiotic, or it can confer a compound-specific metabolic advantage on the host cell. Cells can be transformed with the expression vector using any convenient method known in the art, including chemical transformation, e.g., whereby cells are made competent by treatment with reagents such as CaCl.sub.2; electroporation and other electrical techniques; microinjection and the like.

In embodiments of the method that make use of a tightly regulable expression system derived from the lac operon, the host cell is or has been genetically engineered or otherwise altered to contain a source of Lac repressor protein in excess of the amount produced in wild-type E. coli. A host cell that contains an excess source of Lac repressor protein is one that expresses an amount of Lac repressor protein sufficient to repress expression of the peptide under repressed conditions, i.e., in the absence of an inducing agent, such as isopropyl .beta.-D-thiogalactoside (IPTG). Preferably, expression of Lac repressor protein is constitutive; For example, the host cell can be transformed with a second vector comprising a gene encoding Lac repressor protein, preferably lacI, more preferably lacI.sup.q, to provide an excess source of Lac repressor protein in trans, i.e., extraneous to the tightly regulable expression vector. An episome can also serve as a trans source of Lac repressor. Another option for providing a trans source of Lac repressor protein is the host chromosome itself, which can be genetically engineered to express excess Lac repressor protein Alternatively, a gene encoding Lac repressor protein can be included on the tightly regulable expression vector that contains the peptide-encoding oligonucleotide so that Lac repressor protein is provided in cis. The gene encoding the Lac repressor protein is preferably under the control of a constitutive promoter.

The invention is not intended to be limited in any way by the type of host cell used for screening. The host cell can be a prokaryotic or a eukaryotic cell. Preferred mammalian cells include human cells, of any tissue type, and can include cancer cells or hybridomas, without limitation. Preferred bacterial host cells include gram negative bacteria, such as E. coli and various Salmonella spp., and gram positive bacteria, such as bacteria from the genera Staphylococcus, Streptococcus and Enterococcus. Protozoan cells are also suitable host cells. In clear contrast to conventional recombinant protein expression systems, it is preferable that the host cell contains proteases and/or peptidases, since the selection will, as a result, be advantageously biased in favor of peptides that are protease- and peptidase-resistant. More preferably, the host cell has not been modified, genetically or otherwise, to reduce or eliminate the expression of any naturally expressed proteases or peptidases. The host cell can be selected with a particular purpose in mind. For example, if it is desired to obtain peptide drugs specific to inhibit Staphylococcus, peptides can be advantageously expressed and screened in Staphylococcus.

There is, accordingly, tremendous potential for the application of this technology in the development of new antibacterial peptides useful to treat various pathogenic bacteria. Of particular interest are pathogenic Staphylococci, Streptococci, and Enterococci, which are the primary causes of nosocomial infections. Many of these strains are becoming increasingly drug-resistant at an alarming rate. The technology of the present invention can be practiced in a pathogenic host cell to isolate inhibitor peptides that specifically target the pathogenic strain of choice. Inhibitory peptides identified using pathogenic microbial host cells in accordance with the invention may have direct therapeutic utility; based on what is known about peptide import, it is very likely that small peptides are rapidly taken up by Staphylococci, Streptococci, and Enterococci. Once internalized, the inhibitory peptides identified according to the invention would be expected to inhibit the growth of the bacteria in question. It is therefore contemplated that novel inhibitor peptides so identified can be used in medical treatments and therapies directed against microbial infection. It is further contemplated that these novel inhibitor peptides can be used, in turn, to identify additional novel antibacterial peptides using a synthetic approach. The coding sequence of the inhibitory peptides is determined, and peptides are then chemically synthesized and tested in the host cell for their inhibitory properties.

Novel inhibitor peptides identified in a pathogenic microbial host cell according to the invention can also be used to elucidate potential new drug targets. The protein target that the inhibitor peptide inactivated is identified using reverse genetics by isolating mutants that are no longer inhibited by the peptide. These mutants are then mapped in order to precisely determine the protein target that is inhibited. New antibacterial drugs can then be developed using various known or yet to be discovered pharmaceutical strategies.

Following transformation of the host cell, the transformed host cell is initially grown under conditions that repress expression of the peptide. Expression of the peptide is then induced. For example, when a lac promoter/operator system is used for expression, IPTG is added to the culture medium. A determination is subsequently made as to whether the peptide is inhibitory to host cell growth, wherein inhibition of host cell growth under induced but not repressed conditions is indicative of the expression of a bioactive peptide.

Notably, the bioactive peptides identified according to the method of the invention are, by reason of the method itself, stable in the intracellular environment of the host cell. The method of the invention thus preferably identifies bioactive peptides that are resistant to proteases and peptidases.

Resistance to proteases and peptidases can be evaluated by measuring peptide degradation when in contact with appropriate cell extracts or purified peptidases and/or proteases, employing methods well-known in the art. A protease- or peptidase-resistant peptide is evidenced by a longer half-life in the presence of proteases or peptidases compared to a control peptide.

Randomized peptides used in the screening method of the invention can be optionally engineered according to the method of the invention in a biased synthesis to increase their stability by making one or both of the N-terminal or C-terminal ends more resistant to proteases and peptidases, and/or by engineering into the peptides a stabilizing motif.

In one embodiment of the screening method of the invention, the putative bioactive peptide is stabilized by adding a stabilizing group to the N-terminus, the C-terminus, or to both termini. To this end, the nucleic acid sequence that encodes the randomized peptide in the expression vector or the expression vector itself is preferably modified to encode a first stabilizing group comprising the N-terminus of the peptide, and a second stabilizing group comprising the C-terminus of the peptide.

The stabilizing group can be a stable protein, preferably a small stable protein such as thioredoxin, glutathione sulfotransferase, maltose binding protein, glutathione reductase, or a four-helix bundle protein such as Rop protein, although no specific size limitation on the protein anchor is intended. Proteins suitable for use as a stabilizing group can be either naturally occurring or non-naturally occurring. They can be isolated from an endogenous source, chemically or enzymatically synthesized, or produced using recombinant DNA technology. Proteins that are particularly well-suited for use as a stabilizing group are those that are relatively short in length and form very stable structures in solution. Proteins having molecular weights of less than about 50 kD are preferred for use as a stabilizing group; more preferably the molecular weight of the small stable protein is less than about 25 kD, most preferably less than about 12 kD. For example, E. coli thioredoxin has a molecular weight about 11.7 kD; E. coli glutathione sulfotransferase has a molecular weight of about 22.9 kD, and Rop from the ColE1 replicon has a molecular weight of about 7.2 kD); and maltose binding protein (without its signal sequence) is about 40.7 kD. The small size of the Rop protein makes it especially useful as a stabilizing group, fusion partner, or peptide anchor, in that it is less likely than larger proteins to interfere with the accessibility of the linked peptide, thus preserving its bioactivity. Rop's highly ordered anti-parallel four-helix bundle topology (after dimerization) and slow unfolding kinetics (see, e.g., Betz et al, Biochemistry 36, 2450 2458 (1997)) also contribute to its usefulness as a peptide anchor according to the invention. Other proteins with similar folding kinetics and/or thermodynamic stability (e.g., Rop has a midpoint temperature of denaturation, T.sub.m, of about 71.degree. C., Steif et al., Biochemistry 32, 3867 3876 (1993)) are also preferred peptide anchors. Peptides or proteins having highly stable tertiary motifs, such as a four-helix bundle topology, are particularly preferred.

Alternatively, the stabilizing group can constitute one or more prolines (Pro). Preferably, a proline dipeptide (Pro-Pro) is used as a stabilizing group, however additional prolines may be included. The encoded proline(s) are typically naturally occurring amino acids, however if and to the extent a proline derivative, for example a hydroxyproline or a methyl- or ethyl-proline derivative, can be encoded or otherwise incorporated into the peptide, those proline derivatives are also useful as stabilizing groups.

At the N-terminus of the peptide, the stabilizing group can alternatively include an oligopeptide having the sequence Xaa-Pro.sub.m-, wherein Xaa is any amino acid, and m is greater than 0. Preferably, m is about 1 to about 5; preferably m=2 or 3, more preferably, m=2. Likewise, at the C-terminus of the peptide, the stabilizing group can alternatively include an oligopeptide having the sequence -Pro.sub.m-Xaa, wherein Xaa is any amino acid, and m is greater than 0. Preferably, n is about 1 to about 5; preferably n=2 or 3, more preferably, m=2. In a particularly preferred embodiment of the method of the invention, the nucleic acid sequence that encodes the randomized peptide in the expression vector is modified to encode each of a first stabilizing group comprising the N-terminus of the peptide, the first stabilizing group being selected from the group consisting of small stable protein, Pro-, Pro-Pro-, Xaa-Pro- and Xaa-Pro-Pro-, and a second stabilizing group comprising the C-terminus of the peptide, the second stabilizing group being selected from the group consisting of a small stable protein, -Pro, -Pro-Pro, Pro-Xaa and Pro-Pro-Xaa. The resulting peptide has enhanced stability in the intracellular environment relative to a peptide lacking the terminal stabilizing groups.

In another preferred embodiment of the screening method of the invention, the expression vector encodes a four-helix bundle protein fused, at either the C-terminus or the N-terminus, to the randomized peptide. Preferably, the four-helix bundle protein is E. coli Rop protein or a homolog thereof. The non-fused terminus of the randomized peptide can, but need not, comprise a stabilizing group. The resulting fusion protein is predicted to be more stable than the randomized peptide itself in the host intracellular environment. Where the four-helix bundle protein is fused to the N-terminus, the randomized peptide can optionally be further stabilized by engineering one or more prolines, with or without a following undefined amino acid (e.g., -Pro, -Pro-Pro, -Pro-Xaa, -Pro-Pro-Xaa, etc.) at the C-terminus of the peptide sequence; likewise, when the four-helix bundle protein is fused to the C-terminus, the randomized peptide can be further stabilized by engineering one or more prolines, with or without a preceding undefined amino acid (e.g., Pro-, Pro-Pro-, Xaa-Pro-, Xaa-Pro-Pro-, etc.) at the N-terminus of the peptide sequence.

In yet another embodiment of the screening method of the invention, the putative bioactive peptide is stabilized by engineering into the peptide a stabilizing motif such as an .alpha.-helix motif or an opposite charge ending motif. Chemical synthesis of an oligonucleotide according to the scheme [(CAG)A(TCAG)] yields an oligonucleotide encoding a peptide consisting of a random mixture of the hydrophilic amino acids His, Gin, Asn, Lys, Asp, and Glu (see Table 14). Except for Asp, these amino acids are most often associated with .alpha.-helical secondary structural motifs; the resulting oligonucleotides are thus biased in favor of oligonucleotides that encode peptides that are likely to form .alpha.-helices in solution. Alternatively, the putative bioactive peptide is stabilized by flanking a randomized region with a region of uniform charge (e.g., positive charge) on one end and a region of opposite charge (e.g., negative) on the other end, to form an opposite charge ending motif. To this end, the nucleic acid sequence that encodes the randomized peptide in the expression vector or the expression vector itself is preferably modified to encode a plurality of sequential uniformly charged amino acids comprising the N-terminus of the peptide, and a plurality of sequential oppositely charged amino acids comprising the C-terminus of the peptide. The positive charges are supplied by a plurality of positively charged amino acids consisting of lysine, histidine, arginine or a combination thereof; and the negative charges are supplied by a plurality of negatively charged amino acids consisting of aspartate, glutamate or a combination thereof. It is expected that such a peptide will be stabilized by the ionic interaction of the two oppositely charges ends. Preferably, the putative bioactive peptide contains at least three charged amino acids at each end. More preferably, it contains at least four charged amino acids at each end. In a particularly preferred embodiment, the larger acidic amino acid glutamate is paired with the smaller basic amino acid lysine, and the smaller acidic amino acid aspartate is paired with the larger basic amino acid arginine.

It is to be understood that novel bioactive peptides identified using the method for identification of bioactive peptides described herein are also included in the present invention.

The present invention further provides a bioactive peptide containing one or more structural features or motifs selected to enhance the stability of the bioactive peptide in an intracellular environment. During development and testing of the intracellular screening method of the present invention, it was surprisingly discovered that several bioactive peptides identified from the randomized peptide library shared particular structural features. For example, a disproportionately high number of bioactive peptides identified using the intracellular screening method contained one or more proline residues at or near a peptide terminus. A disproportionate number also contained sequences predicted, using structure prediction algorithms well-known in the art, to form secondary structures such as .alpha. helices or .beta. sheets; or a hydrophobic membrane spanning domain. Bioactive fusion proteins comprising the randomized peptide sequence fused to the Rop protein, due to a deletion event in the expression vector, were also identified.

Accordingly, the invention provides a bioactive peptide having a stabilizing group at its N-terminus, its C-terminus, or at both termini. In a bioactive peptide stabilized at only one terminus (i.e., at either the N- or the C-terminus) the stabilizing group is preferably either a four-helix bundle protein, such as Rop protein, a proline (Pro), or a proline dipeptide (Pro-Pro). It should be understood that in any synthetic peptide having a stabilizing group that includes one or more prolines according to the present invention, the proline is preferably a naturally occurring amino acid; alternatively, it can be a synthetic derivative of proline, for example a hydroxyproline or a methyl- or ethyl- proline derivative. Accordingly, where the abbreviation "Pro" is used herein in connection with a stabilizing group that is part of a synthetic peptide, it is meant to include proline derivatives in addition to a naturally occurring proline.

A peptide stabilized at both termini includes a first stabilizing group comprising the N-terminus, and a second stabilizing group stabilizing the C-terminus, where the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. The stabilizing group is covalently attached to the peptide. The bioactive peptide of the invention includes a bioactive peptide that has been detectably labeled, derivatized, or modified in any manner desired prior to use, provided it contains one or more terminal stabilizing groups as provided herein. In one preferred embodiment of the bioactive peptide of the invention, the first stabilizing group, comprising the N-terminus, is Xaa-Pro-Pro-, Xaa-Pro-, Pro- or Pro-Pro-; and second stabilizing group, comprising the C-terminus, is Pro-Pro-Xaa, -Pro-Xaa, -Pro or -Pro-Pro; preferably -Pro-Pro. In another preferred embodiment, the first (N-terminal) stabilizing group is a small stable protein, preferably a four-helix bundle protein such as Rop protein; and the second (C-terminal) stabilizing group is Pro-Pro-Xaa, -Pro-Xaa, -Pro or -Pro-Pro; preferably -Pro-Pro. In yet another preferred embodiment, the second (C-terminal) stabilizing group is a small stable protein, preferably a four-helix bundle protein such as Rop protein, and the first (N-terminal) stabilizing group is Pro-, Pro-Pro-, Xaa-Pro- or Xaa-Pro-Pro-.

The invention further provides a peptide stabilized by flanking the amino acid sequence of a bioactive peptide with an opposite charge ending motif, as described herein. Preferably, the resulting stabilized peptide retains at least a portion of the biological activity of the bioactive protein. The stabilized peptide includes a peptide that has been detectably labeled, derivatized, or modified in any manner desired prior to use.

It should be understood that any bioactive peptide, without limitation, can be stabilized according to the invention by attaching a stabilizing group to either or both of the N- and C-termini, or by attaching oppositely charged groups to the N- and C-termini to form an opposite charge ending motif. Included in the present invention are any and various antimicrobial peptides, inhibitory peptides, therapeutic peptide drugs, and the like, as, for example and without limitation, those listed in Tables 1 and 2, that have been modified at one or both peptide termini to include a stabilizing group, for example a four-helix bundle protein such as Rop protein, proline (Pro-), a proline-proline dipeptide-(Pro-Pro-), an Xaa-Pro- dipeptide, or an Xaa-Pro-Pro-tripeptide at the N-terminus, and/or a four-helix bundle protein such as Rop protein, proline (-Pro), or a proline-proline dipeptide (-Pro-Pro), a Pro-Xaa dipeptide, or a Pro-Pro-X tripeptide at the C-terminus; or that have been modified to contain an opposite charge ending motif according to the invention. In this aspect the invention is exemplified by peptides such as Pro-Pro-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Pro-Pro (SEQ ID NO: 3) and Glu-Asp-Glu-Asp-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Arg-Lys-Arg-Lys (SEQ ID NO: 4), wherein the middle nine amino acids (-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-; SEQ ID NO: 5) constitute the sequence of angiotensin.

Modification of a bioactive peptide to yield a stabilized bioactive peptide according to the invention can be achieved by standard techniques well-known in the arts of genetics and peptide synthesis. For example, where the peptide is synthesized de novo, as in solid state peptide synthesis, one or more prolines can be added at the beginning and the end of the peptide chain during the synthetic reaction. In recombinant synthesis, for example as described in Example III herein, one or more codons encoding proline can be inserted into the peptide coding sequence at the beginning and/or the end of the sequence, as desired. Preferably, codons encoding N-terminal prolines are inserted after (i.e., 3' to) the initiation site ATG (which encodes for methionine). Analogous techniques are used to synthesize bioactive peptides having an opposite charge ending motif. When a known bioactive peptide is modified to yield a stabilized bioactive peptide according to the invention, the unmodified peptide can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm, if desired, that the modified peptide exhibits increased stability.

The present invention also provides a cleavable polypeptide comprising a stabilized, bioactive peptide either immediately preceded by (i.e., adjacent to the N-terminus of the bioactive peptide) a cleavage site, or immediately followed by (i.e., adjacent to the C-terminus of the bioactive peptide) a cleavage site. Thus, a bioactive peptide as contemplated by the invention can be part of a cleavable polypeptide. The cleavable polypeptide is cleavable, either chemically, as with cyanogen bromide, or enzymatically, to yield the bioactive peptide. The resulting bioactive peptide either includes a first stabilizing group comprising its N-terminus and a second stabilizing group comprising its C-terminus, or it includes an opposite charge ending motif, both as described hereinabove. The cleavage site immediately precedes the N-terminal stabilizing group or immediately follows the C-terminal stabilizing group. In the case of a bioactive peptide having an opposite charge ending motif, the cleavage site immediately precedes the first charged region or immediately follows the second charged region. The cleavage site makes it possible to administer a bioactive peptide in a form that could allow intracellular targeting and/or activation.

Alternatively, a bioactive peptide of the invention can be fused to a noncleavable N-terminal or C-terminal targeting sequence wherein the targeting sequence allows target,ed delivery of the bioactive peptide, e.g., intracellular targeting or tissue-specific targeting of the bioactive peptide. In one embodiment of this aspect of the invention, the free terminus of the bioactive peptide comprises a stabilizing group as described hereinabove in connection with the screening method for identifying bioactive peptides, for example one or more prolines. The targeting sequence forming the other peptide terminus can, but need not, contain a small stable protein such as Rop or one or more prolines comprising its terminus, as long as the targeting function of the targeting sequence is preserved. In another embodiment of this aspect of the invention, the bioactive peptide comprises a charge ending motif as described hereinabove, wherein one charged region occupies the free terminus of the bioactive peptide, and the other charged region is disposed between the targeting sequence and the active sequence of the bioactive peptide.

The invention further includes a method for using an antimicrobial peptide that includes covalently linking a stabilizing group, as described hereinabove, to the N-terminus, the C-terminus, or to both termini, to yield a stabilized antimicrobial peptide, then contacting a microbe with the stabilized antimicrobial peptide. Alternatively, the stabilized antimicrobial peptide used in this aspect of the invention is made by covalently linking oppositely charged regions, as described hereinabove, to each end of the antimicrobial peptide to form an opposite charge ending motif. An antimicrobial peptide is to be broadly understood as including any bioactive peptide that adversely affects a microbe such as a bacterium, virus, protozoan, or the like, as described in more detail hereinabove. An example of an antimicrobial peptide is an inhibitory peptide that inhibits the growth of a microbe. When the antimicrobial peptide is covalently linked to a stabilizing group at only one peptide terminus, any of the stabilizing groups described hereinabove can be utilized. When the antimicrobial peptide is covalently linked to a stabilizing group at both peptide termini, the method includes covalently linking a first stabilizing group to the N-terminus of the antimicrobial peptide and a second stabilizing group to the C-terminus of the antimicrobial peptide, where the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. In a preferred embodiment of the method for using an antimicrobial peptide, one or more prolines, more preferably a proline-proline dipeptide, is attached to at least one, preferably both, termini of the antimicrobial peptide. Alternatively, or in addition, an Xaa-Pro- or an Xaa-Pro-Pro sequence can be attached to the N-terminus of a microbial peptide, and/or a Pro-Xaa or a Pro-Pro-Xaa sequence can be attached to the C-terminus, to yield a stabilized antimicrobial peptide.

The antimicrobial peptide thus modified in accordance with the invention has enhanced stability in the intracellular environment relative to an unmodified antimicrobial peptide. As noted earlier, the unmodified peptide can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm, if desired, that the modified peptide exhibits increased stability. Further, the antimicrobial activity of the antimicrobial peptide is preferably preserved or enhanced in the modified antimicrobial peptide; modifications that reduce or eliminate the antimicrobial activity of the antimicrobial peptide are easily detected and are to be avoided.

The invention further provides a method for inhibiting the growth of a microbe comprising contacting the microbe with a stabilized inhibitory peptide. In one embodiment of this aspect of the invention, the stabilized inhibitory peptide has a stabilizing group at the N-terminus, the C-terminus, or to both. Preferably, the inhibitory peptide has a first stabilizing group comprising the N-terminus of the inhibitory peptide, and a second stabilizing group comprising the C-terminus of the inhibitory peptide; the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. In another embodiment of this aspect of the invention, the inhibitory peptide is stabilized by the addition of oppositely charged regions to each end to form an opposite charge ending motif, as described hereinabove.

Also included in the present invention is a method for treating a patient having a condition treatable with a peptide drug, comprising administering to the patient a peptide drug that has been stabilized as described herein. Peptide drugs for use in therapeutic treatments are well-known (see Table 1). However, they are often easily degraded in biological systems, which affects their efficacy. In on embodiment of the present method, the patient is treated with a stabilized drug comprising the peptide drug of choice and a stabilizing group attached at either the N-terminus, the C-terminus of, or at both termini of the peptide drug. In another embodiment of the present method, the patient is treated with a stabilized drug comprising the peptide drug of choice and stabilized by attachment of oppositely charged regions to both termini of the peptide drug. Because the peptide drug is thereby stabilized against proteolytic degradation, greater amounts of the drug should reach the intended target in the patient.

In embodiments of the method involving administration of a peptide drug that is covalently linked to a stabilizing group at only one peptide terminus, the stabilizing group is preferably a four-helix bundle protein such as Rop protein, provided that attachment of the four-helix bundle protein to the peptide terminus preserves a sufficient amount of efficacy for the drug. It is to be nonetheless understood that group or groups used to stabilize the peptide drug are as defined hereinabove, without limitation. In embodiments involving administration of a peptide drug covalently linked to a stabilizing group at both peptide termini, the peptide drug includes a first stabilizing group comprising the N-terminus of the peptide drug and a second stabilizing group comprising the C-terminus of the peptide drug. Thus, in another preferred embodiment of the treatment method of the invention, the stabilized peptide drug comprises one or more prolines, more preferably a proline-proline dipeptide, attached to one or both termini of the peptide drug. For example, the peptide drug can be stabilized by covalent attachment of a Rop protein at one terminus, and by a proline or proline dipeptide at the other terminus; in another preferred embodiment, the peptide drug can be stabilized by proline dipeptides at each of the N-terminus and C-terminus. Alternatively, or in addition, the stabilized peptide drug used in the treatment method comprises an Xaa-Pro- or an Xaa-Pro-Pro- sequence at the N-terminus of the peptide drug, and/or a -Pro-Xaa or a -Pro-Pro-Xaa sequence at the C-terminus. Optionally, prior to administering the stabilized peptide drug, the treatment method can include a step comprising covalently linking a stabilizing group to one or both termini of the peptide drug to yield the stabilized peptide drug.

If desired, the unmodified peptide drug can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm that the stabilized peptide drug exhibits increased stability. Further, the therapeutic efficacy of the peptide drug is preferably preserved or enhanced in the stabilized peptide drug; modifications that reduce or eliminate the therapeutic efficacy of the peptide drug are easily detected and are to be avoided.

The present invention further includes a fusion protein comprising a four-helix bundle protein, preferably Rop protein, and a polypeptide. Preferably the polypeptide is bioactive; more preferably it is a bioactive peptide. The fusion protein of the invention can be used in any convenient expression vector known in the art for expression or overexpression of a peptide or protein of interest. Optionally, a cleavage site is present between four helix bundle protein and the polypeptide to allow cleavage, isolation and purification of the polypeptide. In one embodiment of the fusion protein, the four helix bundle protein is covalently linked at its C-terminus to the N-terminus of the polypeptide; in an alternative embodiment, the four helix bundle protein is covalently linked at its N-terminus to the C-terminus of the polypeptide. Fusion proteins of the invention, and expression vectors comprising nucleic acid sequences encoding fusion proteins wherein the nucleic acid sequences are operably linked to a regulatory control element such as a promoter, are useful for producing or overproducing any peptide or protein of interest.
 

Claim 1 of 22 Claims

1. A method for making a protease resistant bioactive peptide comprising: attaching a first stabilizing group to the N-terminus of a bioactive peptide; and attaching a second stabilizing group to the C-terminus of the bioactive peptide to yield the protease resistant bioactive peptide; wherein the first stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase, Pro-, Pro-pro-, Xaa-Pro- and Xaa-Pro-Pro-, and wherein the second stabilizing group is selected from the group consisting of a small stable protein selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase, -Pro, -Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa, wherein Xaa is any amino acid residue.

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