Note: Descriptions are shown in the official language in which they were submitted.
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PAREVINS AND TACHYTEGRINS
This invention was made with fi~nding from NIH Grant No. A122839. The U.S.
Government has certain rights in this invention.
Techni~l Field
The invention relates to the field of a,~Lil,i~,lic peptides. In particular, the invention
concerns short peptides with unique patterns of cysteine type residues and cor~llllaLions that
have a wide range of ~ntim:crobial activities.
Bacl~:loulld Art
One of the defense m~c.~l~ni~m~ against infection by both animals and plants is the
production of peptides that have ~ntimirrobial and antiviral activity. Various classes of these
peptides have been isolated from tissues of both plants and ~nim~l~ PCT application
WO 95/03325 published 2 February 1995 contains a review ofthe li~e.~lul~ on this subject.
Such peptides inc!ude tachyplesins, which are 17-18 amino acid peptides co.,~ g four
invariant cysteines, the d~re,-si--s, ~-d~:re--sins, and insect der~ sins, which are somewhat
longer peptides characterized by six invariant ~,y~,L~illes, and antifi~ng~l and ~ntibacterial
peptides and proteins which have been found in plants.
The applications in the series of which WO 95/03325 is a part provide a new class of
antimicrobial and antiviral peptides, deei~n~ted "protegrins", representative members of ~,vhich
have been isolated from porcine leukocytes. These peptides are useful as antibacterial antiviral
and ~ntifi-ng~l agents in both plants and ~nim~lc
The isolation of some of the protegrin peptides was reported in a paper by Kokryakov,
V.N. et al. FEBS (1993) 337:231-236 (July issue). A later publication described the presence
of a new protegrin, whose sequence and that of its ple~iul~or were ded~lced from its isolated
cDNA clone. Zhao, C et al, FEBSLetters (1994) 346:285-288. An additional paper
disclosing cationic peptides from porcine neutrophils was published by Mirgorodskaya, O.A.
et al. FEBS (1993) 330:339-342. Storici, P. et al. Biochem Biophys Res Comm (1993)
196: 1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte
antirnicrobial peptide with a cathelin-like prosequence. The peptide is reported to be one of
the protegrins. ~ 1ition~1 publications related to pLotegrills are Harwig, S. S.L., et al. J
Peptide Sci (1995) in press; Zhao, C., et al. FEBS Lett (1995) 376: 130-134; Zhao, C. et al.
FEBS Lett (1995) 368: 197-202.
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The protegrins have also been found to bind to endotoxins -- i.e., the
lipopolysaccharide (LPS) compositions derived from gram-negative bacteria which are
believed .t;~on~;l,le for gram-negative sepsis. The protegrins are also effective in hlhibiLi.lg
the growth of or~ni~mx that are associated with sexually ll~ led ~ P~es such as
Chlamydia t,.lc~lv".atis and Neisseria g~,~.J"hoeae.
Protegrins are also effective against the microolg~ a~sori~ted with oral mucositis,
a signific~nt side effect of cancer therapy and bone marrow transplantation that is not
adequately m~n~gecl by current approaches (Sonis, S.T. In. J.L. Holland et al. Cancer
Medicine~ 3rd ed. Lea and Febiger, Philadelphia (1993a) pp. 2381-2388; Sonis, S.T. In: V.
DeVitta et al. (ed.), Principles and Practice of Oncologv. J.B. Lip~,hlcoLL, Phil~-lçlrhi~
(1993b) pp. 2385-2394). Oral mucositis is ;..;~ ecl by the cytotoxic effects of these therapies
on the rapidly dividing epitheli~l cells ofthe olophhlyngeal mucosa, and is exacerbated by
infection with both endogenous oral flora and opportunistic bacterial and fungal pathogens.
Oral mucositis is associated with extreme discon~l L and pain, especially when eating. In the
case of head and neck cancers, oral mucositis is frequently a dose limiting toxicity causing
delay in the completion of the therapeutic regimen. Such a delay in trç~tment can have a
negative impact on the final therapeutic outcome. Tnfectecl oral lesions are also increasingly
recognized as a ~i~nific~nt portal of entry of microolg~l-ix...~ to the immnnosupplessed patient
rçslllting in sepsis (Sonis, 1993 a,b, supra).
The invention described below relates to peptide type compounds that are related to
the protegrins described above, but reflect displ~ce. . .~ of the protegrin cysteines at positions
6 and 15. The availability ofthese compounds, the ple~"ed forms of which are clecign~te~
parevins and tachytegrins, ç~r~n~ls the lept;,Lc,ile of antimicrobial peptides and permits more
exquisite m~tching of inrlic~tion~ to antimicrobial formulations. Although at least one of C4 ',
C5, C 16 or C 17 in the formula set forth below must be cysteine, the common name terminlogy
of these components reflects particularly perferred situations wherein both of C4 and Cl7 are
cysteine type residues (the tacllyleglins) or where both C 5 and C 16 are cysteine type residues
(the parevins).
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Disclosure of the Invention
The invention provides compounds which retain generally the antimicrobial activity of
the ploLe~ ,s ~ c~lqse~ above, but differ in conrolllldlion due to the dislocation ofthe
cysteine residues at positions 6 and/or 15 of these protegrins. Surprisingly, these modified
compounds exhibit activity spectra which are analogous to those ofthe plol~,ins, but offer
the oppo~tunity to fine-tune the biological activity of antibiotics and antivirals. All of these
peptides can be produced synthetic~lly and those that contain only gene-encoded amino acids
can also be produced reco~ A~ y. These compounds are usefi~l as preservatives or in
pharm~celltic~l compositions in treating or preventing infection in anim~l~ Alternatively, the
peptides can be form~ ted into compositions which can be applied to plants to protect them
against viral or rnicrobial infection. In still another approach, the DNA encoding the peptides
can be expressed in situ, in animals or plt;Çt;,~bly in plants, to combat infections. The peptides
are also useful as standards in antimicrobial assays and in binding endotoxins.
Accoldingly, in one aspect, the invention is directed to a purified and isolated or
;collll)inhlllly or synthetically produced compound which contains the amino acid sequence
Al-A2-A3-C-C~C~A7-C8-Ag-AIo-Al ,-A,2-C,3-A,4-C~5-C~6-C~;-A,8 ( 1 )
said compound co~ g 11-24 amino acid residues. The sequence shown as (I) can
be e~ctçndecl at the N and/or C terminus with non-intelrelling amino acids or sequence.
The compounds also include the N-terminal acylated and/or C-terminal ami~l~ted or
esterified forms and may be either in the, optionally -SH stabilized, linear or in a tli~ fi~e-
bridged forrn.
In the amino acid seq~l~nt~e shown, each of Al-A3 is independently present or not
present, and if present each is independently a basic, hydlophobic, polar/large, or small amino
acid;
each of C4*, C5 *, C6*, C,5*, C,6* and Cl7* is independently cysteine, homocysteine or
p~nicill~min~ or a basic, hydrophobic, polar/large, or small amino acid, and C4* and/or Cl~*
may be present or not pleselll, C6* and/or Cl5* may also be acidic;
each of C8 and Cl3 is independently cysteine, homocysteine or pPniç~ mine;
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each of A~ and Al4 is in~epen-l~.ntly a hydrophobic or a small amino acid;
Ag-Al2 must be capable of ~jl ;..g a 13-turn when contained in the compound and at
Ieast one of Ag-Al2 must be a basic amino acid;
Al8 is present or not present, and if present, is a basic, hydrophobic, polar/large or
small amino acid.
The compounds of the invention may, in the alternative, contain a lnodified form of
formula (1) wherein one or both of Cg and Cl3 is indep~n-lently replaced by a basic,
hydrophobic, polar/large, acidic, or small amino acid.
In all of the compounds of the invention at least about 15% and no more than about
50% of the amino acids must be basic amino acids, and the compounds must have a net charge
of +1 at physiological pH;
with the proviso that at least one of C~ 6 arld C~; must be cysteine, homocysteine
or p~nicill~mine; and
only one of C4, C5, and C6, and only one of Cl5, Cl6 and Cl, can be cysteine,
homocysteine or penicill~mine.
A particular advantage of some of the peptides of the invention, especially those which
contain fewer amino acids, lies in their reduced size. As a result of this, they are less costly to
produce, generally are expected to provide better distribution in tissue, and are less
imml~nogenic. As they provide alternative structures, they are likely to have di~e
pharmacokinetic and toxicological profiles.
In still other aspects, the invention is directed to reco-,-bi.la..l materials useful for the
production of the peptides of the invention as well as plants or animals modified to contain
~.es~ion systems for the production ofthese peptides. The invention is also directed to
pharm~celltic~l compositions and compositions for application to plants co..~ ;..g the
peptides of the invention as active ingredients or compositions which contain c~l~-ession
systems for production of the peptides or for in si~u ~ es~ion of the nucleotide sequence
encoding these peptides. The invention is also directed to methods to prepare the invention
peptides synthetic~lly~ to antibodies specific for these peptides, and to the use ofthe peptides
as preservatives.
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Specific infections against which some of the peptides, especially the parevins and
tachytegrins are particularly effective are those associated with oral mucositis, infections, such
as stomach ulcers associated with H. pylori, and infections caused by Pseudomonas or MRSA.
In other aspects, the invention is directed to the use of the compounds of the invention
as ~Lan~alds in antimicrobial assays. The compounds many also be used as antirnicrobials in
solutions useful in eye care, such as contact lens solutions, and in topical or other
pharns~sce~lfic~sl compositions for tre~stmpnt of sexually L~ ed t~i~ç~eps (STDs). The
invention is also directed to use of the invention compounds as preservatives for foods or
other perishables. As the invention peptides can inactivate endotoxin, the invention is also
directed to a method to inactivate endotoxins using the compounds of the invention and to
treat gram-negative sepsis by taking advantage of this pl opc:l ~y.
Brief Description of the Drawin~s
Figure 1 shows antibacterial activity of two of the parevins against E. Coli;
Figure 2 shows ~sntibactPrial activity of two of the parevins against Listeria
monocytogenes;
Figure 3 shows antifi-ng~l activity of two of the parevins against Candida albicans;
Figure 4 shows ~ntihacterial activity of a tachytegrin against E. coli.
Figure 5 shows ~nfil~actPrial activity of a tachytegrin against ~. subtilis.
Figure 6 shows ,snti~acterial activity of a tachytegrin against S. typhimurium.
Modes of Carrying Out the Invention
The peptides of the invention are characterized by the amino acid sequence:
Al-A2-A3-C4 -C5 -C6 -A7-c8-A9-Alo-All-Al2-cl3-Al4-cl5 -Cl6 -Cl7 -Al8- (1)
and its defined modified forms. Any of these peptides which may coin-;ident~lly occur
in nature must be in purified and isolated form or plep~ed recombinantly or synthetically.
The dç~i~n~tiQn An in each case ,eplese-lls an amino acid at the specified position in
the peptide. As defined Al-A3~ C4, Cl7 and/or Al8 may or may not be present. However,
the peptides of the invention contain 1 1-24 amino acids. Thus, the sequence sho wn as ( 1 ) can
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be extended at the N and/or C terrninus with non~ Lc-rcl~ing amino acids or sequence. The
positions ofthe cysteine, homocysteine or pçnirill~minl? r~idtlee, shown as C in formula (1),
are invariant in one embodiment of the peptides of the invention; however, in the modified
forms ofthe peptides co~ g the sequence offormula (1), also inclnded within the scope of
the invention, one or more of these cysteines may be replaced by a small, basic acidic or
hydlophobic amino acid. However, at least one of C, C~ C~ and (~; must be cysteine,
homocysteine peniçill~min~
All of the peptides of the invention, however, have a net positive charge of at least + 1
at physiological pH; appl~J~ill.alely 15%-50% ofthe amino acid residues in~l~lded in the
sequence should be basic. For embo~imente having as few as 11 amino acids, there may be
only one basic amino acid residue; however, at least two basic resid~çs, even in this short-
chain residue, are pl crel I ed. If the peptide contains as many as 15 amino acid residues, two
basic residues are required. It is plcrcl I cd that at least 20% of the amino acids in the sequence
be basic, more preferably 30%, but not more than 50%.
The active peptides also preferably contain a ~ turn bracketed by two strands that form
a ,~ sheet. While not intP.n~ling to be bound by any theory, applicants believe that antimicrobial
activity ofthe compounds co..~;";..g the sequence offormula (1) is associated with such a ~-
turn bracketed by two strands that form a ,~ sheet structure. The amino acids A9-AI2 must be
capable of e~ g a 13 turn, which can be encouraged by the cystine bond between C8 and
Cl3 as well as by hydrogen bonding between Ag and Al2. The presence of proline at Alo
and/or All does not interfere with the ~-tum stabilized by the presence of a hydrophobic
amino acid at positions Ag or Al2.
As used herein, "~-turn" refers to a recognized sub-class of reverse-turns. Typically, a
",~-turn" is a four amino acid residue peptide segm~nt that reverses the direction of a
polypeptide chain so as to allow a single polypeptide chain to adopt an anti-parallel ,13-sheet
secondary structure. Generally, the two internal amino acid residues of the ,B-turn are not ~
involved in the hydrogen-bonding of the ~-sheet; the two amino acid residues on either side of
the internal residues are inclllded in the hydrogen-bonding ofthe 13-sheet. The terrn "~-turn"
C~.~JI cs~ly incllldes all types of peptide ,13-turns commonly known in the art inci~ ing~ but not
limited to, type-I, type-II, type-III, type-I', type-II', and type-III' ,~-turns (see, Rose ef al.,
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1985, Adv. Protein Chem. 37: 1-109; Wilmer-White et al., 1987, Trends Biochem. Sci.
12- 189 192; Wilmot et al., 1988, J. Mol. Biol. 206:759-777; Tr~mont~no et al., 1989,
o~eins; Struct. Funct. Genet. 6:382-394).
The presence ofthe four invariant cysteines ofthe proteg~ s or ofthe C8 and Cl3
cysteines, homocysteine or peniç~ mine of the compounds of the present invention is helpful
in e~e~ g the 13-turn co,~""a~ion; however, by p,o~e,ly choosing the s~bstit~ltions~ one or
both of the cysteine, homocysteine or penirill~mine residues at C8 or Cl3 can be replaced
~,vithout subst~nti~lly disturbing the three-dimensional shape of the molecule.
The ,B sheets are believed to be ~ffected by the sequences surrounding C8 and Cl3, and
are inclusive of these residues. Thus, in the unmodified forms of the compound, A, and Al4
are preferably hydrophobic amino acids. The cysteine residues may also, then, be replaced by
other residues which do not affect the m~inten~nce of the ,~ sheet formation; these
substitutions would include acidic, basic, hydrophobic polar or small amino acids.
The amino terminus of the peptide may be in the free amino form or may be acylated
by a group ofthe formula RCO-, wherein R lep,ese,lls a hydrocarbyl group of 1-6C. The
hydrocarbyl group is saturated or unsaturated and is typically, for example, methyl, ethyl,
i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, and the
like.
The C-terminus of the peptides of the invention may be in the form of the
underivatized carboxyl group, either as the free acid or an acceptable salt, such as the
pot~eeil~m sodium, calcium, m~n~eillm, or other salt of an inorganic ion or of an organic ion
such as caffeine. In some embo~1imçnts7 it is difficult to make salts since the rçm~inrler of the
molecule bears a positive charge which may repel the relevant cation. The carboxyl terminus
may also be derivatized by formation of an ester with an alcohol of the formula ROH, or may
be ~mid~ted by an amine of the formula NH3, or RNH2, or R2NH, wherein each R is
independently hydrocarbyl of 1-6C as defined above. Amidated forms of the peptides wherein
the C-terminus has the formula CONH2 are pl~re"t;d.
As the peptides of the invention contain substantial numbers of basic amino acids, the
peptides of the invention may be supplied in the form of the acid addition salts. Typical acid
addition salts include those of i"o,~ a,fic ions such as chloride, bromide, iodide, fluoride or the
SU85TITUTE SHEET (RULE 26)
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like, sulfate, nitrate, or phosphate, or may be salts of organic anions such as acetate, formate,
bel~zoale and the like. The acceptability of each of such salts is dependent on the intçnded
use, as is co~ lolllyunderstood.
The peptides of the invention that contain at least two cysteines, homocysteine or
pPnir.ill~minç may be in straight-chain or cyclic form. The straight-chain forms are convertible
to the cyclic forms, and vice versa. Methods for rulll~il~ ~ielllfide bonds to create the cyclic
peptides are well known in the art, as are methods to reduce di.elllfides to form the linear
compounds. The linear compounds can be stabilized by addition of a suitable alkylating agent
such as iodo~cet~mide.
The cyclic forms are the result of the formation of die~llfide link~ges among all or some
of the four cysteine, homocysteine or p~n:~ill~mine residues that may be present. Cyclic forms
of the invention include all possible permutations of ~liel~lfi(le bond formation; if the -SH
co."~il.;..g amino acids are numbered in order oftheir occurrence starting at the N-terminus as
C6 C8, Cl3, C~6, (~; or ~8, these permutations include, when two ~lielllfid~os are present:
a) C4-CI7 and C8-CI3;
b) C4-C~6 and C8-CI3;
c) C4-CIs and C8-CI3;
d) C5-C~7 and C8-CI3,
e) C5-CI6 and C8-CI3;
f) C5-CI5 and C8-CI3;
g) C6-CI7 and C8-CI3;
h) C6-CI6 and C8-CI3;
i) C4-C8 and Cl3-C17;
j) C4-C8 and C,3-CI6;
k) C5-C8 and Cl3-CI7; and
1) C5-C8 and Cl3-CI6;
When one fli.elllfide is present, these pelllluL~Lions include:
C4-CI7;
C4-CI6;
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C4-CI5;
C5-C I7;
C5-C16;
C5-CI5;
C6-C 17;
C6-C16;
C8-CI3;
C4-C8;
C5-C8;
Cl3-CI7; and
Cl3-CI6.
In the modified forms of the peptides, where 1 or 2 ~;y~leilles, homoey~Lei"e orp~nicill~mine are replaced, similar permutations are available as in when 2-3 cysteines,
homocysteine or p~n~ mine are present.
The linearalized forms of the native cyclic peptides have valuable activities, even when
chemically stabilized to preserve the sulfhydryl form of cysteine, homocysteine or
penicillamine for example, by reaction with iodo~cet~mide. The compounds of the invention
also include linearalized forms which are stabilized with suitable reagents. As defined herein,
"SH-stabilized" forms ofthe peptides ofthe invention contain sulfhydryl groups reacted with
standard reagents to prevent lerc~ ion into di~--lfide linkages.
An alternative approach to providing linear forms of the invention compounds
comprises use of the ~odifie~ form of the peptides where residues at Cg and/or Cl3 are
replaced by amino acia~s which do not form cystine linkages, in cc,ml,inaLion with st~bili7.~tion
of any cysteine, homocysteine or peniçill~mine residues at C~ or C~and/or (~, C~, C~;.
Forms of the invention compounds which have only one ~ fide bond are
conveniently obtained by replacing the cysteine, homocysteine or p~nicill~mine residues at C8
and/or Cl3, preferably both, with amino acids which do not form di~llfide link~ges
The amino acids denoted by An may be those encoded by the gene or analogs thereof,
and may also be the D-isomers thereof. One pler~"ed embodiment of the peptides of the
invention is that form whel eill all of the residues are in the D-configuration thus co~" hlg
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resi~t~nee to protease activity while ~ ntimicrobial or antiviral propt;,Lies. The
resulting peptides are enantiomers ofthe native L-amino acid-co..~ g forrns.
In one class of peptides described herein, either one or both of the residues found at
C5 and/or C,6 is a basic amino acid and/or at least one of Al-A3 and C4 iS hydrophobic
and/or at least one, and pler~l~ly all four ofthese amino acids are deleted. By suitable
m~nipul~ti~n of these and other features, the range of con~litions under which the class of
peptides ofthe present invention are effective can be varied. F~ llnole, the s~e.illul~, of
microbes against which they are effective can also be modified. This is further described
hereinbelow.
The amino acid notations used herein are conventional and are as follows:
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One-Letter Three Letter
Amino AcidSymbol Symbol
Alanine A Ala
Argilune R Arg
~ J~ ;"r. N Asn
Aspartic acid D Asp
Cysteine C Cys
.... ;.. e Q Gln
t'rl~t~mic acid E Glu
Glycine G Gly
IIi~li~LR H His
i~olc~lrin~ I lle
Leucine L Leu
Lysine K Lys
~cthinnine M Met
Phenylalanine F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Tly~Jtc~h~ W Trp
Tyrosine Y Tyr
Valine V Val
The amino acids not encoded genetically are abbreviated as indicated in the discussion
below.
In the specific peptides shown in the present application, the L-form of any amino acid
residue having an optical isomer is intçn-le~i unless the D-form is expressly indicated by a
dagger sul)e~,lipl (t)
The compounds of the invention are peptides which are partially defined in terrns of
amino acid residues of de~ign~ted classes. Amino acid residues can be generally subclassified
into major subclasses as follows:
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Acidic: The residue has a negative charge due to loss of H ion at physiological
pH and the residue is attracted by aqueous solution so as to seek the surface positions in the
co"ru""alion of a peptide in which it is co~ d when the peptide is in aqueous mç~ m at
physiological pH.
Basic: The residue has a positive charge due to association with H ion at
physiological pH and the residue is attracted by aqueous solution so as to seek the surface
positions in the co"ru"llalion of a peptide in which it is contained when the peptide is in
aqueous merlil-m at physiological pH.
Hydrophobic: The residues are not charged at physiological pH and the
residue is repelled by aqueous solution so as to seek the inner positions in the co,~l",ation of
a peptide in which it is col~ ed when the peptide is in aqueous me~ m
Polar/large: The residues are not charged at physiolûgical pH, but the residue
is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the
col~ll"alion of a peptide in which it is conlai"ed when the peptide is in aqueous mP~illm
This description also characterizes certain neutral amino acids as "small" since their
side chains are not sufficiently large, even if polar groups are l~rL in~ to confer
hydrophobicity. "Small" amino acids are those with four carbons or less when at least one
polar group is on the side chain and three carbons or less when not.
It is understood, of course, that in a st~ti~t~ collection of individual residuemolecules some molecules will be charged, and some not, and there will be an attraction for or
repulsion from an aqueous merlillm to a greater or lesser extent. To fit the definition of
"charged," a ~ignific~nt percentage (at least a~)pro~;...~tçly 25%) of the individual molecules
are charged at physiological pH. The degree of attraction or repulsion required for
rl~.cific~ti~ n as polar or nonpolar is ~bill~y and, therefore, amino acids specifically
colllelllplated by the invention have been classified as one or the other. Most amino acids not
specifically named can be çl~sified on the basis of known behavior.
Amino acid residues can be further s~b~ ified as cyclic or noncyclic, and aromatic or
nonalû",aLic, self-explanatory çl~c.cific~fions with respect to the side-chain substituent groups
of the rç~ ç~, and as small or large. The residue is con~idçred small if it contains a total of
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four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar
~ substituent is plesellL, three or less if not. Small residues are, of course, always non~u~aLic.
For the naturally occurring protein amino acids, sub~l~.c.~ifi~tiQn acco,-lh~g to the
rO~goil~g scheme is as follows.
Acidic Aspartic acid and Glutamic acid
Basic l'~l~,y~,lic. A~ginine, Lysine
Cyclic: T-Tictit1in.o
Small Glycine, Serine, Alanine, Threonine
Po~ arge ~ TI-~
Hy~ G Tyrosine, Valine, T! ~ Leucine,
Pl~ c~ T.,~
The gene-encoded secondary amino acid proline is a special case due to its knowneffects on the seCon~ry co"ru...,ation of peptide chains, and is not, the~ro,e, in~ ded in a
group. Cysteine and other -SH co.~l~;..;ng amino acid residues are also not in~ ded in these
çl~ceific~tions since their capacity to form ~liclllfi~e bonds to provide secondary structure is
critical in the compounds ûf the present invention.
Certain cornrnonly encountered arnino acids, which are not encoded by the genetic
code, include, for example, 13-Alanine (~-Ala), or other omega-amino acids, such as
3-aminopropionic, 2,3-~ minopropionic (2,3-diaP), 4-aminobutyric and so forth,
a-aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), t-butylalanine
(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and
cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine (2-Nal); 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (Tic); J-2-thienylalanine (Thi); methionine sulfoxide
(MSO); and homoa-~inine (Har). These also fall conveniently into particular categories.
Based on the above clçfinitions,
Sar, ~-AIa, and Aib are small,
t-BuA, t-BuG, N-MeIle, Me, Mvl, Cha, Phg, Nal, Thi and Tic are
hydrophobic;
Orn, 2,3-diaP and Har are basic;
Cit, Acetyl Lys, and MSO are polar/large.
SUBSTITUTE 5H EET (RULE 26)
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The various omega-amino acids are classified accoldil~, to size as small (,B-Ala and
3-all.i.,o~ )ionic) or as large and L~dlc"ohobic (all others).
Other amino acid substitutions ofthose encoded in the gene can also be inrllldrd in
peptide compounds within the scope of the invention and can be çl~c~ifie(l within this general
scheme accol~lh~g to their structure.
In all of the peptides of the invention, one or more amide link~es (-CO-NH-) mayoptionally be replaced with another linkage which is an isostere such as -CH2NH-, -CH2S-,
-CH2CH2, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2- and -CH2SO-. This
repl~cçm~nt can be made by methods known in the art. The following references describe
ple;~al~LiOn of peptide analogs which include these alternative-linking m~ie~ies Spatola, A.F.,
Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general
review); Spatola, A.F., in ChellllsLly and Bioçh~mistry of Arnino Acids Peptides and Proteins.
B. Wein~t~in~ eds., Marcel Dekker, New York, p. 267 (1983) (general review); Morley, J.S.,
Trends Pharm Sci ( 1980) pp. 463-468 (general review); Hudson, D., et al., Int JPept Prot
Res (1979) 14:177-185 (-CH2NH-, -CH2CH2-); Spatola, A.F., etal., Life Sci (1986) 38:1243-
1249 (-CH2-S); Hann, M.M., J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH-, cis and
trans); Almquist, R.G., etal., JMed Chem (1980) 23:1392-1398 (-COCH2-); Jennings-White,
C., et aL, Tetrahedron Lett (1982) 23:2533 (-COCH2-); Szelke, M., et al., European
Application EP 45665 (1982) CA:97:39405 (1982) (-CH(OH)CH2-); Holladay, M.W., et al.,
Tetrahedron Lett (1983) 24:4401-4404 (-C(OH)CH2-); and Hruby, V.J., Life Sci (1982)
UB:189-199 ~-CH2-S-).
In addition to analogs which contain isosteres in place of peptide linkages, the peptides
or proteins of the invention include peptide mimetics in general, such as those described by
Olson, G.L. etal. JMed Chem (1993) 36:3039-3049 and retro-inverso type peptides as
described by Chorev, M. et al. Science (1979) 204: 1210-1212; and Pallai, P.V. et al., Int J
Pept Protein Res (1983) 21 :84-92.
One class of plere-,~d embo~limentc ofthe compounds invention includes the
"unrnodified" forms where positions 8 and 13 are independently cysteine, homocysteine or
penirill~mine residues, especially in the r~ lfide bonded form.
SUBSTITUTE SHEET (RULE ~6)
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In addition, or alternatively, each of A7 and Al4is a hydl ophobic acid, p,ere.~bly Ile,
Val, Leu, NLe, Trp, Tyr or Phe, or is a small amino acid, Ala, Gly, Ser or Thr.
In another set of plGrelled embot~im~nts, all of Al-A3 are not present or at least one,
and preferably two of Al-A3 is a hydlophol~ic amino acid, preferably Ile, Val, Leu, NLe, Trp,
Tyr or Phe.
In another set of plerélled embo~im~nt~C4 and/or C1, is not present or, if present, is
a cysteine, homocysteine or p~nicill~mine or a llydlophobic amino acid, plërel~bly Ile, Val,
Leu,.NLe, Trp, Tyr or Phe, or a small amino acid, preferably S, A, G or T.
In another set of plerélled embo-limPnt~, C5$ and/or Cl6* is a cysteine, homocysteine
or p~nic.ill~mine or a hydrophobic amino acid, preferably Ile, Val, Leu,.NLe, Trp, Tyr or Phe,
or a small amino acid, plere.~bly S, A, G or T.
In another set of plerelled embo~1im~nte7 Ag-Al2 contain at least one hydlOphobic
amino acid residue, plerel~bly Phe, Tyr or Trp.
Other pleÇelled embodiments include those wherein each of Al and A9isindepPn(lently
selected from the group consisting of R, K and Har; more preferably, both Al and A9 are R;
however, each of Al may be absent.
In another class of p~ert:--ed embo-iim~nte, each of A2 and A3is independently selected
from the group consisting of G, A,S and T; more preferably, A2 and A3 are G; however, A2
and/or A3 may be absent.
In another set of plerellèd embodim~nte, one of A9 and Al2 is R, K, Har, Orn or H
plerel~bly R and the other is I, V, L, NLe, W, Y or F, preferably R, F or W, or is S, G, A or
T.
In another set of plefelled embo~1im~nt~ each of Alo and All is independently proline
or a small, basic or hydrophobic amino acid, preferably R, G, W or P.
Al8 is preferably absent, but when present, is preferably E~, K or Har, most preferably
R.
Also preferably when all four amino acids Al-A3 and C4 are present, Alis basic, C4 iS
C or basic, and A2 and A3 are small amino acids, or at least one of Al-A3 and C4 iS
Lydlophobic. ~lèrelled embodiments of Al-A3 include R-G-G, K-G-S, K-S-G, and the like.
As described above, the compounds of Formula (1 ) are either in cyclic or noncyclic
SUBSTITUTE SH EET (RULE 26)
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16
(linearalized) form or may be modified wherein 1 or 2 of the cysteines, homocysteine or
p~nir.ill~mine at C8 and Cl3 are replaced by a small, hydrophobic, or a basic amino acid
residue. Such modification is plert;~ d when compounds con~ ;..g only one ~iisulfide bond
are prepared. If the linearalized forms ofthe compound of Forrnula (1) are prep~ed, or if
linearalized forms of those modified peptides which contain at least two cysteines are
pl ~ ~d, it is pl c;rt;llt;d that the sulfhydryl groups be stabilized by addition of a suitable
reagent. Plerellt;d embodim~nt~ for the hydrophobic amino acid to replace cysteine,
homocysteine or p~nirill~min~ residues at C8 and/or C13 are I, V, L and Me, plerel~bly I, V or
L. Preferred small amino acids to replace the cysteine, homocysteine or p~ni~.ill~mine residues
include G, A, S and T, more preferably G. Pl~rt;ll~;d basic amino acids are R and K.
Where the compounds of the invention have two ~ lfide bridges, particularly
pler~lled are the pairs of bridges:
a) C5-C16 and C8-Cl3;
b) C5-C17 and C8-CI3;
C) C5-C8 and Cl3-CI6;
d) C4-CI6 and C8-CI3; and
e) C4-Cl7 and C8-CI3.
F.speçi~lly pl~rt;lled are the bridges:
C5-CI6 and C8-CI3; and
C4-CI7 and C8-CI3.
Where the compound has only one di~l~lfide bridge, particularly plefelled are:
C4-CI7and C5-CI6.
Particularly plt;rt;lled compounds ofthe invention, incl~in~ the N-terminal acylated
and C-terminal ~mi~l~ted forms thereof are the parevins, wherein C~and C~6 are both cysteine,
homocysteine or penic~ mine and the tachytegrins wherein both C;and (:~; are cysteine,
homocysteine or peni~ min~o Also prerelled are the di~ llfide forms ofthese compounds,
cis-parevins wherein the two diel~!fide bridges are C5-C8 and Cl3-CI6; the trans-parevins
wherein the ~lielllfi(le bridges are C5-CI6 and C8-CI3; and the trans-tachytegrins wherein the
ll1fide bridges are C4-CI7 and C8-Cl3. Particularly prt;~:llc;d are the following parevins and
tachytegrins:
SUBSTITUTE SH EET (RULE 26)
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Parevin-ltrans_ and cis
R-G-G-R-i~ L-Y l-R
R
R
R-G-C-V-V-C-F
L...... l
Parevin-2trans_ and cis
R-G-G-R-l-L-Y l-R
R
R
R-G-C-V-I-C-F
L...... l
Parevin-3trans_ and cis
R-G-G-G-l L-Y-l-R
R
R
R-G-C-V-V-C-F
SUBSTITUTE SHEET (RULE 26)
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Parevin-4 trans_ and cis
R-G-G-R-~ L-Y-~-R
\
R
R-G-C-V-V-C-F
1...... 1
Parevin-S trans_ and cis
R-G-G-R-l-L-Y-~-R
\
R
R-G-C-V-V-C-F
1...... 1
Tachyte~rin- 1 trans_
R-G-G-C-R-L-Y-C-R
R
R
R-C-G-V-V-C-F
SUBSTITUTE SHEET (RULE 26)
. CAo22261211998-01-05
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Tachyte~rin-2 trans_
- R-G-G-C-R-L-Y-C-R
R
R
R-C-G-V-I-C-F
Tachyte~rin-3 trans_
R-G-G-C-G-L-Y-C-R
R
R
R-C-G-V-V-C-F
Tachyte~rin-4 trans_
R-G-G-C-R-L-Y-C-R
G
W
R-C-G-V-F-C-I
Tachyte~rin-S trans_
R-G-G-C-R-L-Y-C-R
p
R
R-C-G-V-V-C-F
Particularly prt;r~,.ed are cis- and trans-parevin-l and trans-tachytegrin-l.
Typical compounds of the invention include:
Unmodified forms
R-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-R-R-F-C-I-V-C-G;
SUBSTITUTE SHEET (RULE 26)
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WO 97/02287
R-G-G-C-R-L-Y-C-R-R-R-F-C-I-V-G-C;
R-G-G-G-C-L-Y-C-R-R-R-F-C-V-V-C-G-R;
R-G-G-C-G-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-G-W-I-C-F-V-C-G-R;
R-G-G-C-R-L-Y-C-R-G-W-I-C-F-V-G-C-R;
R-G-G-R-C-L-Y-C-R-P-R-F-C-V-V-C-G-R;
R-a G-C-R-L-Y-C-R-P-R-F-C-V-V-G-C-R;
R-G-G-R-C-V-Y-C-R-R-R-F-C-V-V-C-G;
R-G-G-C-R-V-Y-C-R-R-R-F-C-V-I-G-C;
K-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G;
K-G-G-C-R-I-Y-C-R-R-R-F-C-V-I-G-C;
R-G-G-Har-C-L-Y-C-R-R-R-F-C-V-V-C;
R-G-G-C-Har-L-Y-C-R-R-R-F-C-V-I-C;
R-G-G-Har-C-L-Y-C -Har-R-R-F-C-V-V-C-G-R;
R-G-G-C-Har-L-Y-C-Har-R-R-F-C-V-I-G-C-R;
R-G-G-R-C-V-Y-C-R-Har-R-F-C-V-V-C-G-R;
R-G-G-C-R-V-Y-C-R-Har-R-F-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-K-K-W-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-K-K-W-C-V-V-G-C-R;
R-G-G-R-C-L-Y-C-R-Har-R-Y-C-V-V-C-G-R;
R-G-G-C -R-L-Y-C-R-Har-R-Y-C-V-V-A-C-R;
R-G-S-G-C-L-Y-C-R-R-K-W-C-V-V-C-G-R;
R-G-S-C-G-L-Y-C-R-R-K-W-C-V-V-G-C-R;
R-A-T-R-C-I-F-C-R-R-R-F-C-V-V-C-G-R;
R-A-T-C-R-I-F-C-R-R-R-F-C-V-I-G-C-R;
R-G-G-K-C-V-Y-C-R-Har-R-F-C-V-V-C-G-R;
R-G-G-C-K-V-Y-C-R-Har-R-F-C -V-I-G-C-R;
R-A-T-R-C-I-F-C-Rt-R-R-F-C-V-V-C-G-Rt;
R-A-T-C-R-I-F-C-Rt-R-R-F-C-V-V-G-C-Rt
R-G-G-K-C-V-Y-C-R-Hart-R-F-C-V-V-C-G-R;
SU8STITUTE SH EET (RULE 26)
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R-G-G-C-K-V-Y-C-R-Hart-R-F-C-V-V-G-C-R;
- R-G-G-R-C-L-Y-C-R-R-R-F-C-V-V-C-G-R (all t);
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-R-R-F-C-I-V-C-G (all t);
R-G-G-C-R-L-Y-C-R-R-R-F-C-I-V-G-C (all t);
R-G-G-G-C-L-Y-C-R-R-R-F-C-V-V-C-G-R (all t);
R-GG-C-G-L-Y-C-R-R-R-F-C-V-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-G-W-I-C-F-V-C-G-R (all t);
R-G-G-C-R-L-Y-C-R-G-W-I-C-F-V-G-C-R (all t);
R-G-G-R-C-L-Y-C-R-P-R-F-C-V-V-C-G-R;
R-G-G-C-R-L-Y-C-R-P-R-F-C -V-V-G-C-R;
R-G-G-C-L-R-Y-C-R-P-R-F-C-V-R-V-C-R
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C-R;
R-G-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-R;
R-G-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-F-R;
R-W-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-L-R;
R-G-W-R-V-C-L-K-Y-C-R-G-R-F-C-V-K-L-C-L-R;
R-G-G-R-V-C-L-R-Y-C-R-G-K-F-C -V-R-L-C-L-R;
both the linear and mono- and bicyclic forms thereof, and incl-l-lin~ the N-terminal
acylated and C-terminal ami(~ted forms;
Particularly pl~re-led are the cyclic forms and C-terminal ~mid~ted forms of
R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C -R
R-G-G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-C-R-P-R-F-C-V-R-V-C-R
R-G-G-C-R-L-Y-C-R-R-R-F-C-V-V-G-C -R;
R-G-V-C-L-R-Y-C-R-G-R-F-C -V-R-L-C-R;
R-G-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C -F-R;
R-W-R-V-C-L-R-Y-C-R-G-R-F-C-V-R-L-C-L-R;
SUBSTIl UTE SHEET (RULE 26)
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R-G-W-R-V-C-L-K-Y-C-R-G-R-F-C-V-Kl-C-L-R;
R-G-G-R-V-C-L-R-Y-C-R-G-K-F-C-V-R-L-C-L-R;
Modified forms
R-G-G-R-C-L-Y-A-R-R-R-F-A-V-V-C-G-R;
R-G-aR-C-L-Y-A-R-R-R-F-S-I-V-C;
R-G-G-G-C-L-Y-S-R-R-R-F-A-V-V-C-G-R;
R-G-G-R-C-L-Y-A-R-R-R-F-G-V-V-C;
K-G-G-R-C-L-Y-V-R-R-R-F-I-V-V-C;
R-G-G-Har-C-L-Y-A-R-R-R-F-V-G-C-V;
R-G-G-Har-C-L-Y-A-Har-R-R-F-S-V-V-C-G-R;
R-G-G-C-Har-L-Y-A-Har-R-R-F-S-V-V-G-C-R;
R-G-G-R-C-V-Y-V-R-Har-R-F-L-V-C-V-G-R;
R-G-G-R-C-L-Y-S-R-K-K-W-A-V-S-C-G-R;
R-G-G-R-C-L-Y-S-R-Har-R-Y-S-V-I-C-G-R;
R-G-S-G-C-I-Y-C-R-R-K-W-G-V-V-aC-R;
R-A-T-R-C-I-F-S-R-R-R-F-S-V-V-C-G-R;
R-G-G-K-C-V-Y-G-R-Har-R-F-S-V-V-C-G-R;
R-A-T-R-C-I-F-G-Rt-R-R-F-G-V-V-C-G-Rt;
R-G-G-K-C-V-Y-L-R-Hart-R-F-L-V-V-C-G-R;
R-G-G-R-C-V-F-L-R-P-R-I-G-V-V-C-G-R;
R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-F -C -L-R-Y-T-V-P-R-F-T-V-R-F-C -V-R;
R-G-F-C-L-R-Y-K-V-GR-F-K-V-R-F-C-V-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG);
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C -R;
SUBSTITUTE SHEET (RULE 26)
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R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X---NMeG)
both the linear and cyclic (where possible) forms thereof, and inclllding the N-terrninal
acylated and C-terminal ~mi~l~ted forms.
Particularly plert;l-ed are the cyclic forms and C-terrninal ~mit~te~ forms of
R-G-G-C-L-R-Y-A-V-P-R-F-A-V-R-V-C-R
R-a G-C-L-R-Y-T-K-P-K-F-T-V-R-V-C-R
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (~=NMeG);
R-G-F-C-L-R-Y-T-V-P-R-F-T-V-R-F-C-V-R;
R-G-F-C-L-R-Y-K-V-G-R-F-K-V-R-F-C-V-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG);
R-G-G-C-L-R-Y-A-R-X-R-F-A-V-R-V-C-R (X=NMeG);
R-G-G-C-L-R-Y-A-V-G-R-F-A-V-R-V-C-R;
R-G-F-C-L-R-Y-X-V-G-R-F-X-V-R-F-C-V-R (X=NMeG)
Pl ~al ~Lion of the Invention Compounds
The invention compounds are ess~nti~lly peptide backbones which may be modified at
the N- or C-terminus and also may contain one or two cystine diclllfide lin~s The peptides
may first be synthP~i~ed in noncyclized forrn. These peptides may then be converted to the
cyclic peptides if desired by standard methods of cystine bond forrnation. As applied to the
compounds herein, "Gyclic forms" refers to those forms which contain cyclic portions by virtue
of the formation of dielllfide linkages between cysteine residues in the peptide. If the straight-
chain forrns are prerel I ed, it is pl ert;l ~ble to stabilize the sulfhydryl groups for any peptides of
the invention which contain two or more cysteine residues.
- Standard methods for synthesis of peptides can be used. Most commonly used
currently are solid phase synthesis techniques; indeed, automated equipment for syst~m~tic~lly
constructing peptide chains can be purchased. Solution phase synthesis can also be used but is
considerably less convenient. When synth~ei~ed using these standard techniques, amino acids
not encoded by the gene and D-lo.n~ntiom~rs can be employed in the synthesis. Thus, one very
SUBSTITUTE SHEET (RULE 26)
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24
practical way to obtain the compounds of the invention is to employ these standard chemi~
sy,l~lle~is techniques.
In addition to providing the peptide backbone, the N- and/or C-terminus can be
de~iva~i~ed, again using conv~ontion~l rh~m;-~l techniques. The compounds of the invention
may optionally contain an acyl group, ~ ;rel~bly an acetyl group at the amino terrninus.
Methods for acetylating or, more generally, acylating, the free amino group at the N-terrninus
are generally known in the art; in addition, the N-terminal amino acid may be supplied in the
syll~hesis in acylated form.
At the carboxy terrninus, the carboxyl group may, of course, be present in the form of
a salt; in the case of pharm~ce~lti~ l compositions this will be a pharm~ce~-tic~lly acceptable
salt. Suitable salts include those formed with inol ~,anic ions such as NH4+, Na+, K+, Mg++,
Ca~+, and the like as well as salts formed with organic cations such as those of caffeine and
other highly substituted amines. However, when the compound of formula 1 contains a
m~ltipli~.ity of basic resi~ e~, salt formation may be difficult or impossible. The carboxy
terminus may also be esterified using alcohols of the formula ROH wherein R is hydrocarbyl
(1-6C) as defined above. Similarly, the carboxy terminus may be ~rni(l~ted so as to have the
formula -CONH2, -CONHR, or -CONR2, wherein each R is independently hydrocarbyl (1 -6C)
as herein df~fine~ Techniques for esterification and amidation as well as neutralizing in the
presence of base to form salts are all standard organic chemical techniques.
If the peptides of the invention are prepared under physiological conditions, the side-
chain amino groups of the basic amino acids will be in the form of the relevant acid addition
salts.
Formation of ~ -lfi(le link~es, if desired, is conrl~lcted in the presence of rnild
oxitli7:in~ agents. Chemical oxidizing agents may be used, or the compounds may simply be
exposed to the oxygen of the air to effect these link~es. Various methods are known in the
art. Processes useful for ~ lfide bond formation have been described by Tam, J.P. et al.,
Synthesis (1979) 955-957; Stewart, J.M. et al., "Solid Phase Peptide Synthesis" 2d Ed. Pierce
Chemic~l Company Rockford, IL (1984); Ahmed A.K. et al., J Biol Chem (1975) 250:8477-
8482 and Pennin~on M.W. et al., Peptides 1990, E. Giralt et al., ESCOM Leiden, The
Netherlands (1991) 164-166. An additional alternative is described by Karnber, B. et al., Helv
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Chim Acta (1980) 63 :899-915. A method con~ cted on solid supports is described by
Albericio Int J Pept Protein Res (1985) 26:92-97.
A particularly prer~ d method is solution oxidation using molecular oxygen. Thismethod has been used by the inventors herein to refold the compounds of the invention.
If the peptide backbone is comprised entirely of gene-encoded amino acids, or if some
portion of it is so composed, the peptide or the relevant portion may also be synthPci7~d using
recollll)inalllDNA techniques. The DNA en~o-ling the peptides of the invention may itself be
,y"l1~ee;,.ed using collllllelc;ally available equipl-,ell~, codon choice can be illlegl,lLed into the
synthesis depending on the nature of the host.
Synfh~ci7ed and lecol.,b;l.al.Lly produced forms ofthe compounds may require
subsequent derivatization to modify the N- and/or C-terminus and, depending on the isolation
procedure, to effect the formation of cystine bonds as described hereinabove. Depending on
the host organism used for recombi.~ production, some or all of these conversions may
already have been effected.
For I eco..ll~;nal.L production, the DNA encoding the peptides of the invention is
incl~ldecl in an ~ ,ression system which places these coding sequences under control of a
suitable promoter and other control sequences colllpalible with an intçn~led host cell. Types of
host cells available span almost the entire range of the plant and animal kingdoms. Thus, the
compounds of the invention could be produced in bacteria or yeast (to the extent that they can
be produced in a nontoxic or refractile form or utilize resi~ strains) as well as in animal
cells, insect cells and plant cells. Indeed, modified plant cells can be used to regenel~e plants
co.,~ g the relevant e,.,u~es~ion systems so that the resulting transgenic plant is capable of
self protection vis-à-vis these infective agents.
The compounds of the invention can be produced in a form that will result in their
secretion from the host cell by fusing to the DNA encoding the peptide, a DNA encoding a
suitable signal peptide, or may be produced intrac~ rly. They may also be produced as
fusion proteins with additional amino acid seq~l~nce which may or may not need to be
subsequently removed prior to the use of these compounds as ~ntimiçrobials or antivirals.
Thus, the compounds of the invention can be produced in a variety of modalities
in~.hlrling ~h~mic~l synthesis, recolllbin~l~ production, isolation from natural sources, or some
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26
co...l)i..~tion ofthese techniques.
Any In~ Cl:i ofthe invention class which coinsidpnt~lly occur naturally must be
supplied in purified and i~ol~ted form. By "purified and isolated" is meant free from the
en~,i,ol,..,~;"l in which the peptide normally occurs (in the case of such naturally occurring
peptides) and in a form where it can be used practically. Thus, "purified and isolated" form
means that the peptide is s~ lly pure, i.e., more than 90% pure, pr~fe.~bly more than
95% pure and more preferably more than 99% pure or is in a completely di~ltillL context
such as that of a pharm~ce~ltic~l preparation.
Antibodies
Antibodies to the peptides of the invention may also be produced using standard
immllnological techniques for production of polyclonal antisera and, if desired, immortalizing
the antibody-producing cells ofthe i"....~.;,e(l host for sources of monoclonal antibody
production. Teçhniqllec for producing antibodies to any substance of interest are well known.
It may be necessa,y to e l~h~nse the immnnogenicity of the substance, particularly as here,
where the material is only a short peptide, by coupling the hapten to a carrier. Suitable
carriers for this purpose include substances which do not Ihe,,,selves produce an immnne
l~spollse in the .. A~.. ~I to be a-lmini~t~red the hapten-carrier conjugate. Common carriers
used include keyhole limpet hemocyanin (KLH), diphtheria toxoid, serum ~lknmin~ and the
viral coat protein of rotavirus, VP6. Coupling of the hapten to the carrier is ~ffected by
standard techniques such as cont~ting the carrier with the peptide in the presence of a
dehydrating agent such as dicyclohexylcarbodiimide or through the use of linkers such as those
available through Pierce Chemical Company, Chicago, IL.
The peptides ofthe invention in immllnogenic form are then injected into a suitable
",A.. ~ n host and antibody titers in the serum are monitored. It should be noted, however,
that some forrns of the peptides require modification before they are able to raise antibodies,
due to their reei~t~nse to antigen processing. For example, peptides co..~ g two cystine
bridges may be nG~ n-)genic when ~ el ed without coupling to a larger carrier and
may be poor imml-nogens even in the presence of potent adjuvants and when coupled in
certain formats such as using glutaraldehyde or to KLH. Any lack of immnnogenicity may
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ll~elerc"e result from rÇ~iet~nre to procç~ing to a linear form that can fit in the antigen-
plese~ g pocket ofthe ~lese~l;..g cell. Tmmllnogenicity ofthese forrns ofthe peptides can
be enh~ncecl by cleaving the ~ fi~le bonds.
Polyclonal antisera may be harvested when titers are s -ffiei~ntly high. Alternatively,
antibody-producing cells ofthe host such as spleen cells or peli~hel~l blood lymphocytes may
be harvested and hlllllol Lalized. The immortalized cells are then cloned as individual colonies
and s~ elled for the production of the desired monoclonal antibodies.
Reco".l,i.lallL techniques are also available for the production of antibodies, and thus,
the antibodies of the invention include those that can be made by genetic ç~ ee~ ;,-g
techniques. For eY~m~le, single-chain forms, such as Fv forms, chimeric antibodies, and
antibodies modified to mimic those of a particular species, such as hllm~n~ can be produced
using sLalldaud metho~5 Thus, the antibodies ofthe invention can be prepared by i~ol~ting or
modifying the genes encoding the desired antibodies and producing these through e,~les~ion
in recombill~,L host cells, such as CHO cells.
The antibodies ofthe invention are, of course, useful in immllnoassays for dele.l.~ g
the amount or presence of the peptides. Such assays are ~c~.nti~l in quality controlled
production of compositions co..l~ g the peptides ofthe invention. In addition, the
antibodies can be used to assess the efficacy of l~co--.bi"~,l production of the peptides, as
well as sclee,fillg eAp,~ssion libraries for the presence of peptide encoding genes.
Compositions Co..~ the Invention Peptides and Methods of Use
The peptides of the invention are effective in inactivating a wide range of microbial and
viral targets, in~ tling gram-positive and gram-negative bacteria, yeast, protozoa and certain
strains of virus. Acco~L"gly, they can be used in di~i"re-;La,lL compositions and as
preservatives for materials such as foodstuffs, co~metiÇs7 medic,....~ i, or other materials
~ cor.l~;.. i~-g nutrients for o~ . For use in such contexts, the peptides are supplied either
as a single peptide, in admixture with several other peptides of the invention, or in admixture
with additional antimicrobial agents or both. In general, as these are preservatives in this
context, they are usually present in relatively low amounts, of less than 5%, by weight of the
total composition, more pr~elably less than 1%, still more preferably less than 0.1%.
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The peptides ofthe invention are also useful as ~L~Idalds in ~ntimir.robial assays and in
assays for delellllillaLion of capability of test compounds to bind to endotoxins such as
lipopolysaccharides.
For use as ~ntimirrobials or antivirals for lle.,~ of ani~nal subjects, the peptides of
the invention can be form~ ted as pharm~ce-ltic~l or veterinary compositions. Depending on
the subject to be treated, the mode of ~ lion, and the type of 11 e ~ desired -- e.g.,
prevention, prophylaxis, therapy; the invention peptides are forrmll~ted in ways consollarl with
these p~ clers. A summary of such teçhni~ es is found in Remin~on's Pharrn~- e~ltic~
Sçiencee, latest edition, Mack Publishillg Co., Easton, PA.
The peptides of the invention can also be used as active ingredients in pharrn~ceuti
compositions useful in l, ~ of sexually L~ ed ~liceA~es~ inr l~lding those caused by
Chlamydiatrachomatis, Treponemap~ ~ Neisseria~ J"hoeae, Trichomonas
vaginalis, Herpes simplex type 2 and HIV. Topical formulations are ~crc~ed and include
creams, salves, oils, powders, gels and the like. Suitable topical excipient are well known in
the art and can be adapted for particular uses by those of ordinary skill.
In general, for use in L,~ or prophylaxis of STDs, the peptides of the invention
may be used alone or in col,lbhlalion with other antibiotics such as erythlolllycill, tetracycline,
macrolides, for example azill~lul"yci" and the cephalosporins. Depending on the mode of
arl-";--;~ lion, the peptides will be formlll~ted into suitable compositions to permit facile
delivery to the affected areas. The tachytegrins may be used in forms c~ ;..;..g one or two
disulfide bridges or may be in linear form. In addition, use of the enantiomeric forms
co..~ g all D-amino acids may confer advantages such as r~eict~nce to those proteases,
such as trypsin and chymol,y~ , to which the peptides co~ L-amino acids are less
reci~t~nt
The peptides of the invention can be ~ , ed singly or as mixtures of severalpeptides or in co",bh~dLion with other pharm~t~e~lti~ lly active components. The formulations
may be prep~ed in a manner suitable for systemic ~mini~tration or topical or local
lion Systemic formulations include those designed for injection (e.g.,
intr~mll~clll~r, intravenous or subc~lt~n~oll~ injection) or may be p,eya,ed for transdermal,
tr~n~mllcosal, or oral a~ lion. The formulation will generally include a diluent as well
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as, in sorne cases, adjuvants, buffers, preservatives and the like. The tachytegrins can be
mini~tçred also in liposomal compositions or as microemulsions.
If ~imini~tration is to be oral, the peptidles of the invention must be plote~;Led from
degradation in the stomach using a suitable enteric coatin~ This may be avoided to some
extent by lltili7ing amino acids in the D-configuration, thus providing ~ nre to protease.
However, the peptide is still susceptible to hydrolysis due to the acidic con~iition~ of the
stom~ch; thus, some degree of enteric coating may still be required.
The peptides of the invention also retain their activity against microbes in the context of
borate solutions that are commonly used in eye care products. Also, it is i..,po~ that the
peptides retain their activity under physiological conditions incl~ltling relat*ely high saline and in
the presence of serum. In addition, the peptides are dr~m~tir.~lly less ~;ylolo~ic with respect to
the cells of higher or~ni~m~ as colllpal ~d with their toxicity to microbes. These pl.~pel lies,
make them particularly suitable for in vivo and the-~l~eu~ic use.
By app. up.iately choosing the member or ~ )tl :i of the peptide class of the
invention, it is possible to adapt the ~ntimir.robial activity to .~ .;...;7e its effectiveness ~,-vith
respect to a particular target microbe. As used herein, "microbe" will be used to include not
only yeast, bacteria, and other unicellular org~nicm~, but also viruses. The particular peptide
used can also be chosen to be advantageous in a particular context, such as low salt or
physiological salt, the presence or human serum, or conditions that mimic the conditions found
in blood and tissue fluids.
The peptides ofthe invention may also be applied to plants or to their en-vho--l--e--~ to
prevent virus- and microbe-inrl-lced ~ e~eçs in these plants. Suitable compositions for this use
will typically contain a diluent as well as a spreading agent or other ancillary a~ l eç~ s
b~nPfici~l to the plant or to the ellvilulllllc;llL.
Thus, the peptides of the invention may be used in any context wherein an
antimicrobial and/or antiviral action is required. This use may be an entirely in vitro use, or
the peptides may be ~(lmini~tered to ol~
In addition, the ~ntimi~robial or antiviral activity may be generated in situ bymini~t~ring an ~ u- es~ion system suitable for the production of the peptides of the invention.
Such e~,-es~ion systems can be supplied to plant and animal subjects using known
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te~hniq~les. For example, in ~nim~le, pox-based c,.l~lession vectors can be used to generate
the peptides in situ. Similarly, plant cells can be ~ r~,.,.ed with ~ s:,ion vectors and then
ene~led into whole plants which are capable oftheir own production ofthe peptides.
The peptides of the invention are also capable of inactivating endotoxins derived from
gram-negative bacteria -- i.e., lipopolysaccharides (LPS) and may be used under any
circ~ ee where inactivation of LPS is desired. One such eit~l~tiQn is in the llc~ .r~ll or
amelioration of gram-negalive sepsis.
Conditions Relevant to AntimicrobiaVAntiviral Activity
It has been stated above that as used herein "antimicrobial" activity refers to inhibition
with respect both to traditional m-icroorg~ llle and to viruses, although occasionally,
"5mtimjcrobial" and "antiviral" are both specifically in-1ic~ted.
Media for testing antimicrobial activity are de~ei~ned to m-imic certain specific
conditions. The standard buffer metiillm, me~ m A, uses an underlay agar with the following
composition: 0.3 mg/ml oftrypticase soy broth powder, 1% w/v agarose and 10 mM sodium
phosphate buffer (final pH 7.4). This will be c~e.ei~n~ted either "merlillm A" or "standard in
vitro conditions" herein.
All ofthe ~c~ ;..g media contain these same components. However, in addition:
A second metlillm co.-L~i--s 100 mM NaCI in order to mimic the salt levels in blood and
tissue fluids. This will be dçei~n~ted "medillm B" or "salt mP~ lm" herein.
A third merlillm is supplemented with 2.5% normal human serum; however, it is of low
ionic strength and thus does not mimic body fluids. This me-iillm will be cleei n~ted "me~illm
C" or "serum-co..~ me~illm~ herein.
A fourth me~ m co.-L~ns 80% RPMI-1640, a standard tissue culture m~dium which
co..lai..s the principal ions and amino acids found in blood and tissue fluids. In addition, it
cont~ine 2.5% norrnal human serum. This will be desi~n~ted "medi~lm D" or "physiological
m~ m" herein.
Particularly prere,.ed is the ~mi~ted form ofthis peptide.
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Summary
The peptides ofthe invention thelerole lep,ese ll a peculiarly useful class of
compounds because of the following prope. lies:
1) They have an ~ntimicrobial effect with respect to a broad SpC~,~IUlll oftarget
microbial systems, inr.lllrling viruses, in~ din~ retroviruses, bacteria, fungi, yeast and
pro~ozoa.
2) Their ~ c~ ol)ial activity is effective under physiological con-iitiQns - i.e.,
physiological saline and in the presence of serum.
3) They are much less toxic to the cells of higher or~nicmcth~n to microbes.
4) They can be prepared in no~immllnogenic form thus ~,Yt~ntlin~ the number of
species to which they can be ~i., .;.,i~lp., ed.
S) They can be plepaled in forms which are resistant to certain proteases
s~ gesting they are ~ntimicrobial even in Iysosomes.
6) They can be prepared in forrms that resist degradation when autoclaved, thus
simplifying their prep~lion as components of pharm~ce~ltic~lc.
7) They can be modified in amino acid sequence so as to oplillll e the specificity
with respect to target.
8) They can be modified structurally so as to accommodate the conditions under
which ~ntimicrobial activity is to be exhibited.
The following examples are int~n~1ecl to illustrate but not to limit the invention.
Example I
Synthesis of the Invention Compounds
The peptides of the invention are synthçci~ed using conventional Fmoc ~.h~.mictry on
solid-phase supports. The crude synthetic peptides are refolded, purified and characterized as
follows.
The crude synthetic peptide is reduced by adding an amount of dithiothreitol (DTT)
equal in weight to that of the synthetic peptide, which has been dissolved at 10 mg/ml in a
solution cc"~li.;..;..g 6M ~l~ni~ine HCI, 0.5M Tris buffer and 2 mmol EDTA, pH 8.05 and
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incl~h2ted for 2 hours at 52~C under nitrogen. The rnixture is passed through a 0.45 um, filter,
~ci~ified with 1/20 v/v glacial acetic acid and subjected to conventional RP HPLC purification
with a C 18 column.
The HPLC-purified, reduced peptides are partially concentrated by vacuum
centrifugation in a Speed Vac and allowed to fold for 24 hours at room telll?~ re and air.
The folding is accomplished in 0. lM Tris, pH 7.7 at 0.1 mg peptide/rnl to .~ e formation
of i,l~elcl~ cystine di~ ~lfides. The folded compounds are concentrated and acidified with 5%
acetic acid. The purity of the final products is verified by AU-PAGE, analytical HPLC and
FAB-mass spec.
Using this procedure, the compounds trans-parevin-l (or the "hairpin" isoform), cis-
parevin-l ~or "cloverleaf" isoforrn) and trans-tachytegrin-l were prepared. These compounds
are of the formulas
Trans-parevin- 1:
R-G-G-R-C-L-Y-C-R
R
R
R-G-C-V-V-C-F
Cis-parevin- 1:
R-G-G-R-C-L-Y-~-R
R
R
R-G-C-V-V-C-F
L...... l
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Trans-tachyte~rin- 1:
R-G-G-C-R-L-Y-C-R
R
R
R-C-G-V-V-C-F
Example 2
Antimicrobial Activity
The radial diffusion assay in agarose gels is con~ ted using radiodiffusion and gel
overlay techniques as described by Lehrer, R.I. et al. J Tmm~lnol Meth (1991) 137:167-173.
Briefly, the underlay agars used for all o~ had a final pH of 7.4 and co..~ çd lO mM
sodium phosphate buffer, 1% w/v agarose and 0.30 ug/ml tryptocase soy broth powder (BBL
Cockeysville, MD). In some cases, the underlay was suppllo.m~.nted with 100 mM NaCI. The
units of activity in the radial diffusion assay were measured as described; 10 units COIIespond
to a 1 mm ~ meter clear zone around the sample well. Figures 1-6 show the results against
five test or~ni~m~ in units des~,libed as above. A synthetic protegrin (PG-l) cont~inin~ two
cystines (sPG-l) or PG-l in linear form were used as controls.
Figure 1 shows the results for trans-parevin and tachytegrin with respect to E coli
both with and without the addition of l OO mM NaCI. Both of these peptides were slightly
more effective than sPG-1 although slightly less effective than linear PG-1 in the absence of
salt. However, in the presence of l OO mM NaCl, all four peptides were colllpal ~bly effective.
Figure 2 shows the results of the same detel.n,llalion with respect to
L. monocytogenes. With respect to this organism, all four peptides were roughly similarly
effective in the absence of salt; the presence of 100 mM NaCI, however, greatly reduced the
effectiveness of linear PG-1. The Ir.."~il.;..g three peptides rem~ined effective under these
conditions.
Figure 3 shows the results of the same experiment using C. albicans as the target
olg~islll. All four peptides were conlpal~bly effective in the absence of salt; again, the
effectiveness of linear PG- l was greatly reduced in the pl esence of 100 mM NaCl, while the
,~."~ g three peptides ..-~ ed their effectiveness under these conditions.
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Figures 4-6 show the results of similar expelinlcllls using, as test peptides, the two
isomers of parevin, trans-parevin (hairpin) and cis-parevin (cloverleaf). sPG- 1 was used as a
control. As shown in Figure 4, the two parevins were colllpal ~bly effective in the absence of
salt and both were more effective than sPG- 1. In the presellce of 100 mM NaCI, all three
peptides ...~ d their effectiveness and were col~ ble.
Figure S shows results of the same e;A~lhllent con~ cte(~ with B. subtilis as target
olg~islll. Again, both forms of parevin were colllp~bly effective and both were slightly
more effective than sPG-l; in the presence of 100 mM NaCI, all three peptides r~.m~ined
effective ~ntimicrobials and had about the same activity.
The results obtained with respect to S. typ*imurium are also similar, as shown in
Figure 6. Again, the two parevins were more effective than sPG1 in the absence of salt and
all three peptides had collll al~ble effectiveness when 100 mM NaCI was added.
Example 3
Ability to Bind Endotoxin
The compounds of the invention are tested for their ability to bind the lipid
polysaccharide (LPS) of the gram-negative bacterium E. coli strain 0.55B5, using the Limulus
amebocyte Iysate (LAL) test for endotoxins con~ucte~l in the presence and absence of the test
compounds. The test is cond~lcted using the procedure desclibed in Sigma Technical Bulletin
No. 210 as revised in December 1992 and published by Sigma Chemic~l Company, St. Louis,
MO.
The LAL test is based on the ability of LPS to effect gelation in the commercialreagent E-ToxateO which is plepaled from the Iysate of circ~ ting amebocytes of the
Horseshoe Crab ~int~ pol.~ ",us. As described in the teçhnicAI bulletin, when exposed to
minute quantities of LPS, the Iysate h~ eases in opacity as well as viscosity and may gel
depending on the concentration of endotoxin. The technical bulletin goes on to speculate that
the mec.l1~..i.c.-- appears analogous to the clotting of ".A~",-AliAn blood and involves the steps
of activation of a trypsin-like preclotting enzyrnes by the LPS in the presence of calcium ion,
followed by enzymic modifications of a "coagulogen" by proteolysis to produce a clottable
protein. These steps are believed tied to the biologically active or "pyrogenic" portion of the
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molecllle. It has been shown previously that detc xified LPS (or endotoxin) gives a negative
LAL test.
The test compounds are used at various conce~ LLions from 0.25 ug-10 ug in a final
volume of 0.2 ml and the test llli~lu.es conlahled LPS at a final conc~ Lion of 0.05
endotoxin unit/ml and E-Toxate~M at the sarne cQncf;~ lion. The test co~ oullds are
in-.~tb~tecl together with the LPS for 15 mimltes before the E-ToxateTM is added to a final
volume af'~er E-ToxateTM addition of 0.2 ml. The tubes are then inc~lb~ted for 30 rninutes at
37~C and ex~mined for the formation of a gel.
In a ffillow-up cA~e,illlent, the concentration of LPS is varied from 0.05-0.25
endotoxin units (E.U.).
Example 4
Antimicrobial Activit,v Under Conditions
Suitable for Tl e~ of the Eye
Contact lens solutions are typically form~ ted with borate buffered physiological
saline and may or may not contain EDTA in addition. The compounds of the invention are
tested generally in the assay described in E~ ,le 2 ~Leleill all underlay gels contain 25 mM
borate buffer, pH 7.4, 1% (v/v) trypticase soy broth (0.3 ug/ml TSB powder) and 1% agarose.
Additions include either 100 mM NaCI, 1 mM EDTA or a co",l)illalion thereof. Other test
compounds used as controls are the defensin NP-l and lysozyme, and dose response curves
are dete",lined.
Example 5
Plep~lion of Enanfio Trans-parevin
Using standard solid phase techniques, a peptide having the amino acid sequence of
~ trans-parevin, but wherein every amino acid is in the D form is prepared. This form is tested
against E. coli, L. monocytogenes, C. albicans and other microbes in the absence and presence
of protease and otherwise as described for the radiodiffusion assay in agarose gels set forth in
Example 2.
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Example 6
Activity A~ainst STD Patho~ens
The colllpounds of the invention are tested for ~ntimic robial activity against various
STD pathogens. These include E~V-1, Chlamydia l,dc~lv,,.atis, Treponema palli~um,
Neisseriag~J"u"h~eae, Trichomonas vaginalis, Herpes simplex type 2, Herpes simplex type
1, Hemophilus ducreyi, and Human papilloma virus. The results are provided in a form
whelc;ill "active" means that the peptide is effective at less than 10 ug/ml; moderately active
in~iç~tes that it is active at 10-25 ug/ml; and slightly active means activity at 25-50 ug/ml. If
no effect is obl~ined at 50-200 ug/ml the compound is corl~id~red inactive.
The compounds of the invention are tested for their ~ntimirrobial activity against
Chlamydia using the "gold standard" chlamydial culture system for clinical ~,l,e~
described by Clarke, L.M. in Clinical Microbiology Procedures Handbook II (1992), Isenberg,
H.T. Ed. Am. Soc. Microbiol. W~hin~on, D.C.; pp. 8Ø1 to 8.24.3.9.
In the assays, C. trachomatis serovar L2 (L2/434Bu) described by Kuo, C.C. et al. in
Nongl,~nococcal Urethritis and Related Infections (1977), Taylor-Robinson, D. et al. Ed. Am.
Soc. Microbiol. Washington, D.C., pp. 322-326 is used. The seed is pl~paled from a
sonic~ted culture in L929 mouse fibroblast cells, and partially purified by centrifugation. Since
host protein is still present in the seed aliquots, each seed batch is titered at the time of
pl~ion with serial ten-fold dilutions to 2 x 10~. The seed co..~ ng 9.2 x 106 IFlJ/ml is
thawed quickly at 37~C and diluted to 10-2 with sucrose/phosphate salts/glycine to produce
IFU of about 200 after room temperature pr.?inc~b~tion and to dilute background eukaryotic
protein.
In the initial assays, the peptides to be tested are prepared as stock solutions in 0.01%
glacial acetic acid. 100 ul ofthe diluted chlall,y-lial seed are aliquoted into 1.~ ml eppendorf
tubes and 200 ul of the antibiotic peptide was added per tube. Aliquots of the peptide stock
(and controls) are inc~lb~ted with the seed at room te.,.,)e-~ re for one hour, two hours and
four hours. About 10 mimltes before the end of each incub~tion period, ,,.~ el~ ce media
are aspi~led from the McCoy vials in p-e~a ~lion for standard inoculation and culture.
Culture is then pelrc,lllled in the presence and absence of the peptides; in some cases, the
peptides are added to final concentration in the culture media in addition to the preculture
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in~ b~tion. The test is evaluated microscopically.
- In another series of cAI~G~ lcllL~, various collc~ntrations of tachyleg,in (1 ug, 12.5 ug,
25 ug and 50 ug) are used in the two-hour pr~inc~ ation.
The effect ofthe presence of serum is also tested. The Chlamydia seed is pr~inrl~b~ted
for two hours with and without 10% FBS and also with or without test compound at 25 ug.
The expe~ lellLs are re~eaLed but adding 25 ug of compound after the start of the
chlamydial culture, i.e., after centrifugation and final medil~m mix and one hour into the
beginning of the 48-hour culture period. Finally, the colllpo~nd (at 25 ug) is added to the
chlamydial seed and the mix then immçdi~t~ly cultured.
The effect of serum is particularly important since for a topical agent to be effective in
c~ Chl~mydia infection~ it must act in the pl~sGnce of serum.
In addition, there are several mouse-based models for Chlamydia infection which can
be used to assess the efficacy of the tachytegrins. These include those described by Patton,
D.L. et al. in Chlamydial Infections (1990) Bowie, W.R. et al. Eds. Cambridge
Universproviding a source of complement. Ten ul of a suspension of T. pallidum co.~ -g
about 5 x 107/ul or~nicm.C is added to each tube and the mixtures with the apploplia~e
peptides are inrllb~ted at 34~C under 95% N2 and 5% CO2. At time zero, just prior to
in-,ub~tion, 4 hours and 16 hours, 25 randomly s~lected orf~;~ni.cmc are ~ ..;..ed for the
presence or absence of motility. The 50% immobilizing end point (IE50) iS r~ ted to
inrlic~te the conct;n~ ion needed to immobilize 50% ofthe spirochetes. Tacl.y~Jle~i-- IE50s are
5.231 ug and 2.539 ug for 0 and 4 hours, in contrast to HNP and NP plt;p~alions which show
little immobilizing ability.
For Herpes Simplex Virus, using viral stocks p. t;pal ed in VERO cells, grown inminim~l es~enti~l m~ m (~M) with 2% fetal calf serum, the effect of various peptides on
HSV 1 MacIntyre strain, a pool of ten clinical HSV 1 isolates, HSV-2G, and a pool of ten
~ clinical HSV 2 isolates, all sensitive to 3 uM acyclovir are tested. Two fibroblast cell lines,
human W138 and equine CCL57, are used as targets and tests are done by direct viral
neutr~li7~tic-n and delayed peptide addition.
In the direct neutralization format, the virus is pr~inc~lbated with the peptides for 90
min before it is added to the tissue culture monolayers. In the delayed peptide addition
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38
format, the virus is added and allowed 50 min to adsorb to the target cells, then the
monolayers are washed and peptides are added for 90 min. Finally, the monolayer is washed
to remove the peptide and the cells are fed with peptide-free MEM and cultured until the
untreated h~iled monolayers exhibit 4+ ~iylopalllic effect (CPE) (about 60 hours).
For Trichomonas vaginallis, strain Cl (ATCC 30001) is grown as described by
Gorrell, T.E. etal., C~l~be.~, Res Comm (1984) 49:259-268. In ~,~elilllc;llL~ pelrulllled in
RPMI + 1% heat-activated fetal calf serum, within a few mim~t~s after exposure to S0 ug/ml
PG-l, T. vaginallis (heretofore vigolously motile) becollles ~Liona,y. Soon thereafter, the
Olg~ become permeable to trypan blue, and, over the ensuing 15-30 min~lte~ Iyse. As
c,~l.e.;Led, such org~ .C fail to grow when introduced into their customary growth m~ lm
(Diamond's medium). o~ ",.c exposed to 25 ug/ml of PG-3 retain their motility.
Example 6
~ ;Llo~iral Activity
The invention compounds are tested for antiviral activity against strains of HIV using
the method described in Miles, S.A. et al., Blood (1991) 78:3200-3208. Briefly, the
mononuclear cell fraction is recovered from normal donor leukopacs from the Arnerican Red
Cross using a Ficoll-hypaque density gradient. The mononuclear cells are resuspended at 1 x
106 ce11s per ml in RPMI 1640 me~ m with 20% fetal bovine serum, 1% penn/strep with
fullgiGol1e and 0.5% PHA and inrllb~ted 24 hours at 37~C in 5% COz. The cells are
centrifuged, washed and then exp~n-led for 24 hours in growth me~ lm
Non-laboratory adapted, cloned HIV~R~SF and ~V.1R-FL are ele~llopol~led into thehuman peripheral blood mononllr.le~r cells plepared as described above. Titers are delel,l,illed
and in general, multiplicities of infection (MOI) of about 4,000 infectuous units per cell are
used (which cc,llt;s~ollds to 25-40 picograms per ml HIV p24 antigen in the S~ ;lllaLa"l).
In the assay, the HIV stocks prepared as above are diluted to the correct MOI and the
PBM are added to 24 well plates at a concentration of 2 x 106 per ml. One ul total volume is
added to each well. The peptide to be tested is added in growth merli-lm to achieve the final
desired conce~ lion. Then the a~ rc,plial~ number of MOI are added. To assay viral
growth, 200 ul of supelllalalll is removed on days 3 and 7 and the concentration of p24
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39
antigen is d~ ed using a commercial assay (Coulter Tmml-nology, Hialeah, Florida).
Controls include ~llrlic~te wells co~ cells alone, cells plus peptide at S ug/ml cells with
virus but not peptide and cells with virus in the pre~nce of AZT at 10-5 M - 10-8 M.
The time of addition of peptide can be varied. Cells preLl~,a~ed for 2 hours prior to
addition of virus, at the time of ad(litiQn of virus, or 2 hours after infection show antiviral
activity for the peptide.
Example 7
P~ Lion and Activity of Tachytegrins
Several illustrative tachytegrins were synth~ ed as described in Example 1 and tested
for activity against Staphylococcus aureus (~SA), Ps~ nnas (Psa), VREF, Candic~a and
E.coli as described in Example 2. The results shown in Table 1 as minim~l inhibitory
concentration (MIC) in ug/ml were obtained with the C-terminal ~mitl~te~l forms except for
the last two which were tested as the free acids, as in~ ted by *.
TABLE 1
SEQUENCE MRSA Psa ¦VREF¦Candida¦ E. Coli
RGGCLRYAVPRFAVRVCR >128 0.05
RGCCLRYTKPKFTVRVCR
RCCCLRYAVGRFAVRVCR
RC(CLRYARXRFAVRVCR (X=NMeG) >32 5.7
RC-:'CLRYTVPRFTVRFCVR 1.88 0.57 0.99
RCFCLRY~XVGRFKVRFCVR >64 2.7
R(FCLRYXVt--RFXVRFCVR (X=NMeG)
RC-GCLRYCRPRFCVRVC:. 9.8 0.18 9.68 0.2
RC-GCRLYCR:.RFCW GC~ 53.3 3.3 4 4
RGVCLRYCRGRFCVRLC............. 8 2
RGRVCLRYCRCRFCV.LCF:. 6.7
RWRVCLRYCRtRFCV.LCL:. 4 4
R~WRVCLKYC.~R:'CJKLC_R
RC-GRVCL:.YCRt-K'CVRLC_R 8 0.75
:.CGCLRY~RXR:'AVRVC. (X=NMeG)
.. GGCLRYAVGR:'AVRVC. >32 5.3
:.GFCLRYXVGR:'XVRFCVR(X=NMeG) >32 12
SUBSTITUTE SHEET (RULE 26)
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