U.S. patent application number 11/280988 was filed with the patent office on 2006-05-25 for structured peptide scaffold for displaying turn libraries on phage.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Andrea G. Cochran, Nicholas J. Skelton, Melissa A. Starovasnik.
Application Number | 20060110777 11/280988 |
Document ID | / |
Family ID | 22484754 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110777 |
Kind Code |
A1 |
Cochran; Andrea G. ; et
al. |
May 25, 2006 |
Structured peptide scaffold for displaying turn libraries on
phage
Abstract
The invention is directed to a model system for
structure-activity analysis of peptide or protein molecules
involved in important biological processes. Provided by the
invention are combinatorial peptide libraries comprising
disulfide-constrained cyclic peptides with sequences favorable for
energy stabilized conformations. One aspect of the invention is
directed to cyclic peptide scaffolds that present .beta.-hairpin
structure in solution. Methods of selecting and using such peptide
scaffolds are provided herein, which are useful for mimicking in
vivo molecular interactions and designing therapeutic agents. Thus,
the invention has profound utility for biological studies and drug
development.
Inventors: |
Cochran; Andrea G.; (San
Francisco, CA) ; Skelton; Nicholas J.; (San Mateo,
CA) ; Starovasnik; Melissa A.; (San Francisco,
CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
22484754 |
Appl. No.: |
11/280988 |
Filed: |
November 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10271343 |
Oct 15, 2002 |
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11280988 |
Nov 15, 2005 |
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09592695 |
Jun 13, 2000 |
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10271343 |
Oct 15, 2002 |
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60139017 |
Jun 14, 1999 |
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Current U.S.
Class: |
435/7.1 ;
530/317 |
Current CPC
Class: |
C07K 14/70535 20130101;
C07K 1/047 20130101; C07K 7/06 20130101; C07K 14/70514 20130101;
C07K 7/08 20130101; C12N 15/1037 20130101; C40B 40/02 20130101;
C07K 14/71 20130101 |
Class at
Publication: |
435/007.1 ;
530/317 |
International
Class: |
C40B 40/10 20060101
C40B040/10; C07K 7/64 20060101 C07K007/64; C07K 5/12 20060101
C07K005/12 |
Claims
1. A cyclic peptide scaffold for presenting a .beta.-turn hairpin
structure, said cyclic peptide scaffold comprising an amino acid
sequence C1-A1-A2-(A3).sub.n-A4-A5-C2 (SEQ ID NO: 1), wherein C1
and C2 are cysteines; A1, A2, A3, A4, and A5 are naturally
occurring L-amino acids; the N'-terminus of the scaffold is
optionally protected with an amino protecting group; the
C'-terminus of the scaffold is optionally protected with a carboxy
protecting group; A1 and A5 are selected from the group consisting
of amino acids W, Y, F, H, I, V and T; A2 and A4 are selected from
the group consisting of amino acids W, Y, F, L, M, I, and V; A3 is
any naturally occurring L-amino acid and n is an integer that is 3,
4, 5, 6, 7, 8, 9, 10, 11, or 12; and C1 and C2 are joined together
by a disulfide bond thereby forming a cyclic peptide.
2. The cyclic peptide scaffold of claim 1, wherein A1 or A5 is a
.beta.-branched residue having two non-hydrogen substituents on the
.beta.-carbon of the amino acid residue.
3. The cyclic peptide scaffold of claim 1, wherein A1 or A5 is
T.
4. The cyclic peptide scaffold of claim 1, wherein A1 or A5 is
amino acid W, F, H or Y.
5. The cyclic peptide scaffold of claim 1, wherein A1 is H and A5
is V.
6. The cyclic peptide scaffold of claim 1, wherein A2 or A4 is
amino acid W, F or Y.
7. The cyclic peptide scaffold of claim 6. wherein A2 or A4 is
W.
8. The cyclic peptide scaffold of claim 7, wherein both A2 and A4
are W.
9. The cyclic peptide scaffold of claim 1, wherein n is at least
4.
10. The cyclic peptide scaffold of claim 9, wherein n is no greater
than 10.
11. The cyclic peptide scaffold of claim 9, wherein n is 4.
12. The cyclic peptide scaffold of claim 11, wherein (A3).sub.4 is
EGNK, ENGK, QGSF or VWQL.
13. The cyclic peptide scaffold of claim 12, wherein A1 is T or H,
A2 is W or L, A4 is W or L and A5 is T or V.
14. The cyclic peptide scaffold of claim 1, wherein (A3).sub.n is
DLLVRH.
15. The cyclic peptide scaffold of claim 14, wherein the amino acid
sequence is CHWDLLVRHWVC (SEQ ID NO:33).
16. The cyclic peptide scaffold of claim 13, wherein the amino acid
sequence is CTWEGNKLTC (SEQ ID NO:2).
17. The cyclic peptide scaffold of claim 13, wherein the amino acid
sequence is CHWEGNKLVC (SEQ ID NO:30).
Description
[0001] This application is a continuation-in-part application of
the application U.S. Ser. No. 09/592,695, filed Jun. 13, 2000,
which claims priority under 35 U.S.C. 119(e) to the U.S.
provisional application Ser. No. 60/139,017, filed Jun. 14,
1999.
TECHNICAL FIELD
[0002] The present invention relates in general to protein
structure-activity relationship studies, and in particular to
combinatorial libraries of conformationally-constrained peptides
and methods of generating and screening such libraries for
biological and pharmaceutical use.
BACKGROUND ART
[0003] Structure-Activity Relationship (SAR) study provides
valuable insights for understanding intermolecular interactions
between a protein or peptide and other biologically active
molecules. In their natural environment, peptides or proteins adopt
unique, conformationally-constrained structures in order to
recognize and bind to their binding partners, and to form a
molecular complex therewith, which in turn elicit particular
activities. Examples of protein-protein binding partners include
enzyme-substrate, ligand-receptor, and antigen-antibody.
Determination of the conformation of a peptide in its native form,
therefore, become crucial for closely mimicking its in vivo
activity and rationally designing its analogues which may be useful
as drugs.
[0004] Most small peptides are highly flexible and do not typically
adopt unique solution conformations; in particular, they do not
maintain the structure that the same sequence adopts in the native
protein. The lack of fixed structure reduces the affinity the
peptide might have for a target (for entropic reasons) and makes
determination of the active conformation of the molecule extremely
difficult. Because of this, many strategies have been described to
introduce constraints into peptides (such as D-amino acids,
disulfide or other crosslinks), or to replace parts of the peptide
with more rigid non-peptide scaffolds. Indeed, such peptidomimetics
have been widely used to perform structure-activity studies in a
systematic way to provide information about the specific amino acid
residues or functional groups in a peptide that are adaptable to a
particular conformation and are important to biological
activities.
[0005] Several constrained protein scaffolds, capable of presenting
a protein of interest as a conformationally-restricted domain have
been identified, including minibody structures (Bianchi et al.
(1994) J Mol Biol 236:649-659), loops on .beta.-sheet turns,
coiled-coil stem structures (Myszka & Chaiken (1994) Biochem
33:2363-2372), zinc-finger domains, cysteine-linked (disulfide)
structures, transglutaminase linked structures, cyclic peptides,
helical barrels or bundles, leucine zipper motifs (Martin et al.
(1994) EMBO J 13:5303-5309), and etc. Of the identified protein
scaffolds, .beta.-turns have been implicated as an important site
for molecular recognition in many biologically active peptides.
Smith & Pease (1980) CRC Crit Rev Biochem 8:315-399.
Consequently, peptides containing conformationally constrained
.beta.-turns are particularly desirable. The great majority of the
identified .beta.-turn bearing peptides are cyclopeptides which
have been generated by the cyclization of a peptide similar to a
sequence in the natural substrate. Milner-White (1989) Trends
Pharmacol Sci 10:70-74. These cyclopeptides, however, may still
retain significant flexibility. For this reason, many studies have
attempted to introduce rigid, nonpeptide compounds which mimic the
.beta.-turn. Peptides with such nonpeptide .beta.-turn mimic
provide useful leads for drug discovery. Ball & Alewood (1990)
J Mol Recog 3:55-64; WO 94/03494 (Kahn).
[0006] One of the revolutionary advances in drug discovery is the
development of combinatorial libraries. Combinatorial libraries are
a collection of different molecules, such as peptides, that can be
made synthetically or recombinantly. Combinatorial peptide
libraries contain peptides in which all amino acids have been
incorporated randomly into certain or all positions of the peptide
sequence. Such libraries have been generated and used in various
ways to screen for peptide sequences which bind effectively to
target molecules and to identify such sequences.
[0007] Many methods for generating peptide libraries have been
developed and described. For example, members of the peptide
library can be created by split-synthesis performed on a solid
support such as polystyrene or polyacrylarnide resin, as described
by Lam et al. (1991) Nature 354:82 and PCT publication WO 92/00091.
Another method disclosed by Geysen et al., U.S. Pat. No. 4,833,092
involves the synthesis of peptides in a methodical and
predetermined fashion, so that the placement of each library member
peptide gives information concerning the synthetic structure of
that peptide.
[0008] Considerable effort has been devoted to introducing
structural constraints into combinatorial peptide libraries so that
the member peptides represent more closely to their native
counterparts. Houston et al. U.S. Pat. No. 5,824,483 describes a
synthetic peptide library containing peptides featuring a-helical
conformation and thus capable of forming coiled-coil dimers with
each other. McBride et al. (1996) J Mol Biol 259:819-827 describe a
synthetic library of cyclic peptides mimicking the anti-tryptic
loop region of an identified proteinase inhibitor.
[0009] A complementary method for peptide library-based lead
discovery is display of libraries on filamentous bacteriophage.
This method allows the preparation of libraries as large as
10.sup.10-10.sup.12 unique peptide members, many orders of
magnitude larger than libraries that may be prepared synthetically.
In addition to large library sizes, advantages of phage display
include ease of library construction (Kunkel mutagenesis), coupling
of the binding entity (displayed peptide) to a unique identifier
(its DNA sequence), a selection protocol for amplifying rare
binding clones in a pool, and the high fidelity of biosynthesis
(compared to synthetic methods). Furthermore, rapid and inexpensive
selection protocols are available for identifying those library
members that bind to a target of interest. However, only natural
peptides composed of L-amino acids may be displayed on phage, so
the problem of defining three-dimensional structure-activity
relationships is more difficult than it might be for a constrained
peptidomimetic containing non-naturally occurring peptides or
nonpeptide compounds. One possible solution to this problem is to
use the structural constraints of a folded protein to present small
variable peptide segments. Indeed, several small, stable proteins
have been proposed as peptide display scaffolds. Nygren & Uhlen
(1997) Curr. Opin. Struct. Biol. 7:463-469; Vita et al. (1998)
Biopolymers 47:93-100; Vita et al. (1999) Proc. Natl. Acad Sci. USA
96:13091-13096; Smith et al. (1998) J. Mol. Biol. 277:317-332;
Christmann et al. (1999) Protein Engng. 12:797-806. Unfortunately,
it is not clear that protein ligands obtained by this approach
could be transformed to small-molecule drug leads. Epitope transfer
from proteins to small peptides or to non-peptide small molecules
remains an extremely challenging problem. Cochran (2000) Chem.
Biol. 7:R85-R94.
[0010] Therefore, despite extensive studies of the rules governing
conformational preferences in natural peptides and the existence of
several peptide library systems, those features necessary for
structural stability of natural peptides remain poorly understood.
In particular, there has been little systematic or quantitative
assessment of the effect of residue substitutions and non-covalent
interactions on structure.
DISCLOSURE OF INVENTION
[0011] The present invention provides a novel model system for
assessing individual residue contributions to the stability of a
defined peptide scaffold and for evaluating a series of
substitutions presented in a combinatorial peptide library. The
peptides of the invention are cyclized via disulfide bond between
two cysteines within the peptide sequences. Amino acid
substitutions at various defined residue sites influence the
conformation of the cyclic peptides and their structural
stabilities. The invention also provides methods of screening for
and analyzing cyclic peptides with a specific secondary structure,
.beta.-turn, which provides further structural constraints to the
peptides. The subject peptide library comprising a collection of
.beta.-turn bearing cyclic peptides can be used in screening for
candidate biologically active molecules through molecular binding
assays. Methods for such screenings are also provided by the
present invention. The compositions and methods of the invention
can be used in analyzing the structure-activity relationships of
peptides of interest, thereby providing useful information for
studies of molecular interactions involved in particular biological
processes, as well as for rational design of therapeutic
agents.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1 depicts the design of bhp, a 10-amino acid model
.beta.-hairpin peptide. (A) Superimposed protein structures
illustrate packing between disulfides and side chains of the
closest non-hydrogen-bonded residues; (B) Schematic representation
of the bhp model .beta.-hairpin peptide with the side chains of the
non-hydrogen-bonded residues 1, 3, 8 and 10 shown. X represents the
varied residue selected from 19 of the 20 natural L-amino acids
(excluding Cys).
[0013] FIG. 2 shows the relative hairpin stability for substitution
X in the bhp peptide sequence. (A) Cysteine effective
concentrations (Ceff) relative to glutathione. Error bars are for
.+-.one standard deviation; (B) Equilibrium free energy differences
relative to the alanine peptide.
[0014] FIGS. 3A-3B show two views of the minimized mean NMR
structure of disulfide-cyclized .beta.-hairpin bhpW. (A) Non
hydrogen-bonded (NHB) strand residues Trp3 and Leu8 are
highlighted. (B) Hydrogen-bonded (HB) strand residues are
highlighted (Thr2, Thr9, Glu4 and Lys7).
[0015] FIGS. 4A-4B depict NMR analysis of CD4 peptides. (A) Overlay
of the fingerprint region of the COSY spectra for cd1 and cd2. (B)
NMR structure ensemble for cd2 (20 models; two orthogonal views)
shown superimposed on CD4 residues 37-46 from the crystal structure
of gp120-bound CD4 (PDB entry IGC1).
[0016] FIG. 5 shows circular dichroism spectra of three peptide
pairs of Example 2.
[0017] FIG. 6 shows effective concentration (C.sub.eff) values for
substitutions X in the peptides of Example 3. The strand
substitutions X are shown at the top of the graph, and the central
residues of the turns are indicated to the right.
[0018] FIG. 7 depicts minimized mean structures of the tryptophan
analogs of peptides in Example 3 overlaid on the backbone atoms of
residues 1-3 and 8-10 (RMSD of 0.36 and 0.30 .ANG. for 1 with
respect to 2 and 3, respectively). Peptide 1 is in grey; peptide 2
is in black; and peptide 3 is in white. For clarity, non-proline
side chain atoms are not shown for the four turn residues.
[0019] FIGS. 8A-8B show effective concentration (C.sub.eff) values
for peptides with hydrophobic pairs in non hydrogen-bonded (NHB)
strand positions as described in Example 4. Values for
substitutions paired with a cross-strand leucine are shown in (A);
those for tryptophan pairs are shown in (B).
[0020] FIG. 9 depicts a Hammett plot comparing substitution free
energy differences between the peptides of Example 4.
[0021] FIG. 10 shows double mutant analysis of the stability of
W3Y8 relative to L3L8.
[0022] FIGS. 11A-11B illustrate relative stabilities of bhpw
analogues. Free energy changes (293 K) are calculated from the
ratio of the cysteine effective concentration (Ceff) for each
peptide to that of bhpW (TT pair) using the relationship
-.DELTA..DELTA.G=RT ln (C.sub.eff,XX/C.sub.eff,TT). (A)
Substitutions at position 2 (X2 series). (B) Substitutions at
position 9 (X9 series).
[0023] FIG. 12 illustrates relative stabilities of bhp hairpins
with valine at position 2 or 9. Solid bars indicate substitutions
in the background of bhpW, while hatched bars correspond to
peptides with the altered turn sequence KGNE. Free energy changes
(293 K) are calculated from the ratio of peptide C.sub.eff to that
of the appropriate TT analogue (-.DELTA..DELTA.G=RT ln
(C.sub.eff,XX/C.sub.eff,TT)).
[0024] FIG. 13 shows correlation of position 3 substitution free
energy differences for TT bhp analogues and for the HV, X3 series.
The residue substituted at position 3 is indicated, and the slope
of the plot is 1.96 (R=0.95).
[0025] FIG. 14 depicts position-specific strand twists
(.psi..sub.i+.phi..sub.i+1) for the 20 structures in the ensembles
determined for bhpW (circles), VH (triangles), and HV
(diamonds).
[0026] FIGS. 15A-15C depict sequences of BR3 variants and structure
of bhpBR3. (A) Amino acid sequences of BR3 variants used in this
study. (B and C) Three-dimensional structure of bhpBR3 determined
by NMR spectroscopy. The backbone atoms of 20 models are shown
superposed with residue labels positioned in the direction of the
side chain (B); one representative structure highlighting the BR3
turn residues (C) in the same orientation as in B.
[0027] FIGS. 16A-16B depict binding of BR3 variants to BAFF. (A)
Competitive displacement of biotinylated miniBR3 measured by ELISA
(see methods). Data are shown for BR3 extracellular domain (filled
triangles), miniBR3 (open squares), and bhpBR3 (filled circles).
IC.sub.50 values from the fitted curves are 70 nM, 65 nM, and 15
.mu.M, respectively. (B) HeLa cells expressing a chimeric receptor
composed of the extracellular ligand-binding domain of BR3 fused to
the death domain of DR4 were seeded 16 hr before treatment. BAFF (2
nM) was added to cells alone or after being preincubated with
miniBR3 (3 .mu.M), bhpBR3 (100 .mu.M) or a control hairpin peptide
with an unrelated turn sequence in the same bhp scaffold (bhpC, 100
.mu.M) for 30 min at room temperature. Apoptosis was assessed 24 hr
later. The turn sequences of bhpBR3 and bhpC are shown above the
corresponding bars.
MODE(S) FOR CARRYING OUT THE INVENTION
I. Definitions
[0028] The term ".beta.-turn" refers to a protein secondary
structure consisting of a tetrapeptide sequence which causes the
peptide chain to reverse direction, and which often contains a 4'
to 1' hydrogen bond, forming a pseudo 10-membered ring. The most
widely accepted classification of the different conformations of
the .beta.-turn is described in Chou and Fasman (1977) J Mol Biol
115:135-175, the disclosure of which is expressly incorporated by
reference herein. Various .beta.-turn types have been defined,
including for example, type I, I', II, and II'. For the purpose of
this invention, the term "reverse-turn" is used in a general sense
to encompass well known protein secondary structures including
.beta.-turns, .gamma.-turns, .beta.-hairpins and .beta.-bulges.
[0029] "Cell," "cell line," and "cell culture" are used
interchangeably herein and such designations include all progeny of
a cell or cell line. Thus, for example, terms like "transformants"
and "transformed cells" include the primary subject cell and
cultures derived therefrom without regard for the number of
transfers. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. Where distinct designations are
intended, it will be clear from the context.
[0030] The terms "competent cells" and "electoporation competent
cells" mean cells which are in a state of competence and able to
take up DNAs from a variety of sources. The state may be transient
or permanent. Electroporation competent cells are able to take up
DNA during electroporation.
[0031] "Control sequences" when referring to expression means DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. The control sequences that
are suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancers.
[0032] The term "coat protein" means a protein, at least a portion
of which is present on the surface of the virus particle. From a
functional perspective, a coat protein is any protein which
associates with a virus particle during the viral assembly process
in a host cell, and remains associated with the assembled virus
until it infects another host cell. The coat protein may be the
major coat protein or may be a minor coat protein. A "major" coat
protein is a coat protein which is present in the viral coat at 10
copies of the protein or more. A major coat protein may be present
in tens, hundreds or even thousands of copies per virion.
[0033] The terms "electroporation" and "electroporating" mean a
process in which foreign matter (protein, nucleic acid, etc.) is
introduced into a cell by applying a voltage to the cell under
conditions sufficient to allow uptake of the foreign matter into
the cell. The foreign matter is typically DNA.
[0034] A "fusion protein" is a polypeptide having two portions
covalently linked together, where each of the portions is a
polypeptide having a different property. The property may be a
biological property, such as activity in vitro or in vivo. The
property may also be a simple chemical or physical property, such
as binding to a target molecule, catalysis of a reaction, etc. The
two portions may be linked directly by a single peptide bond or
through a peptide linker containing one or more amino acid
residues. Generally, the two portions and the linker will be in
reading frame with each other.
[0035] "Heterologous DNA" is any DNA that is introduced into a host
cell. The DNA may be derived from a variety of sources including
genomic DNA, cDNA, synthetic DNA and fusions or combinations of
these. The DNA may include DNA from the same cell or cell type as
the host or recipient cell or DNA from a different cell type, for
example, from a mammal or plant. The DNA may, optionally, include
selection genes, for example, antibiotic resistance genes,
temperature resistance genes, etc.
[0036] "Ligation" is the process of forming phosphodiester bonds
between two nucleic acid fragments. For ligation of the two
fragments, the ends of the fragments must be compatible with each
other. In some cases, the ends will be directly compatible after
endonuclease digestion. However, it may be necessary first to
convert the staggered ends commonly produced after endonuclease
digestion to blunt ends to make them compatible for ligation. For
blunting the ends, the DNA may be treated in a suitable buffer for
at least 15 minutes at 15.degree. C. with about 10 units of the
Klenow fragment of DNA polymerase I or T4 DNA polymerase in the
presence of the four deoxyribonucleotide triphosphates. The DNA may
then purified by phenol-chloroform extraction and ethanol
precipitation. The DNA fragments that are to be ligated together
are put in solution in about equimolar amounts. The solution will
generally also contain ATP, ligase buffer, and a ligase such as T4
DNA ligase at about 10 units per 0.5 .mu.g of DNA. If the DNA is to
be ligated into a vector, the vector is first linearized by
digestion with the appropriate restriction endonuclease(s). The
linearized fragment is then treated with bacterial alkaline
phosphatase or calf intestinal phosphatase to prevent self-ligation
during the ligation step.
[0037] A "mutation" is a deletion, insertion, or substitution of a
nucleotide(s) relative to a reference nucleotide sequence, such as
a wild type sequence.
[0038] "Operably linked" when referring to nucleic acids means that
the nucleic acids are placed in a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence
or secretory leader is operably linked to DNA for a polypeptide if
it is expressed as a preprotein that participates in the secretion
of the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous and, in the case of a secretory leader, contiguous and
in reading phase. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accord with conventional
practice.
[0039] "Phage display" is a technique by which variant polypeptides
are displayed as fusion proteins to a coat protein on the surface
of phage, e.g. filamentous phage particles. A utility of phage
display lies in the fact that large libraries of randomized protein
variants can be rapidly and efficiently sorted for those sequences
that bind to a target molecule with high affinity. Display of
peptides and proteins libraries on phage has been used for
screening millions of polypeptides for ones with specific binding
properties. Polyvalent phage display methods have been used for
displaying small random peptides and small proteins through fusions
to either gene III or gene VIII of filamentous phage. Wells and
Lowman (1992) Curr. Opin. Biotech. 3:355-362 and references cited
therein. In monovalent phage display, a protein or peptide library
is fused to a gene III or a portion thereof and expressed at low
levels in the presence of wild type gene III protein so that phage
particles display one copy or none of the fusion proteins. Avidity
effects are reduced relative to polyvalent phage so that sorting is
on the basis of intrinsic ligand affinity, and phagemid vectors are
used, which simplify DNA manipulations. Lowman and Wells (1991)
Methods: A companion to Methods in Enzymology 3:205-216. In phage
display, the phenotype of the phage particle, including the
displayed polypeptide, corresponds to the genotype inside the phage
particle, the DNA enclosed by the phage coat proteins.
[0040] A "phagemid" is a plasmid vector having a bacterial origin
of replication, e.g., ColE1, and a copy of an intergenic region of
a bacteriophage. The phagemid may be based -on any known
bacteriophage, including filamentous bacteriophage. The plasmid
will also generally contain a selectable marker for antibiotic
resistance. Segments of DNA cloned into these vectors can be
propagated as plasmids. When cells harboring these vectors are
provided with all genes necessary for the production of phage
particles, the mode of replication of the plasmid changes to
rolling circle replication to generate copies of one strand of the
plasmid DNA and package phage particles. The phagemid may form
infectious or non-infectious phage particles. This term includes
phagemids which contain a phage coat protein gene or fragment
thereof linked to a heterologous polypeptide gene as a gene fusion
such that the heterologous polypeptide is displayed on the surface
of the phage particle. Sambrook el al. 4.17.
[0041] The term "phage vector" means a double stranded replicative
form of a bacteriophage containing a heterologous gene and capable
of replication. The phage vector has a phage origin of replication
allowing phage replication and phage particle formation. The phage
is preferably a filamentous bacteriophage, such as an M13, f1, fd,
Pf3 phage or a derivative thereof, a lambdoid phage, such as
lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative
thereof, a Baculovirus or a derivative thereof, a T4 phage or a
derivative thereof, a T7 phage virus or a derivative thereof.
[0042] "Preparation" of DNA from cells means isolating the plasmid
DNA from a culture of the host cells. Commonly used methods for DNA
preparation are the large- and small-scale plasmid preparations
described in sections 1.25-1.33 of Sambrook et al. After
preparation of the DNA, it can be purified by methods well known in
the art such as that described in section 1.40 of Sambrook et
al.
[0043] "Oligonucleotides" are short-length, single- or
double-stranded polydeoxynucleotides that are chemically
synthesized by known methods (such as phosphotriester, phosphite,
or phosphoramidite chemistry, using. solid-phase techniques such as
described in EP 266,032 published 4 May 1988, or via
deoxynucleoside H-phosphonate intermediates as described by
Froehler et al. (1986) Nucl. Acids Res., 14:5399-5407). Further
methods include the polymerase chain reaction defined below and
other autoprimer methods and oligonucleotide syntheses on solid
supports. All of these methods are described in Engels et al.
(1989) Agnew. Chem. Int. Ed. Engl. 28:716-734 . These methods are
used if the entire nucleic acid sequence of the gene is known, or
the sequence of the nucleic acid complementary to the coding strand
is available. Alternatively, if the target amino acid sequence is
known, one may infer potential nucleic acid sequences using known
and preferred coding residues for each amino acid residue. The
oligonucleotides are then purified on polyacrylamide gels.
[0044] "Polymerase chain reaction" or "PCR" refers to a procedure
or technique in which minute amounts of a specific piece of nucleic
acid, RNA and/or DNA, are amplified as described in U.S. Pat. No.
4,683,195 issued 28 Jul. 1987. Generally, sequence information from
the ends of the region of interest or beyond needs to be available,
such that oligonucleotide primers can be designed; these primers
will be identical or similar in sequence to opposite strands of the
template to be amplified. The 5' terminal nucleotides of the two
primers may coincide with the ends of the amplified material. PCR
can be used to amplify specific RNA sequences, specific DNA
sequences from total genomic DNA, and cDNA transcribed from total
cellular RNA, bacteriophage or plasmid sequences, etc. See
generally Mullis et al. (1987) Cold Spring Harbor Symp. Quant.
Biol. 51:263; Erlich, ed., PCR Technology, (Stockton Press, NY,
1989). As used herein, PCR is considered to be one, but not the
only, example of a nucleic acid polymerase reaction method for
amplifying a nucleic acid test sample comprising the use of a known
nucleic acid as a primer and a nucleic acid polymerase to amplify
or generate a specific piece of nucleic acid.
[0045] DNA is "purified" when the DNA is separated from non-nucleic
acid impurities. The impurities may be polar, non-polar, ionic,
etc.
[0046] "Recovery" or "isolation" of a given fragment of DNA from a
restriction digest means separation of the digest on polyacrylamide
or agarose gel by electrophoresis, identification of the fragment
of interest by comparison of its mobility versus that of marker DNA
fragments of known molecular weight, removal of the gel section
containing the desired fragment, and separation of the gel from
DNA. This procedure is known generally. For example, see Lawn et
al. (1981) Nucleic Acids Res., 9:6103-6114, and Goeddel et al.
(1980) Nucleic Acids Res., 8:4057.
[0047] A "transcription regulatory element" will contain one or
more of the following components: an enhancer element, a promoter,
an operator sequence, a repressor gene, and a transcription
termination sequence. These components are well known in the art.
U.S. Pat. No. 5,667,780.
[0048] A "transformant" is a cell which has taken up and maintained
DNA as evidenced by the expression of a phenotype associated with
the DNA (e.g., antibiotic resistance conferred by a protein encoded
by the DNA).
[0049] "Transformation" or "transforming" means a process whereby a
cell takes up DNA and becomes a "transformant". The DNA uptake may
be permanent or transient.
[0050] A "variant" or "mutant" of a starting polypeptide, such as a
fusion protein or a heterologous polypeptide (heterologous to a
phage), is a polypeptide that 1) has an amino acid sequence
different from that of the starting polypeptide and 2) was derived
from the starting polypeptide through either natural or artificial
(manmade) mutagenesis. Such variants include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequence of the polypeptide of
interest. Any combination of deletion, insertion, and substitution
may be made to arrive at the final variant or mutant construct,
provided that the final construct possesses the desired functional
characteristics. The amino acid changes also may alter
post-translational processes of the polypeptide, such as changing
the number or position of glycosylation sites. Methods for
generating amino acid sequence variants of polypeptides are
described in U.S. Pat. No. 5,534,615, expressly incorporated herein
by reference.
[0051] The term "peptide analog" refers to a molecule or part
thereof which is comprised of amino acids and resembles, with
regard to its binding ability and/or specificity, a specific
molecule, as defined above. Such peptide analogs may be found or
constructed by protein engineering techniques, such methods being
well known to those of skill in the art. Alternatively, such
peptide analogs may be found by a screening process, for example
wherein a natural binding partner of the specific molecule (which
specific molecule is not necessarily a protein or peptide), or a
portion thereof, is used as described herein (i.e. in a chimeric
protein) to screen peptide compounds for the ability to bind to it.
In a second screening step, the newly found peptide compound (or a
portion thereof) may itself be used as a peptide analog of the
specific molecule in a chimeric protein to screen for analogs of
the natural binding partner. Other methods for finding or making
peptide analogs will be apparent to those of skill in the art.
[0052] The term "epitope" means an antigen or portion thereof which
is capable of binding with an antibody as an antigenic
determinant.
[0053] By "binding partner complex" is meant the association of two
or more molecules which are bound to each other in a specific,
detectable manner; thus the association of ligand and receptor,
antibody and antigen, and chimeric protein and the compound to
which it binds.
[0054] The term "directly or indirectly labeled" refers to a
molecule may contain a label moiety which moiety emits a signal
which is capable of being detected, such as a radioisotope, a dye,
or a fluorescent or chemiluminescent moiety, or may contain a
moiety, such as an attached enzyme, ligand such as biotin, enzyme
substrate. epitope, or nucleotide sequence which is not itself
detected but which, through some additional reaction. is capable of
indicating the presence of the compound.
[0055] By "ligand" is meant a molecule or a multimeric molecular
complex which is able to specifically bind another given molecule
or molecular complex. Often, though not necessarily, a ligand is
soluble while its target is immobilized, such as by an anchor
domain imbedded into a cell membrane.
[0056] The term "receptor" refers to at least a portion of a
molecule, or a multimeric molecular complex which has an anchor
domain embedded into a cell membrane and is able to bind a given
molecule or molecular complex. Many receptors have particularly
high affinity for a ligand when either or both the receptor or
ligand are in a homo- or hetero multimeric form, such as a
dimer.
[0057] The term "solid support" refers to an insoluble matrix
either biological in nature, such as, without limitation, a cell or
bacteriophage particle, or synthetic, such as, without limitation,
an acrylamide derivative, cellulose, nylon, silica, and magnetized
particles, to which soluble molecules may be linked or joined.
[0058] By "naturally-occuring" is meant normally found in nature.
Although a chemical entity may be naturally occurring in general,
it need not be made or derived from natural sources in any specific
instance.
[0059] By "non naturally-occurring" is meant rarely or never found
in nature and/or made using organic synthetic methods.
[0060] "Modified" means non naturally-occuring or altered in a way
that deveates from naturally-occurring compounds.
II. General
[0061] The present invention is directed to
conformationally-constrained peptides and peptide libraries that
are useful for structure-activity analysis of bioactive molecules
and for drug lead discovery. The peptide of the invention comprises
two Cysteine residues that are capable of forming disulfide bond
with each other. Thus, the peptide adopts a cyclic form in
solution, which facilitates the formation of a .beta.-hairpin
scaffold. Disulfide cyclization is helpful, although not sufficient
to constrain the structure of many peptides. The rest of the
residues of the peptide are further selected to be significantly
biased toward the formation of the hairpin structure. Moreover, a
subset of the residues within the peptide of the invention is
varied to provide relative diversity for mimicking various
bioactive peptides having a identified secondary structure, such as
.beta.-turn, which has been proven significant in biological
processes.
[0062] In one aspect, the invention encompasses a peptide library
comprising a collection of structurally-constrained cyclic
peptides. Each peptide member of the library comprises amino acid
sequence C1-A1-A2-(A3).sub.n-A4-A5-C2 [SEQ ID NO:1], wherein
[0063] A1, A2, A3, A4, and A5 are naturally occurring L-amino
acids;
[0064] the terminus of Cysteine C1 is optionally protected with an
amino protecting group;
[0065] the terminus of Cysteine C2 is optionally protected with a
carboxy protecting group;
[0066] A1 and A5 are selected from the group consisting of amino
acids W, Y, F, H, I, V and T;
[0067] A2 and A4 are selected from the group consisting of amino
acids W, Y, F, L, M, I, and V;
[0068] A3 is any naturally occurring L-amino acid and n is an
integer that is selected from the group consisting of 3, 4, 5, 6,
7, 8, 9, 10, 11 and 12; and
[0069] C1 and C2 are joined together by a disulfide bond thereby
forming a cyclic peptide.
[0070] In one preferred embodiment, the peptides of the invention
have a .beta.-branched residue having two non-hydrogen substituents
on the .beta.-carbon of the amino acid residue at position A1 or A5
or both. Preferred peptides of the invention have a branched
aliphatic residue I, V or T at A1, A5 or both. More preferably, A1
or A5 is threonine (T). Even more preferably, both A1 and A5 are
threonine residues.
[0071] According to another preferred embodiment, the peptides have
an aromatic residue W, Y, F or H at position A1 or A5 or both. More
preferably, A1 or A5 is W. One preferred peptide contains H at A1
and V at A5.
[0072] In another preferred embodiment, the peptides of the
invention have an aromatic residue W, Y or F at position A2 or A4
or both. More preferably, A2 or A4 is W; and even more preferably,
A2 and A4 are Ws. Another preferred embodiment include peptides
having an unbranched aliphatic residue L or M at position A2 or A4
or both; more preferably A2 or A4 is Leucine. Still other preferred
peptides have a branched aliphatic residue I or V at position A2 or
A4 or both.
[0073] In the peptides of the invention, the number of the A3
residues n can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; preferably 4,
5, 6, 7, 8, 9, or 10; and more preferably 4, 5 or 6. In one
embodiment, n is 4 and the resulting peptides are decamers. In
these decamers, the residue sites A1, A2, A4 and A5 are each from a
selected group of amino acid residues as described above, whereas
the middle (A3).sub.4 is a tetrapeptide sequence with varying amino
acids. In one aspect of the invention, the (A3).sub.4 tetrapeptide
sequence is selected from those favorable to forming a .beta.-turn
structure, including but not limited to EGNK, ENGK, QGSF, VWQL and
GPLT.
[0074] In one aspect, the library of the instant invention contains
at least about 10.sup.2 member peptides, each of which has at least
one amino acid variation from others. Preferably, the library
contains at least about 10.sup.4 peptides, more preferably about
10.sup.10 peptides and even more preferably at least about
10.sup.12 peptides. According to various embodiments, the amino
acid variation occurs at defined positions within the peptides. For
example, variations can occur at hydrogen-bonded (HB) strand sites
(e.g., A1/A5) or non hydrogen-bonded (NHB) strand sites (e.g.,
A2/A4); a residue and its cross-strand counterpart (e.g., A1/A5 or
A2/A4) can have same or different amino acids. Variations can also
occur at the middle (A3).sub.n sites, wherein A3 can be any of the
20 naturally occurring L-amino acids.
[0075] The carboxy terminal end and the amino terminal end of the
cyclic peptide may be protected with any known protecting groups or
may be bonded to other amino acid residues (generally naturally
occurring residues), both in the (L) and in the (D) form through
conventional amide peptide bonds. The protecting groups and
additional residues can be added using conventional peptide
synthesis techniques. Generally from 1 to about 50, preferably from
1 to about 20, amino acid residues may be present on each of the
carboxy and amino terminal positions, independently. These
additional residues may be part of a known protein containing a
beta turn of interest or may be any other desired sequence of
residues. These additional residues may be added to determine the
effect of the beta turn structure on the structure of the overall
polypeptide or to determine the effect of the additional residues
on the binding of the beta turn cyclic peptide with a protein of
interest.
[0076] Alternatively, a library of cyclic peptides of the invention
can be prepared in which one or more of residues A1, A2, A4, and/or
A5 are independently fixed and residues A3 are varied s using known
methods of generating peptide libraries. A preferred method of
generating a library is phage display. Any known method of phage
display, such as those discussed in more detail below, may be used
in the method of the invention.
[0077] In another aspect, the invention encompasses a cyclic
peptide scaffold for presenting a turn hairpin structure. The
cyclic peptide scaffold comprises the amino acid sequence
C1-A1-A2-(A3).sub.n-A4-A5-C2 (SEQ ID NO:1), wherein C1 and C2 are
cysteines; A1, A2, A3, A4, and A5 are naturally occurring L-amino
acids; the terminus of Cysteine C1 is optionally protected with an
amino protecting group; the terminus of Cysteine C2 is optionally
protected with a carboxy protecting group; A1 and A5 are selected
from the group consisting of amino acids W, Y, F, H, I, V and T; A2
and A4 are selected from the group consisting of amino acids W, Y,
F, L, M, I, and V; A3 is any naturally occurring L-amino acid and n
is an integer that is selected from the group consisting of 3, 4,
5, 6, 7, 8, 9, 10, 11 and 12; and C1 and C2 are joined together by
a disulfide bond thereby forming a cyclic peptide.
[0078] In one preferred embodiment, the peptide scaffolds of the
invention have a .beta.-branched residue having two non-hydrogen
substituents on the .beta.-carbon of the amino acid residue at
position A1 or A5 or both. Preferred peptide scaffolds of the
invention have a branched aliphatic residue I, V or T at A1, A5 or
both. More preferably, A1 or A5 is threonine (T). Even more
preferably, both A1 and A5 are threonine residues.
[0079] According to another embodiment, the peptide scaffolds have
an aromatic residue W, Y, F or H at position A1 or A5 or both. More
preferably, A1 or A5 is W. One preferred peptide scaffold of the
invention has H at A1 and V at A5.
[0080] In another preferred embodiment, the peptide scaffolds of
the invention have an aromatic residue W, Y or F at position A2 or
A4 or both. More preferably, A2 or A4 is W; and even more
preferably, A2 and A4 are Ws. Another preferred embodiment include
peptide scaffolds having an unbranched aliphatic residue L or M at
position A2 or A4 or both; more preferably A2 or A4 is Leucine.
Still other preferred peptides have a branched aliphatic residue I
or V at position A2 or A4 or both.
[0081] In the peptides of the invention, the number of the A3
residues n can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; preferably 4,
5, 6, 7, 8, 9, or 10; and more preferably 4, 5 or 6. In one
embodiment, n is 4 and the resulting peptides are decamers. In
these decamers, the residue sites A1, A2, A4 and A5 are each from a
selected group of amino acid residues as described above, whereas
the middle (A3).sub.4 is a tetrapeptide sequence with varying amino
acids. In one aspect of the invention, the (A3).sub.4 tetrapeptide
sequence is selected from those favorable to forming a turn
structure, including but not limited to EGNK, ENGK, QGSF, VWQL and
GPLT.
[0082] The carboxy terminal end and the amino terminal end of the
cyclic peptide scaffold may be protected with any known protecting
groups or may be bonded to other amino acid residues (generally
naturally occurring residues), both in the (L) and in the (D) form
through conventional amide peptide bonds. The protecting groups and
additional residues can be added using conventional peptide
synthesis techniques. Generally from 1 to about 50, preferably from
1 to about 20, amino acid residues may be present on each of the
carboxy and amino terminal positions, independently. These
additional residues may be part of a known protein containing a
beta turn of interest or may be any other desired sequence of
residues. These additional residues may be added to determine the
effect of the beta turn structure on the structure of the overall
polypeptide or to determine the effect of the additional residues
on the binding of the beta turn cyclic peptide with a protein of
interest.
[0083] The present invention also encompasses methods of screening
for peptides having a .beta.-hairpin scaffold that is
conformationally stabilized, comprising the steps of a) providing a
combinatorial library of the invention as described above; b)
selecting at least two peptides from the combinatorial library,
wherein said at least two peptides differ by one amino acid at a
particular position A1, A2, A3, A4 or A5; c) determining the
conformations of the peptides; d) measuring and comparing the
relative stabilities of the peptides; and e) selecting the peptide
having a conformationally stabilized .beta.-hairpin scaffold. The
conformation and stability of the peptides can be determined using
many methods known in the art such as NMR, molecular modeling,
crystallography and free energy calculation. See, for example,
Cavanagh et aL (1995) Protein NMR Spectroscopy, Principles and
Practices (Academic Press, San Diego). Particular methods of
determining peptide conformation and stability are described in
more detail below by way of examples. The .beta.-turn containing
peptides of the invention can be useful for mimicking native
bioactive proteins in their binding activities.
[0084] The identity of the .beta.-turn residues A3 may be
determined by studying known protein structures and then
substituting the known structural sequence into the structured beta
hairpin compound of the invention. In this embodiment, residues A3
are taken from the known protein whereas residues A1, A2, A4 and A5
are as described for the invention. In this way, the fixed residues
of the invention can be used to structure particular turns from
proteins of interest, allowing one to test whether the protein turn
is sufficient for binding to a known protein binding partner, or
for antagonizing the relevant protein-protein interaction.
[0085] The invention also includes methods of identifying a peptide
capable of binding a specific binding partner, comprising the steps
of a) providing a combinatorial library as described above; b)
contacting the combinatorial library with a binding partner; c)
selecting from the library peptides capable of forming a
noncovalent complex with the binding partner; and d) optionally
isolating said peptides of step. Methods and technologies for
assessing peptide binding activity and isolating peptides of
interest are known in the art and described in more detail
below.
[0086] Binding partners of the peptides of the invention can be at
least a portion of any molecules, including any known or unknown
peptides, proteins, other macromolecules or chemical compounds that
are capable of binding to the peptides and optionally exerting
bioactivities. Protein molecules such as receptors, ligands,
antigens, antibodies, enzymes and enzyme substrates and fragments
or portions thereof are encompassed by "binding partners." Other
non-protein chemical compounds, organic or inorganic, can also be
the binding partners of the peptides.
III. .beta.-hairpin Peptides
[0087] One embodiment of the present invention involves a cyclic
peptide scaffold that adopt .beta.-hairpin conformations in
solution. The component parts of .beta.-hairpin structure include
paired antiparallel .beta.-strands and, preferably, pturns. The
preferential placement of disulfide-bonded cysteine pairs at
non-hydrogen bonded sites in the .beta.-strands has been studied,
as has been specific pairs of cross-strand residues that are
statistically favored (in either hydrogen bonded or non-hydrogen
bonded sites), at least in proteins. One study describes
experimental stability measurements of mutant proteins in which
various pairs of residues have been introduced into hydrogen bonded
sites on adjacent antiparallel strands. Smith & Regan (1995)
Science 270: 980-982. Attempts have been made to determine
intrinsic preferences for individual amino acids to adopt
conformations suited to the geometry of a .beta.-strand, either by
analyzing the residue content of .beta.-strands (Chou & Fassman
(1978) Annu. Rev. Biochem. 47:251-276) or by substituting various
amino acids into a .beta.-strand of a protein and measuring the
relative stabilities of the mutants (Kim & Berg (1993) Nature
362:267-270; Minor & Kim (1994) Nature 367:660-663; Minor &
Kim (1993) Nature 371:264-267; Smith et al Biochemistry (1994)
33:5510-5517). A revised statistical method for assigning residue
conformations has improved correlation with the various
experimental propensity scales (Munoz & Serrano (1994) Proteins
20:301-311). The propensity assigned to tryptophan is moderate in
all reported scales.
[0088] It has recently been shown that for some short, linear
peptides (4-16 amino acids), the hairpin conformation is partially
populated in aqueous solution. Both designed peptides and peptides
taken from protein sequences have exhibited this behavior. In
general, these studies involve peptides with statistically strong
turn sequences (e.g., asn-gly at i+1, i+2). Nevertheless, hairpin
populations seldom exceed 40-50% in aqueous solution.
[0089] A 16-mer peptide derived from the protein ubiquitin but with
a statistically more common turn sequence (MQIGVKNPDGTITLEV) did
form a highly populated hairpin in water (ca. 80%), but the hairpin
did not have the same strand register as in the native protein
(Searle et al. (1995) Nat. Struct. Biol. 2:999-1006). Another group
studied a similar peptide in which the turn region was replaced
with several sequences (MQIGVKSXXKTITLKV, wherein XX=pro-ala or
pro-gly; Haque & Gellman (1997) J. Am. Chem. Soc.
119:2303-2304). Evidence for the hairpin structure, with native
strand register, was observed for turns containing D-amino acids
but not for L-amino acid sequences. No population estimates were
given in this study.
[0090] Several groups have studied model peptides based originally
on a sequence from the protein tendamistat. The peptide YQNPDGSQA
shows NMR evidence of a small population of hairpin in water
(Blanco et al. (1993) J. Am. Chem. Soc. 115:5887-5888; de Alba et
al. (1995) Eur. J. Biochem. 233:283-292; Constantine et al. (1995)
J. Am. Chem. Soc. 117:10841-10854; Friedrichs et al. J. Am. Chem.
Soc. (1995) v 117, pp 10855-10864). A variant of this peptide with
strand residues of higher expected .beta.-propensity (IYSNPDGTWT)
was compared to a second peptide with a different turn sequence
(IYSNSDGTWT). Both peptides were estimated by NMR as 30% hairpin in
water (de Alba et al. (1996) Fold. Des. 1:133-144). Further
variation of this peptide, predominantly in the turn sequence,
yielded hairpins of various structures and mixed populations.
Generally no one conformer population exceeded 50% (de Alba et al.
(1997) J. Am. Chem. Soc. 119:175-183). In a final study, the three
N-terminal residues in peptide ITSNSDGTWT were replaced with
various sequences. Again, mixed conformers were frequently observed
and populations of a given hairpin conformer were generally less
than 50%: one peptide (YITNSDGTWT) did form a register-shifted
hairpin that was highly populated (80%; de Alba et al. (1997)
Protein Sci. 6:2548-2560). The authors of these studies conclude
that conformational preferences of the turn residues dominate
cross-strand interactions in determining the stability of hairpins,
at least in these short model peptides.
[0091] Analysis of hairpin sequences in crystal structures has
allowed the design of a different series of .beta.-hairpin
peptides. The target structure was a type I' turn flanked by
three-residue strands. Arg-gly sequences were added to the ends to
improve solubility. The peptide RGITVNGKTYGR is partially folded
into a hairpin conformation (about 30%) as determined by NMR
(Ramirez-Alvarado et al. (1996) Nat. Struct. Biol. 3:604-612). The
importance of strand residues is indicated by replacement of the
ile and val, the lys and tyr, or all four residues with alanine.
None of the alanine-substituted peptides showed any tendency to
form a hairpin. The same authors reported a second series of
experiments in which position i+1 of the turn was varied (asn to
asp, ala, gly or ser). No peptide was more structured than the
original sequence with asn in the turn (Ramirez-Alvarado et al.
(1997) J. Mol. Biol. 273:898-912). A review describing this work
stated that adding glu-lys pairs to the termini of the model
peptide stabilized the hairpin but did not give further details
(Ramirez-Alvarado et aL (1999) Bioorg. Med. Chem. 7:93-103).
[0092] Another model peptide series (RYVEVXGOMKILQ) has yielded
evidence for hairpin formation in water. Residue X as D-pro or
L-asn yields characteristic NOEs and alpha-H shifts, but the I-pro
peptide is unfolded. No population estimates are given, but D-pro
appears to give the more stable hairpin (Stanger & Gellman
(1998) J. Am. Chem. Soc. 120:4236-4237). A later study used a
disulfide-cyclized and back bone-cyclized version of this peptide
as a model for the fully folded hairpin, allowing the estimation of
hairpin population in uncyclized analogs (30-70%). Syud, et al.
(1999) Am Chem Soc. 121:11577-11578.
[0093] A designed 16-residue peptide (KKYTVSINGKKITVSI) based on
the met repressor DNA binding region formed a hairpin structure in
water with an estimated population of 50% at 303 K. Truncation of
one strand showed that the turn was populated without the strand
interactions, although to a lesser degree (35%). An analysis of the
thermodynamic parameters for hairpin formation showed that folding
is enthalpically unfavored and entropically driven, with
.DELTA.G=0.08 kcal/mol at 298 K (Maynard & Searle (1997) Chem.
Commun. 1297-1298; Griffiths-Jones et al. (1998) Chem. Commun.
789-790; Maynard et al. (1998) J. Am. Chem. Soc. 120:1996-2007).
Griffiths-Jones, et al. (1999) J. Mol Biol. 292:1051-1069.
[0094] A final hairpin peptide (GEWTYDDATKTFTVTE) derived from the
B1 domain of protein G (GB1) has some features relevant to the
peptides of the invention. Unlike the above described model
hairpins, the GB1 hairpin has four threonine residues at
hydrogen-bonded sites in the strands, including one thr-thr
cross-strand pair. This is generally believed to be an unfavorable
pairing. In addition, there are trp-val and tyr-phe pairs at
adjacent nonhydrogen-bonded sites that might interact to form a
small hydrophobic core. The reported data indicate that the GBI
peptide formed a well-populated hairpin (about 50%) in water. The
data are consistent with native strand pairing (Blanco et al.(1994)
Nat. Struct. Biol. 1:584-590). A denaturation study of the GB I
peptide allowed estimation of 80% hairpin at 273 K, and analysis of
the data (assuming .DELTA.Cp=0) yielded .DELTA.H=-11.6 kcal/mol,
.DELTA.S=-39 cal/mol K: i.e., folding is enthalpically driven and
entropically disfavored (Munoz et al. Nature (1998) v 390, pp.
196-199). The relative roles of enthalpy and entropy are reversed
compared to the met repressor peptide described above. Similar
results were reported from an NMR study of the thermal denaturation
of the GB1 peptide. Honda et al. (2000) J. Mol. Biol. 295:269-278.
A mutagenesis study from the same group (Kobayashi et al. (2000)
Biochemistry 39:6564-6571) identified the cross-strand
phenylalanine and tyrosine residues as especially important for
hairpin stability. Finally, this group reported a
disulfide-cyclized analog of the GB1 peptide (CEWTYDDATKTFTVTCK;
Kobayashi et al. (1999) Biochemistry 38:3228-3243). The structure
of this analog was not described; however, it bound 7-fold more
tightly to the remainder of the GB1 protein than did the uncyclized
peptide.
[0095] Several designed three-stranded sheets have been reported:
one of these contains only the usual 20 amino acids occurring in
proteins and folds in water (Kortemme et al. (1998) Science
281:253-256). One aspect of the design is addition of a trp at a
nonhydrogen-bonded position (by analogy to WW domains) while also
changing two nonhydrogen-bonded residues on the next strand to
unbranched amino acids. The authors state that the branched
residues would not allow the trp side chain to pack across to the
next strand. Thermodynamic analysis of denaturation data yields a
folding free energy of -0.6 kcal/mol at 278 K (estimated folded
population=80-90%).
[0096] Numerous examples have been reported of
disulfide-constrained peptides intended to mimic protein hairpins
or as de novo designed hairpins. In many cases the designs include
D-cysteines at one or both ends, as it was initially thought that
disulfide bond geometry was not compatible with the cross-strand
geometry of hairpins. However, there are some examples that do use
L-cys.
[0097] Evidence for structure is lacking in most studies of
disulfide-cyclized peptides. Examples listed here are those that
have been experimentally determined, or that use no unusual amino
acids and have potency close to a larger, hairpin-containing
protein in a biological assay.
[0098] The structure of a hexapeptide (Boc-CL-Aib-AVC-NMe) was
determined crystallographically, revealing a type II' turn and
.beta.-sheet geometry (Karle et al. J. Am. Chem. Soc. (1988) v 110,
pp 1958-1963). An octapeptide with the same cysteine spacing
(ACSPGHCE) was studied by NMR, and has a similar structure with a
turn centered on pro-gly (Walse et al. (1996) J. Comput.-Aided Mol.
Des. 10:11-22). Peptides of the form Ac-CXPGXC-NHMe were evaluated
by measurement of disulfide exchange equilibria, which indicated
turn preferences between peptides of as much as 1 kcal/mol (Milburn
et al. (1987) J. Am. Chem. Soc. 109:4486-4496).
[0099] An eleven-residue cyclic peptide (CGVSRQGKPYC) based on the
gene 5 protein from M13 is stably structured in aqueous solution,
as demonstrated by NMR analysis. The cyclic peptide adopts a
structure that is quite similar to the corresponding protein loop.
The authors claim that well-defined .beta.-hairpin structure had
not been previously reported for any unprotected
disulfide-constrained cycle (Rietman et al. (1996) Eur. J. Biochem.
238:706-713). This peptide has a val-pro pair at the nonhydrogen
bonded sites nearest to the cysteines.
[0100] Cyclization of peptides corresponding to loops from Limulus
anti-lipopolysaccharide factor (LALF) based on X-ray structure
yielded potent lipid A binders. There is no evidence for structure
in these peptides. Several of the peptides have aromatic-aromatic
pairs at the nonhydrogen-bonded sites nearest the cysteines;
however, the most potent (GCKPTFRRLKWKYKCG) has a pro-tyr pair
(Ried et al. (1996) J. Biol. Chem. 271:28120-28127).
[0101] Disulfide-cyclized peptides from the hairpin region of a
rabbit defensin have antibacterial activity exceeding (about 5 to
10-fold) that of the linear analogs. Circular dichroism
spectroscopy indicates some non-random structure in phosphate
buffer. The more potent peptide (CAGFMRIRGRIHPLCMRR) has a gly-pro
pair at the nonhydrogen bonded sites nearest to the cysteines
(Thennarasu & Nagaraj (1999) Biochem. Biophys. Res. Commun.
254:281-283).
[0102] A final study describes several peptides from the loops of
domain 1 of human CD4. In addition to a disulfide constraint, the
authors have added exocyclic aromatic amino acids to the peptide
termini. For example, a peptide covering CD4 residues 39-44 was
constrained as FCNQGSFLCY. No evidence for structure is given, but
one cyclic peptide (FCYICEVEDQCY) was reported to antagonize both
normal CD4 interactions and those involved in CD4-mediated cell
entry by HIV (Zhang et al. (1996) Nature Biotechnology 14:472-475;
Zhang et al. (1997) Nature Biotechnology 15:150-154).
IV. Peptide Libraries
[0103] Many methods for generating peptide libraries that are known
in the art can be used to generate the libraries of the invention.
In one embodiment, members of the peptide library can be created by
split-synthesis performed on a solid support such as polystyrene or
polyacrylamide resin, as described by Lam et al. (1991) Nature
354:82 and PCT publication WO 92/00091. In one aspect of the
invention, the library of cyclic peptides can be prepared in which
one or more of residues A1, A2, A4, and/or A5 are independently
fixed and residues A3 are varied.
[0104] A preferred method of generating the library of the present
invention is phage display. In a phage display library, the cyclic
peptide of the invention is fused to at least a portion of a phage
coat protein to form a fusion protein. The fusion protein can be
made by expressing a gene fusion encoding the fusion protein using
known techniques of phage display such as those described below.
The fusion protein may form part of a phage or phagemid particle in
which one or more copies of the cyclic peptide are displayed on the
surface of the particle. A gene comprising a nucleic acid encoding
the cyclic peptide or the fusion protein are within the scope of
the invention.
[0105] In another embodiment, the invention is a method comprising
the steps of constructing a library containing a plurality of
replicable expression vectors, each expression vector comprising a
transcription regulatory element operably linked to a gene fusion
encoding a fusion protein, wherein the gene fusion comprises a
first gene encoding a cyclic peptide of the invention and a second
gene encoding at least a portion of a phage coat protein, where the
library comprises a plurality of genes encoding variant cyclic
peptide fusion proteins. Variant first genes and libraries thereof
encoding variant cyclic peptides are prepared using known
mutagenesis techniques described in more detail below.
[0106] The invention also includes expression vectors comprising
the fusion genes noted above, as well as a library of these
vectors. The library of vectors may be in the form of a DNA
library, a library of virus (phage or phagemid) particles
containing the library of fusion genes or in the form of a library
of host cells containing a library of the expression vectors or
virus particles.
[0107] Also within the invention is a method of selecting novel
binding polypeptides comprising (a) constructing a library of
variant replicable expression vectors comprising a transcription
regulatory element operably linked to a gene fusion encoding a
fusion protein wherein the gene fusion comprises a first gene
encoding the cyclic peptide of the invention, and a second gene
encoding at least a portion of a phage coat protein, where the
variant expression vectors comprise variant first genes; (b)
transforming suitable host cells with the vectors; (c) culturing
the transformed host cells under conditions suitable for forming
recombinant phage or phagemid virus particles containing at least a
portion of the expression vector and capable of transforming the
host, so that the particles display one or more copies of the
fusion protein on the surface of the particle; (d) contacting the
particles with a target molecule so that at least a portion of the
particles bind to the target molecule; and (e) separating the
particles that bind from those that do not. In the method of the
invention, the phage coat protein is preferably the gene III or
gene VIII coat protein of a filamentous phage such as M13. Further,
preferably the culturing of the transformed host cells is under
conditions suitable for forming recombinant phage or phagemid
particles where the conditions are adjusted so that no more than a
minor amount of phage or phagemid particles display one or more
copies of the fusion protein on the surface of the particle
(monovalent display).
[0108] The invention also includes a method of introducing
structural bias into a phage-displayed library, using steps (a)
through (e) described above. The invention further includes a
method of selecting beta hairpin forming peptide structures from a
phage-displayed library, using steps (a) through (e) described
above where the target is known to bind beta hairpin peptide
structures. preferably a protein target known to so bind.
[0109] Bacteriophage (phage) display is a known technique by which
variant polypeptides are displayed as fusion proteins to the coat
protein on the surface of bacteriophage particles (Scott, J. K. and
Smith, G. P. (1990) Science 249: 386). The utility of phage display
lies in the fact that large libraries of selectively randomized
protein variants (or randomly cloned cDNAs) can be rapidly and
efficiently sorted for those sequences that bind to a target
molecule with high affinity. Display of peptide (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6378) or protein (Lowman el
al. (1991) Biochemistry 30:10832; Clackson et al. (1991) Nature
352: 624; Marks et al. (1991). J. Mol. Biol. 222:581; Kang et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8363) libraries on phage have
been used for screening millions of polypeptides for ones with
specific binding properties (Smith, G. P. (1991) Current Opin.
Biotechnol. 2:668). Sorting phage libraries of random mutants
requires a strategy for constructing and propagating a large number
of variants, a procedure for affinity purification using the target
receptor, and a means of evaluating the results of binding
enrichments. U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S.
Pat. No. 5,571,689; U.S. Pat. No. 5,663,143.
[0110] Typically, variant polypeptides, such as the cvclic
compounds of the invention, are fused to a gene III protein, which
is displayed at one end of the virion. Alternatively, the variant
polypeptides may be fused to the gene VIII protein, which is the
major coat protein of the virion. Such polyvalent display libraries
are constructed by replacing the phage gene III with a cDNA
encoding the foreign sequence fused to the amino terminus of the
gene III protein.
[0111] Monovalent phage display is a process in which a protein or
peptide sequence is fused to a portion of a gene III protein and
expressed at low levels in the presence of wild-type gene III
protein so that particles display mostly wild-type gene III protein
and one copy or none of the fusion protein (Bass et al. (1990)
Proteins 8:309; Lowman, H. B, and Wells, J. A. (1991) Methods: a
Companion to Methods in Enzymology 3:205). Monovalent display has
the advantage over polyvalent phage display that progeny phagemid
particles retain full infectivity. Avidity effects are reduced so
that sorting is on the basis of intrinsic ligand affinity, and
phagemid vectors, which simplify DNA manipulations, are used. See
also U.S. Pat. No. 5,750,373 and U.S. Pat. No. 5,780,279. Others
have also used phagemids to display proteins, particularly
antibodies. U.S. Pat. No. 5,667,988; U.S. Pat. No. 5,759,817; U.S.
Pat. No. 5,770,356; and U.S. Pat. No. 5,658,727.
[0112] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. No. 5,723,286; U.S. Pat.
No. 5,432,018; U.S. Pat. No. 5,580,717; U.S. Pat. No. 5,427,908;
and U.S. Pat. No. 5,498,530. See also U.S. Pat. No. 5,770,434; U.S.
Pat. No. 5,734,018; U.S. Pat. No. 5,698,426; U.S. Pat. No.
5,763,192; and U.S. Pat. No. 5,723,323.
[0113] A two-step approach may be used to select high affinity
ligands from peptide libraries displayed on M13 phage. Low affinity
leads are first selected from naive, polyvalent libraries displayed
on the major coat protein (protein VIII). The low affinity
selectants are subsequently transferred to the gene III minor coat
protein and matured to high affinity in a monovalent format.
[0114] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S Pat. No.
5,627,024), T4 phage display systems (Ren et al. (1998) Gene
215:439; Zhu (1997) CAN 33:534; Jiang et al. (1997) CAN 128:44380;
Ren et al. (1997) CAN 127:215644; Ren (1996) Protein Sci. 5:1833;
Efimov et al. (1995) Virus Genes 10:173) and T7 phage display
systems (Smith & Scott (1993) Methods in Enzymology
217:228-257; U.S. Pat. No. 5,766,905) are also known and can be
used to create a library of the cyclic peptides of the
invention.
[0115] Suitable gene III vectors for display of cyclic peptides of
the invention include fuSE5 (Scott, J. K., and Smith G. P. (1990)
Science 249:386-390); fAFFI (Cwirla et al. (1990). Proc. Natl.
Acad. Sci. U.S.A. 87:6378-6382); fd-CAT1 (McCafferty et al. (1990)
Nature (London) 348:552-554); m663 (Fowlkes et al. (1992)
Biotechniques 13:422-427); fdtetDOG, pHEN1 (Hoogenboom et al.
(1991) Nucleic Acids Res. 19:4133-4137); pComb3 (Gram et al. (1992)
Proc. Natl. Acad. Sci. US.A. 89:3576-3580); pCANTAB 5E (Pharmacia);
and LamdaSurfZap (Hogrefe (1993)Gene 137:85-91).
[0116] Phage display methods for proteins, peptides and mutated
variants thereof, including constructing a family of variant
replicable vectors containing a transcription regulatory element
operably linked to a gene fusion encoding a fusion polypeptide,
transforming suitable host cells, culturing the transformed cells
to form phage particles which display the fusion polypeptide on the
surface of the phage particle, contacting the recombinant phage
particles with a target molecule so that at least a portion of the
particle bind to the target, separating the particles which bind
from those that do not bind, are known and may be used with the
method of the invention. See U.S. Pat. No. 5,750,373; WO 97/09446;
U.S. Pat. No. 5,514,548; U.S. Pat. No. 5,498,538; U.S. Pat. No.
5,516,637; U.S. Pat. No. 5,432,018; WO 96/22393; U.S. Pat. No.
5,658,727; U.S. Pat. No. 5,627,024; WO 97/29185; O'Boyle et al.
(1997) Virology 236:338-347; Soumillion et al. (1994) Appl.
Biochem. Biotech. 47:175-190; O'Neil and Hoess. (1995) Curr. Opin.
Struct. Biol. 5:443-449; Makowski (1993) Gene 128:5-11; Dunn (1996)
Curr. Opin. Struct. Biol. 7:547-553; Choo and Klug (1995) Curr.
Opin. Struct. Biol. 6:431-436; Bradbury & Cattaneo (1995) TINS
18:242-249; Cortese et al., (1995) Curr. Opin. Struct. Biol.
6:73-80; Allen et al. (1995) TIBS 20:509-516; Lindquist &
Naderi (1995) FEMS Micro. Rev. 17:33-39; Clarkson & Wells
(1994) Tibtech. 12:173-184; Barbas (1993) Curr. Opin. Biol.
4:526-530; McGregor (1996) Mol. Biotech. 6:155-162; Cortese et al.
(1996) Curr. Opin. Biol. 7:616-621; McLafferty et al. (1993) Gene
128:29-36.
[0117] The gene encoding the coat protein of the phage and the gene
encoding the desired cyclic polypeptide portion of the fusion
protein of the invention (i.e., the cyclic peptide of the invention
fused to at least a portion of a phage coat protein) can be
obtained by methods known in the art (see generally, Sambrook et
al.). The DNA encoding the gene may be chemically synthesized
(Merrfield (1963) J. Am. Chem. Soc. 85 :2149) and then mutated to
prepare a library of variants as described below.
[0118] To ligate DNA fragments together to form a functional vector
containing the gene fusion, the ends of the DNA fragments must be
compatible with each other. In some cases, the ends will be
directly compatible after endonuclease digestion. However, it may
be necessary to first convert the sticky ends commonly produced by
endonuclease digestion to blunt ends to make them compatible for
ligation. To blunt the ends, the DNA is treated in a suitable
buffer for at least 15 minutes at 15.degree. C. with 10 units of
the Klenow fragment of DNA polymerase I (Klenow) in the presence of
the four deoxynucleotide triphosphates. The DNA is then purified by
phenol-chloroform extraction and ethanol precipitation or other DNA
purification technique.
[0119] The cleaved DNA fragments may be size-separated and selected
using DNA gel electrophoresis. The DNA may be electrophoresed
through either an agarose or a polyacrylamide matrix. The selection
of the matrix will depend on the size of the DNA fragments to be
separated. After electrophoresis, the DNA is extracted from the
matrix by electroelution, or, if low-melting agarose has been used
as the matrix, by melting the agarose and extracting the DNA from
it, as described in sections 6.30-6.33 of Sambrook et al.
[0120] The DNA fragments that are to be ligated together
(previously digested with the appropriate restriction enzymes such
that the ends of each fragment to be ligated are compatible) are
put in solution in about equimolar amounts. The solution will also
contain ATP, ligase buffer and a ligase such as T4 DNA ligase at
about 10 units per 0.5 .mu.g of DNA. If the DNA fragment is to be
ligated into a vector, the vector is at first linearized by cutting
with the appropriate restriction endonuclease(s). The linearized
vector is then treated with alkaline phosphatase or calf intestinal
phosphatase. The phosphatasing prevents self-ligation of the vector
during the ligation step.
[0121] After ligation, the vector with the foreign gene now
inserted is purified and transformed into a suitable host cell. A
preferred transformation method is electroporation. Electroporation
may be carried out using methods known in the art and described,
for example. in U.S. Pat. No. 4,910,140; U.S. Pat. No. 5,186,800;
U.S. Pat. No. 4,849,355; U.S. Pat. No. 5,173,158; U.S. Pat. No.
5,098,843; U.S. Pat. No. 5,422,272; U.S. Pat. No. 5,232,856; U.S.
Pat. No. 5,283,194; U.S. Pat. No. 5,128,257; U.S. Pat. No.
5,750,373; U.S. Pat. No. 4,956,288 or any other known batch or
continuous electroporation process. More than one (a plurality)
electroporation may be conducted to increase the amount of DNA
which is transformed into the host cells. Repeated electroporations
are conducted as described in the art. See Vaughan et al. (1996)
Nature Biotechnology 14:309-314. The number of additional
electroporations may vary as desired from several (2,3,4, . . . 10)
up to tens (10, 20, 30, . . . 100) and even hundreds (100, 200,
300, . . . 1000). Repeated electroporations may be desired to
increase the size of a combinatorial library, e.g. an antibody
library, transformed into the host cells.
[0122] Preferably, for library construction, the DNA is present at
a concentration of 25 micrograms/mL or greater. More preferably,
the DNA is present at a concentration of about 30 micrograms/mL or
greater, more preferably at a concentration of about 70
micrograms/mL or greater and even more preferably at a
concentration of about 100 micrograms/mL or greater even up to
several hundreds of micrograms/mL. Generally, the electroporation
will utilize DNA concentrations in the range of about 50 to about
500 micrograms/mL. A time constant during electroporation greater
than 3.0 milliseconds (ms) results in a high transformation
efficiency.
[0123] The DNA is preferably purified to remove contaminants. The
DNA may be purified by any known method, however, a preferred
purification method is the use of DNA affinity purification. The
purification of DNA, e.g., recombinant plasmid DNA, using DNA
binding resins and affinity reagents is well known and any of the
known methods can be used in this invention (Vogelstein, B. and
Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76:615; Callen, W.
(1993) Strategies 6:52-53). Commercially available DNA isolation
and purification kits are also available from several sources
including Stratagene (CLEARCUT Miniprep Kit), and Life Technologies
(GLASSMAX DNA Isolation Systems). Suitable nonlimiting methods of
DNA purification include column chromatography (U.S. Pat. No.
5,707,812), the use of hydroxylated silical polymers (U.S. Pat. No.
5,693,785), rehydrated silica gel (U.S. Pat. No. 4,923,978),
boronated silicates (U.S. Pat. No. 5,674,997), modified glass fiber
membranes (U.S. Pat. No. 5,650,506; U.S. Pat. No. 5,438,127),
fluorinated adsorbents (U.S. Pat. No. 5,625,054; U.S. Pat. No.
5,438,129), diatomaceous earth (U.S. Pat. No. 5,075,430), dialysis
(U.S. Pat. No. 4,921,952), gel polymers (U.S. Pat. No. 5,106,966)
and the use of chaotropic compounds with DNA binding reagents (U.S.
Pat. No. 5,234,809). After purification, the DNA is eluted or
otherwise resuspended in water, preferably distilled or deionized
water, for use in electroporation at the concentrations of the
invention. The use of low salt buffer solutions is also
contemplated.
[0124] Any suitable cells which can be transformed by
electroporation may be used as host cells in the method of the
present invention. Suitable host cells which can be transformed
include gram negative bacterial cells such as E. coli Suitable E.
coli strains include JM101, E. coli K12 strain 294 (ATCC number
31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776
(ATCC number 31,537), E. coli XL-lBlue (Stratagene), and E. coli B;
however many other strains of E. coli, such as XL1-Blue MRF', SURE,
ABLE C, ABLE K, WM100, MC1061, HB101, CJ136, MV1190, JS4, JS5,
NM522, NM538, and NM539, may be used as well. Cells are made
competent using known procedures. Sambrook et al., above,
1.76-1.81, 16.30.
[0125] Cell concentrations of about 10 colony forming units
(cfu)/mL) of viable living cells and greater are preferably used
for electroporation. More preferably, the viable cells are
concentrated to about 1.times.10.sup.11 to about 4.times.10.sup.11
cfu/mL. Preferred cells which may be concentrated to this range are
the SS320 cells described below. Cells are preferably grown in
culture in standard culture broth, optionally for about 6-48 hrs
(or to OD.sub.600=0.6-0.8) at about 37.degree. C., and then the
broth is centrifuged and the supernatant removed (e.g. decanted).
Initial purification is preferably by resuspending the cell pellet
in a buffer solution (e.g. HEPES pH 7.4) followed by
recentrifugation and removal of supernatant. The resulting cell
pellet is resuspended in dilute glycerol (e.g. 5-20% v/v) and again
centrifuged to form a cell pellet and the supernatant removed. The
final cell concentration is obtained by resuspending the cell
pellet in water or dilute glycerol to the desired
concentration.
[0126] A particularly preferred recipient cell for the
electroporation is a competent E. coli strain containing a phage F'
episome. Any F' episome which enables phage replication in the
strain may be used in the invention. Suitable episomes are
available from strains deposited with ATCC or are commercially
available (CJ236, CSH18, DH5alphaF', JM101, JM103, JM105, JM107,
JM109, JM110), KS1000, XL1-BLUE, 71-18 and others ). Strain SS320
was prepared by mating MC1061 cells with XL1-BLUE cells under
conditions sufficient to transfer the fertility episome (F'
plasmid) of XL1-BLUE into the MC1061 cells. In general, mixing
cultures of the two cell types and growing the mixture in culture
medium for about one hour at 37.degree. C. is sufficient to allow
mating and episome transfer to occur. The new resulting E. coli
strain has the genotype of MC1061 which carries a streptomycin
resistance chromosomal marker and the genotype of the F' plasmid
which confers tetracycline resistance. The progeny of this mating
is resistant to both antibiotics and can be selectively grown in
the presence of streptomycin and tetracycline. Strain SS320 has
been deposited with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va., USA on Jun. 18, 1998 and
assigned Deposit Accession No. 98795.
[0127] This deposit of strain SS320 was made under the provisions
of the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure and
the Regulations thereunder (Budapest Treaty). This assures
maintenance of a viable culture for 30 years from the date of
deposit. The organisms will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the cultures to the public upon
issuance of the pertinent U.S. patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes
first, and assures availability of the progeny to one determined by
the U.S. Commissioner of Patents and Trademarks to be entitled
thereto according to 35 USC .sctn.122 and the Commissioner's rules
pursuant thereto (including 37 CFR .sctn.1.14 with particular
reference to 886 OG 638).
[0128] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited cultures is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0129] Oligonucleotide-mediated mutagenesis is a preferred method
for preparing the substitution, deletion, and insertion variants of
the invention. This technique is well known in the art as described
by Zoller et al. (1987) Nucleic Acids Res. 10: 6487-6504. Briefly,
a gene encoding a protein fusion or heterologous polypeptide is
altered by hybridizing an oligonucleotide encoding the desired
mutation to a DNA template, where the template is the
single-stranded form of the plasmid containing the unaltered or
native DNA sequence of the gene. After hybridization, a DNA
polymerase is used to synthesize an entire second complementary
strand of the template which will thus incorporate the.
oligonucleotide primer, and will code for the selected alteration
in the gene. Generally, oligonucleotides of at least 25 nucleotides
in length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on
either side of the nucleotide(s) coding for the mutation. This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al. (1978) Proc. Nat'l. Acad. Sci. USA 75:
5765.
[0130] The DNA template is generated by those vectors that are
derived from the bacteriophage used in the phage display system,
e.g. bacteriophage M13 vectors (the commercially available M13mp18
and M13mp19 vectors are suitable), or those vectors that contain a
single-stranded phage origin of replication; examples are described
by Viera et al. (1987) Meth. Enzymol. 153:3. Thus, the DNA that is
to be mutated can be inserted into one of these vectors in order to
generate single-stranded template. Production of the
single-stranded template is described in sections 4.21-4.41 of
Sambrook et al.
[0131] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA
polymerase or the Klenow fragment of DNA polymerase 1, is then
added to synthesize the complementary strand of the template using
the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the
mutated form of the gene, and the other strand (the original
template) encodes the native, unaltered sequence of the gene. This
heteroduplex molecule is then transformed into a suitable host
cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabelled with 32-Phosphate to identify
the bacterial colonies that contain the mutated DNA.
[0132] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single-stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTTP), is combined with a modified
thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExoIII
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell such as E. coli
JM101, as described above.
[0133] Mutants with more than one amino acid to be substituted may
be generated in one of several ways. If the amino acids are located
close together in the polypeptide chain, they may be mutated
simultaneously using one oligonucleotide that codes for all of the
desired amino acid substitutions. If, however, the amino acids are
located some distance from each other (separated by more than about
ten amino acids), it is more difficult to generate a single
oligonucleotide that encodes all of the desired changes. Instead,
one of two alternative methods may be employed.
[0134] In the first method, a separate oligonucleotide is generated
for each amino acid to be substituted. The oligonucleotides are
then annealed to the single-stranded template DNA simultaneously,
and the second strand of DNA that is synthesized from the template
will encode all of the desired amino acid substitutions. The
alternative method involves two or more rounds of mutagenesis to
produce the desired mutant. The first round is as described for the
single mutants: wild-type DNA is used for the template, an
oligonucleotide encoding the first desired amino acid
substitution(s) is annealed to this template, and the heteroduplex
DNA molecule is then generated. The second round of mutagenesis
utilizes the mutated DNA produced in the first round of mutagenesis
as the template. Thus, this template already contains one or more
mutations. The oligonucleotide encoding the additional desired
amino acid substitution(s) is then annealed to this template, and
the resulting strand of DNA now encodes mutations from both the
first and second rounds of mutagenesis. This resultant DNA can be
used as a template in a third round of mutagenesis, and so on.
[0135] Cassette mutagenesis is also a preferred method for
preparing the substitution, deletion, and insertion variants of the
invention. The method is based on that described by Wells et al.
(1985) Gene 34:315. The starting material is a plasmid (or other
vector) containing the gene to be mutated. The codon (s) in the
gene to be mutated are identified. There must be a unique
restriction endonuclease site on each side of the identified
mutation site(s). If no such restriction sites exist, they may be
generated using the above-described oligonucleotide-mediated
mutagenesis method to introduce them at appropriate locations in
the gene. After the restriction sites have been introduced into the
plasmid, the plasmid is cut at these sites to linearize it. A
double-stranded oligonucleotide encoding the sequence of the DNA
between the restriction sites but containing the desired
mutation(s) is synthesized using standard procedures. The two
strands are synthesized separately and then hybridized together
using standard techniques. This double-stranded oligonucleotide is
referred to as the cassette. This cassette is designed to have 3'
and 5' ends that are compatible with the ends of the linearized
plasmid, such that it can be directly ligated to the plasmid. This
plasmid now contains the mutated DNA sequence of the gene. Vectors
containing the mutated variants can be transformed into suitable
host cells as described above.
[0136] The transformed cells are generally selected by growth on an
antibiotic, commonly tetracycline (tet) or ampicillin (amp), to
which they are rendered resistant due to the presence of tet and/or
amp resistance genes in the vector.
[0137] Suitable phage and phagemid vectors for use in this
invention include all known vectors for phage display. Additional
examples include pComb8 (Gram et al (1992) Proc. Natl. Acad. Sci.
USA 89:3576-3580); pC89 (Felici et al (1991) J. Mol. Biol.
222:310-310); pIF4 (Bianchi et al. (1995) J. Mol. Biol.
247:154-160); PM48, PM52, and PM54 (lannolo. (1995) J. Mol. Biol.
248:835-844); fdH (Greenwood et al (1991) J. Mol. Biol.
220:821-827); pfd8SHU, pfd8SU, pfd8SY, and fdISPLAY8 (Malik &
Perham (1996) Gene 171:49-51); "88" (Smith (1993) Gene 128:1-2);
f88.4 (Zhong et al. (1994) J. Biol. Chem, 269:24183-24188); p8V5
(Affymax); MB1, MB20, MB26, MB27, MB28, MB42, MB48, MB49, MB56:
(Markland et al. (1991) Gene 109:13-19). Similarly, any known
helper phage may be used when a phagemid vector is io employed in
the phage display system. Examples of suitable helper phage include
M13-KO7 (Pharmacia), M13-VCS (Stratagene), and R408
(Stratagene).
[0138] After selection of the transformed cells, these cells are
grown in culture and the vector DNA may then be isolated. Phage or
phagemid vector DNA can be isolated using methods known in the art,
for example, as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[0139] The isolated DNA can be purified by methods known in the art
such as that described in section 1.40 of Sambrook et al., above
and as described above. This purified DNA can then be analyzed by
DNA sequencing. DNA sequencing may be performed by the method of
Messing et al (1981) Nucleic Acids Res. 9:309, the method of Maxam
etal (1980) Meth. Enzymol. 65:499, or by any other known
method.
V. Applications
[0140] The various aspects and embodiments of the present invention
demonstrate the advantages of a novel model system for rationally
designing and analyzing peptides of defined structural features.
The combinatorial libraries comprising such peptides and methods of
using thereof provide useful information and tools for exploring
the basic structure-activity relationships involved in almost all
biological molecular interactions. The peptides disclosed herein or
generated according to the disclosure of the invention can be
candidates for various biological or therapeutic agents, including
but not limited to, enzyme inhibitors, ligand antagonists, ligand
agonists, toxins, and immunogens.
[0141] The following examples are provided by way of illustration
and not by way of limitation. All disclosures of the references
cited herein are expressly incorporated herein by reference in
their entirety.
EXAMPLES
Example 1
Design of a Structured, Disulfide-Constrained .beta.-Hairpin
Peptide Scaffold
[0142] In this example, we chose to investigate
disulfide-constrained .beta.-hairpins of the decamers in the form
of CX8C as scaffolds for .beta.-turn display. For our purpose, it
is essential to design a structure compatible with many turn
sequences. That is, residues other than those in the turn must
significantly bias the peptide toward hairpin structure. Disulfide
cyclization is helpful, although not sufficient to structure many
peptides. Our initial objective was to determine whether the
disulfide bond could be used not only as a covalent constraint, but
also to nucleate a more extended interaction of the
.beta.-strands.
Materials and Methods
[0143] Peptide Synthesis. Peptides were synthesized using standard
Fmoc chemistry on a Pioneer synthesizer (PE Biosystems), cleaved
from resin with 5% triisopropylsilane in trifluoroacetic acid
(TFA), and purified by reversed-phase HPLC
(acetonitrile/H.sub.20/0.1% TFA). Peptide identity was confirmed by
mass spectrometry. Peptides were converted to cyclic disulfides by
dropwise addition of a saturated solution of I.sub.2 in acetic acid
and repurified by HPLC. Purified peptides eluted as single
symmetric peaks on C18 analytical columns (0-40% acetonitrile in 40
minutes).
[0144] Cysteine Effective Concentration Measurements. Glutathione
stock solutions were prepared by mixing 3 volumes of 0.2 M reduced
glutathione (GSH) with I volume of 0.1 M oxidized glutatione
(GSSG). Aliquots were stored at -80.degree. C. and were stable for
several months; use of a single batch eliminated any error in
.DELTA..DELTA.G values that might arise from variability of total
glutathione concentration. Thiol-disulfide equilibria were
established by mixing 50 .mu.L peptide stock (approximately 3 mM in
water) with 50 .mu.L glutathione stock, deoxygenating the acidic
solution with vacuum/argon cycles from a Firestone valve, then
adding 300 .mu.L of deoxygenated buffer by syringe (0.2 M tris, pH
8.0; 1 mM EDTA; 67 mM tris base to titrate glutathione), followed
by further deoxygenation of the mixture. The final pH of all
reaction mixtures was 8.10.+-.0.05. Solutions were stirred under
argon and maintained at 20.degree. C. in a water bath. After 1.5 h,
successive aliquots (100 .mu.L) were removed with a gastight
syringe, immediately quenched by discharge into 400 .mu.L of 31 mM
HCl, and analyzed by HPLC with a minimum of delay. C.sub.eff values
were calculated from the molar ratios of the reduced and oxidized
forms of peptide and glutathione (peak area ratios corrected for
absorbance differences measured by HPLC), assuming 0.025 M total
glutathione monomer (i. e., neglecting the minor amount (<1%) of
glutathione present in mixed disulfides with peptide):
C.sub.eff=([peptide.sub.OX]/[peptide.sub.red]).times.([GSH].sup.2/[GSSG]-
) [GSH]+2[GSSG]=0.025 M [GSSG]=0.025 M/{2+3.26(GSH peak area/GSSG
peak area)} [peptide.sub.OX]/[peptide.sub.red]=equilibrium peak
area ratio/absorbance ratio
[0145] Two or three samples from each reaction mixture were
analyzed; there were no shifts in populations with time, and
calculated C.sub.eff values typically varied by less than 5%
(equivalent to 30 cal/mol uncertainty in .DELTA..DELTA.G).
[0146] NMR Spectroscopy. NMR samples contained 5-10 mM peptide in
92% H.sub.2O/8% D.sub.2O pH 5.1 and 0.1 mM 1,4-dioxane as chemical
shift reference. All spectra were acquired on a Bruker DRX-500 or a
Varian Unity-400 spectrometer at 15.degree. C. 2QF-COSY, TOCSY and
ROESY spectra were acquired as described (Cavanagh et al. (1995)
Protein NMR Spectroscopy, Principles and Practices (Academic Press,
San Diego) with gradient coherence selection (van Zijl et al.
(1995) J. Magn. Reson. 113A:265-270) or excitation sculpting (Hwang
& Shaka, (1995) J. Magn. Reson. 112A, 275-279) for water
suppression. Proton resonances were assigned by standard methods
(Wuthrich (1986) NMR of Proteins and Nucleic Acids (John Wiley and
Sons, New York). .sup.3.sup.JH.sup.N-H.sup..alpha. were obtained by
fitting Lorentzian lines to the antiphase doublets of
H.sup.N-H.sup..alpha. peaks in 2QF-COSY spectra processed to high
digital resolution in F.sub.2. .sup.3.sup.JH.sup.N-H.sup..alpha.
were extracted from COSY-35 spectra acquired on D.sub.2O solutions
of the peptides. Distance and dihedral angle restraints were
generated as described (Skelton et al. (1994) Biochemistry
33:13581-13592). 100 initial structures were calculated using the
hybrid distance geometry/simulated annealing program DGII (Havel et
al. (1991) Prog. Biophys. Mol. Biol. 56:43-78.); 80 of these were
further refined by restrained molecular dynamics using the AMBER
all-atom forcefield implemented in DISCOVER as described previously
(Skelton et al. (1994) Biochemistry 33:13581-13592.). 20
conformations of lowest restraint violation energy were chosen to
represent the solution conformation of each peptide.
[0147] Structure Calculation. Structures were calculated with 78
ROE-derived distance restraints (10 medium- and 28 long-range
restraints; upper bounds of 5.4, 4.3, 3.4 or 3.0 .ANG.) and 12
dihedral angle restraints. The final 20 structures had average
maximum violation of distance and dihedral angle restraints of
0.05.+-.0.02 .ANG. and 0.7.ANG.0.2.ANG., respectively; RMS
deviation from the experimental distance and dihedral angle
restraints were 0.007.+-.0.002 .ANG. and 0.29.+-.0.08.degree.,
respectively. The mean RMSD from the mean structure is 0.28.+-.0.04
.ANG. for N, C.sup..alpha., and C atoms of residues Cys1-Cys10
whilst 75% of residues had .PHI., .PSI.values in the most favored
portions of the Ramachandran plot (none were in the disallowed or
generously allowed region) (Laskowski et al. (1993) J. Appl.
Crystallogr. 26:283-291.).
[0148] NMR Analysis. NMR samples of CD4 peptides contained .about.2
mM peptide in 92% H.sub.2O/8% D.sub.2O, pH 3.5 with 50 .mu.M
3-(trimethylsilyl)-1-propane-1,1,2,2,3,3,-d.sub.6-sulfonic acid
(DSS) as a chemical shift reference. Spectra were acquired and
analyzed as described above. The structure of cd2 was calculated
from 84 (including 13 medium- and 23 long-range) ROE-derived
distance restraints and 13 dihedral angle restraints. The average
maximum violations of distance and dihedral angle restraints are
0.05.+-.0.01 .ANG. and 0.6.+-.0.4.degree., respectively; the RMSDs
from the experimental distance and dihedral angle restraints are
0.009.+-.0.002 .ANG. and 0.2.+-.0.1.degree., respectively. The
covalent geometry is good, with 74% of the .PHI., .PSI. angles
within the most favored and none in the disallowed or generously
allowed regions of the Ramachandran plot (Laskowski et al. (1993)
J. Appl. Crystallogr. 26:283-291).
[0149] Analysis of Sidechain Rotamers. Observation of both
.sup.3J.sub.H.sub..alpha.-.sub.H.beta.1 and
.sup.3J.sub.H.sub..alpha.-.sub.H.beta.2 in the range of 6-9 Hz
indicates that a side chain does not occupy a single classical
rotamer (X1=-60.degree., +60.degree. or 180.degree.), and most
likely samples all three staggered rotamer wells. This is the
situation for Trp3 and Leu8 of bhpW and Gln4, Phe7 and Leu8 of cd2.
ROE peaks observed to these side chains represent a time-average
over the range of X1 values sampled. Not all of these conformations
will give rise to readily observable ROE peaks, hence the structure
calculation process will be biased towards those rotamers for which
restraints could be obtained. For example, ROEs from Phe7 of cd2
are observed to protons in the opposite strand, thereby forcing
Phe7 to lie in the +60.degree. rotamer well. Given the large number
of backbone-backbone distance and .PHI. dihedral angle restraints,
the structures calculated for bhpW and cd2 do accurately represent
the solution conformation of these peptides, except in the over
deterrnination of some side chain orientations.
Results
[0150] In our survey of .beta.-sheets from a set of 928
non-redundant protein structures, the mean
C.sup..beta.-C.sup..beta. distances between hydrogen-bonded and
non-hydrogen bonded pairs of residues in adjacent strands were
4.82.+-.0.58 and 5.37.+-.0.56 .ANG., respectively, while the
average C.sup..beta.-C.sup..beta. distance in disulfide-bonded
cysteines was 3.84 .ANG.. Therefore, the C.sup..beta. atoms of
opposing residues on antiparallel strands are normally too far
apart for disulfide bond formation. Nonetheless, disulfide
crosslinks are sometimes found between cysteines in the
non-hydrogen-bonding register in .beta.-sheets. We found 23
disulfide-bonded cysteine pairs joining adjacent antiparallel
strands. In 14 of 23 cases, the disulfide packs tightly against the
hydrophobic sidechain two residues before one (or both) of the
cysteines (FIG. 1a). In 5 additional cases this hydrophobic site
was occupied by a polar or charged residue with .beta. and
.gamma.-methylenes (E, Q, or R). In particular, the sidechains of
either leucine or an aromatic amino acid provided good shape
complementarity to this characteristic disulfide conformation.
Accordingly, we chose leucine as residue 8 in our model peptide
(FIG. 1b), included threonines at positions 2 and 9 to promote an
extended backbone conformation, and chose the turn sequence EGNK as
a representative, but not overly strong, type II' .beta.-turn. To
deterrnine the best cross-strand pairing with leucine, position 3
was varied.
[0151] The presence of a disulfide provides a convenient probe for
hairpin stability. Thiol-disulfide equilibria were measured
relative to the reference thiol glutathione, yielding effective
concentrations (C.sub.eff) for the cysteine pairs. Larger values of
C.sub.eff indicate an increased proximity, on average, of the
cysteine thiols, consistent with formation of the hairpin
structure. This method has been used to evaluate the effect of
residue substitutions in a .beta.-turn on hairpin stability. Stroup
and Gierasch (1990) Biochemistry 29:9765-9771. Peptide C.sub.eff
values varied significantly for different residues at position 3
(FIG. 2a). Strikingly, tryptophan at position 3 strongly shifted
the peptide equilibrium toward the oxidized form: this behavior was
not caused by peptide aggregation. Scaling of the C.sub.eff values
to that of the alanine analog (-RT ln{C.sub.eff, x/C.sub.eff, ala})
yields free energy differences spanning >0.8 kcal/mol (FIG. 2b)
that can be interpreted as the relative tendencies of the peptides
to fold. These data do not, however, distinguish between effects on
the folded and unfolded states of the peptide. For example, a given
substitution might promote favorable side chain packing in the
oxidized peptide, or simply bias toward an extended backbone
conformation in the reduced peptide.
[0152] To assess whether the peptides were indeed forming
.beta.-hairpins, several of them were evaluated by .sup.1H NMR
spectroscopy (Table 1). The tryptophan peptide (bhpW) exhibited all
the hallmarks of a highly populated .beta.-hairpin in terms of
intense sequential H.sup..alpha.-H.sup.N NOEs, numerous backbone
cross-strand NOEs and large backbone scalar coupling constants
(.sup.3.sup.JH.sup.N-H.sup..alpha.>8.0 Hz) for strand residues.
The H.sup..alpha. chemical shifts for Cys1 and Cys10 were downfield
relative to values observed in unstructured peptides, indicating
that the antiparallel strands encompass these terminal residues.
The other peptides studied were judged to have a lower population
of hairpin structure (see Table 1). Interestingly, the NMR data
correlate well with C.sub.eff (Table 1); thus, the disulfide
exchange assay provides a useful quantitation of the degree of
hairpin structure in the oxidized peptides. TABLE-US-00001 TABLE 1
Comparison of cysteine effective concentrations (C.sub.eff)
and.sup.1H NMR data for selected model hairpin peptides Residue 3
No. of .sup.3J.sub.HN.sub.-H.sup..alpha. > .delta. (Cys1 .delta.
(Cys10 (X, FIG. 1b) C.sub.eff, mM 8 Hz H.sup..alpha.), ppm
H.sup..alpha.), ppm Trp bhpW 210 .+-. 4 7 5.20 5.00 Tyr 98 .+-. 2 7
5.07 4.91 Phe 88 .+-. 0 5 5.07 4.92 Leu 85 .+-. 1 6 5.04 4.89 Val
73 .+-. 0 4 4.97 4.85 Lys 52 .+-. 2 3 4.92 4.82 Asn 52 .+-. 1 3
4.84 4.76 random coil 0 4.71 4.71
The maximum number of strand residues with
.sup.3.sup.JH.sup.N-H.sup..alpha.>8 Hz is 8; for the tryptophan
peptide (bhpW), the Leu8 coupling constant is 7.9 Hz. Random coil
coupling constants are taken from Smith et al. (Smith et al. (1996)
J. Mol. Biol. 225:494-506). Random coil H.sup..alpha. chemical
shifts are taken from Wishart et al. (Wishart et al. (1992)
Biochemistry 31:1647-1651).
Example 2
Transfer of Alternative Tetrapeptide Turn Sequences onto the
Hairpin Scaffold
[0153] Structures calculated for bhpW according to Example 1
revealed a well formed antiparallel hairpin with a type II' turn
(Gly5-Asn6), and hydrophobic contacts between the side chains of
Cys1, Trp3, Leu8 and Cys10 (FIG. 3). Thermodynamic analysis of bhpW
stability was complicated by the failure of the oxidized peptide to
unfold fully, either at high temperature or in the presence of
chemical denaturants. Nevertheless, we estimate the hairpin
conformation to be highly populated, most likely >80%, at
15.degree. C. Because of its structural stability, we have chosen
bhpW for investigation as a turn display scaffold. Accordingly we
tested whether a different turn sequence could be structured by the
bhpw strand sequences.
[0154] A recent crystal structure of HIV gp120 bound to a
neutralizing antibody and to human CD4 revealed details of the
contact surfaces (Kwong et aL (1998) Nature 393:648-659.). As had
been anticipated from numerous mutagenesis studies, the CD4 region
most important for gp120 binding is the C'-C'' hairpin loop
(residues 37-46), with the critical Phe43 side chain extending from
the protein surface. In fact, CD4 residues 40-48 contribute 63% of
the surface area buried in the interface, with 23% of the total
contributed by Phe43 (Kwong et al. (1998) Nature 393:648-659.).
Unexpectedly, there is a large cavity in gp120, behind the Phe43
binding site, that is lined with hydrophobic residues. It seemed
possible that a structured peptide based on the C'-C'' turn might
bind to gp120 and if so, might be a starting point for designing
ligands that extend into the cavity seen in the crystal
structure.
[0155] We synthesized a disulfide-constrained peptide based on the
native sequence of the CD4 hairpin (residues 38-45, cd1 in Table 2)
and found it to be essentially unstructured in solution (FIG. 4a).
We then made the substitutions G2T and N3W, to match the
corresponding residues in bhpW (cd2, Table 2); residues L8 and T9
are already present in the native CD4 sequence. Peptide cd2 is
well-ordered, adopting a hairpin structure with a type II' turn
(FIG. 4). In FIG. 4A, peak assignments for cd2 are shown; arrows
indicate the location of the corresponding crosspeak in cd1. Those
cd2 residues with .sup.3.sup.JH.sup.N-H.sup..alpha.>8.3 Hz are
underlined; all cd1 residues have backbone coupling constants
between 5.9 and 7.7 Hz. From the measured C.sub.eff values, the CD4
turn (QGSF) destabilizes the model hairpin (EGNK turn) by 0.5
kcal/mol, and we found that both the T2 and W3 substitutions were
necessary for stable hairpin structure (Table 2). Importantly,
comparison of the peptide structure with that of CD4 indicates that
the backbone conformations are essentially the same, within the
uncertainties of the structure determinations (0.93 .ANG. RMSD;
FIG. 4B). FIG. 4B shows the NMR structure ensemble for cd2 (20
models; two orthogonal views) shown superimposed on CD4 residues
37-46 (red) from the crystal structure of gp120-bound CD4. The RMSD
for the 20 models, with respect to the mean coordinates for the
backbone atoms of residues 1-10, is 0.50.+-.0.09 .ANG.; comparison
of the mean coordinates of residues 1-10 with residues 37-46 of CD4
from the crystal structure yields an RMSD of 0.93 .ANG.. Note that
the .sup.3.sup.JH.sup..alpha.-H.sup..beta. coupling constants for
Phe7 of cd2 indicate that this sidechain is not fixed in the
rotamer seen in the ensemble; the Phe7 sidechain adopts multiple
conformations in solution, undoubtedly sampling that observed in
the co-crystal structure. This demonstrates that the peptide
scaffold correctly presents the CD4 .beta.-turn. TABLE-US-00002
TABLE 2 Comparison of bhpW and peptides based on the CD4 C'-C''
loop [.theta.]215 No. of .sup.3J.sub.HN.sub.-H.alpha. >
.delta.(Cys.sub.N H.sup..alpha.) .delta. (Cys.sub.C H.sup..alpha.)
Peptide C.sub.eff (mM) (deg cm.sup.2 dmol.sup.-1) 8 Hz (ppm) (ppm)
Ac-CTWEGNKLTC-NH.sub.2 bhpW 210 .+-. 4 -19,800 7 5.20 5.00 (SEQ ID
NO: 2) SCTWEGNKLTCK-NH.sub.2 273 .+-. 2 -17,400 n.d. n.d. n.d. (SEQ
ID NO: 3) Ac-CGNQGSFLTC-NH.sub.2 cd1 n.d. n.d. 0 4.66 4.66 (SEQ ID
NO: 4) Ac-CTWQGSFLTC-NH.sub.2 cd2 n.d. -15,800 6 5.08 4.93 (SEQ ID
NO: 5) SCGNQGSFLTCK-NH.sub.2 cd1a 45 .+-. 4 -1,500 0 4.80 4.72 (SEQ
ID NO: 6) SCTNQGSFLTCK-NH.sub.2 n.d. -5,000 2 4.96 4.79 (SEQ ID N0:
7) SCGWQGSFLTCK-NH.sub.2 48 .+-. 0 -6,100 3 5.00 4.88 (SEQ ID NO:
8) SCTWQGSFLTCK-NH.sub.2 cd2a 120 + 0 -14,000 6 5.36 5.14 (SEQ ID
NO: 9) n.d.: not determined; Cys.sub.N H.sup..alpha.: H.sup..alpha.
chemical shift for the more N-terminal cysteine (Cys1 or Cys2);
Cys.sub.C H.sup..alpha.: H.sup..alpha. chemical shift for the more
C-terminal cysteine (Cys10 or Cys11).
[0156] Terminal serine and lysine residues were added to improve
the solubility of some variants of the CD4 peptide, which are
otherwise uncharged. A similar modification was made to bhpW as a
control. Non-turn residues that differ between bhpw and the CD4
loop are underlined. Coelution of reduced and oxidized peptides
prevented measurement of C.sub.eff for the T2, N3 variant of the
CD4 peptide. Circular dichroism spectra were acquired at 10.degree.
C. with an Aviv Instruments, Inc. Model 202 spectrophotometer;
peptide concentrations were 20 .mu.M in 20 mM potassium phosphate,
pH 7.0.
[0157] Two other turn sequences evaluated were VWQL from the F-G
loop of domain 2 of human Fc-epsilon-RI, and GPLT from the EPO
agonist peptide EMPI. All three turns were evaluated in the trp
peptide scaffold and in cyclized peptides whose sequence matched
more closely the native parent hairpin loops: TABLE-US-00003
SCGNQGSFLTCK-NH.sub.2 CD4 peptides a (SEQ ID NO:10)
SCTWQGSFLTCK-NH.sub.2 b (SEQ ID NO:11) Ac-CTKVWQLWTC-NH.sub.2
Fc-epsilon-RI c (SEQ ID NO:12) peptides SCTWVWQLLTCK-NH.sub.2 d
(SEQ ID NO:13) SCHFGPLTWVCK-NH.sub.2 EMP1 peptides e (SEQ ID NO:14)
SCTWGPLTLTCK-NH.sub.2 f (SEQ ID NO:15)
[0158] Circular dichroism spectra show that in each case, the
designed trp hairpin scaffold yields a more structured peptide
(FIGS. 5a-c). NMR data are consistent with increased hairpin
structure in the peptides, demonstrating that the scaffold can bias
a variety of "difficult" turns toward structured states.
[0159] Other common turns that can be presented on the hairpin
scaffold include gamma-turns (3 amino acids), bulged turns (5 or 6
amino acids), and longer hairpins (8 amino acids). Other turn
lengths are known and are also compatible with the scaffold.
[0160] The results in Example 1 and 2 demonstrate that optimization
of a single strand position in a small disulfide-constrained
hairpin is sufficient to convert a very poorly structured molecule
to one that is highly structured (-.DELTA..DELTA.G>0.8
kcal/mol). The stem portion of the structured hairpin, --CTW . . .
LTC--, does not require an optimized turn sequence; thus, it is a
suitable scaffold for display of .beta.-turn libraries and for
studying particular turns that might not otherwise be highly
populated. Importantly, only natural amino acids are required, so
turn libraries may be displayed on phage.
[0161] It is interesting to compare the substitution energies we
report here with previous studies on .beta.-sheet systems. Although
the magnitude of the energy differences is similar, the rank order
we obtain does not correlate with experimental .beta.-propensity
scales or with observed residue pair frequencies in known
.beta.-sheets (Hutchinson et al. (1998) Protein Sci. 7:2287-2300,
Wouters, M. A. & Curmi, P. M. G. (1995) Proteins 22:119-131).
In particular, tryptophan is unexceptional in such scales. These
differences stress that average trends in typical protein domains
may not apply directly to small peptides in which most residues are
highly solvent exposed, complicating the use of such information in
de novo design. Furthermore, .DELTA..DELTA.G rank order does not
correlate well with increasing non-polar surface area of the side
chains, although the preferred residues are hydrophobic.
[0162] Finally, the hairpin stem is very small, yet the combination
of disulfide and cross-strand tertiary contact imparts a structural
bias exceeding that of a disulfide alone, e. g. CX.sub.4C. Although
it is known that some particular sequences (e. g., VVVV) (Milburn
et al. (1988) Int. J. Peptide Protein Res. 31:311-321.) cannot
adopt turn conformations compatible with our hairpin, it is also
true that very few of the turn sequences observed in proteins have
been shown to adopt well defined turn conformations in isolated
peptides. We have demonstrated a simple strategy to increase this
number. We envision that hairpin libraries with randomized turn
sequences (e.g., XCTWX.sub.4LTCX) might yield structured ligands
whose binding determinants could be transferred readily to small
synthetic turn mimetics or even used directly to identify
small-molecule leads for high-throughput affinity optimization
(Rohrer et al. (1998) Science 282:737-740) .
Example 3
Quantification of the Relative Contributions from Turns and
Cross-Strand Interactions
[0163] In Example 1 above, substitutions were introduced into
position 3 (X) of the model peptide bhp (peptide 1). This guest
site is quite close in space to the type II' turn (gly-asn, FIG.
1). To further investigate whether hairpins with different turn
sequences and geometries would have different residue preferences
at the NHB guest site, the central gly-asn sequence in model
peptide 1 is replaced with the type I' turn asn-gly (peptide 2) and
the type II' turns D-pro-asn and D-pro-gly (peptides 3 and 4).
Substitutions at position 3 (X) were chosen to span the range of
hairpin stabilities we observed in the gly-asn series. C.sub.eff
was measured as previously described in Example 1. The values we
obtained for the different turns are compared in FIG. 6.
TABLE-US-00004 CTXEGNKLTC 1 II' SEQ ID NO:16 CTXENGKLTC 2 I' SEQ ID
NO:17 CTXEpNKLTC 3 II' SEQ ID NO:18 CTXEpGKLTC 4 II' SEQ ID NO:19 X
= W, Y, L, V, T, or D; p = D-pro.
[0164] In all cases, tryptophan at position 3 yields the largest
C.sub.eff value for a given turn, demonstrating that its
stabilizing influence is general. The large changes in C.sub.eff
for the different cross-strand interactions (horizontal axis) and
turn sequences (vertical axis) show that both can contribute
significantly to stability in these cyclic hairpin peptides.
Finally, there are striking linear correlations between data sets,
indicating that substitutions at strand position 3 and the turn
replacements make independent contributions to stability of the
cyclic hairpin. These data suggest that the hairpin fold may be
quite modular, which would significantly simplify hairpin
design.
[0165] Relative turn energies can be calculated by comparing
C.sub.eff for the appropriate pairs of peptides. However, the
correlation in FIG. 6 allow the calculation of relative turn
energies from the slopes, which should be less sensitive to
experimental error. These values are listed in Table 3. Compared to
asn-gly (type I'), gly-asn (type II') is less stablizing, while the
D-pro-containing turns (also type II') enhance hairpin stability.
In the one case where a comparison may be made, asn-gly vs.
D-pro-gly, the AAG value obtained here agrees reasonably well with
that obtained by NMR. This suggests that the reference states
assigned by Syud et al. (1999) J Am Chem Soc 121:11577 and their
assumption of two-state folding are appropriate for their model
system; however, defining such reference states is not always
feasible. TABLE-US-00005 TABLE 3 Turn Energies Relative to Asn-Gly
in peptides 1-4 C.sub.eff correlation turn sequence slope R.sup.2
.DELTA..DELTA.G, kcal mol.sup.-1 a asn-gly repeat .sup.b 1.19 0.99
-0.10 gly-asn 0.29 0.97 0.72 D-pro-asn 2.72 0.98 -0.58 D-pro-gly
.sup.c 3.01 0.99 -0.64 .sup.a .DELTA..DELTA.G = -RT ln(slope), T =
293 K. Slopes are from the plot in FIG. 2. .sup.b Two completely
independent sets of measurements were made for the asn-gly peptides
in order to assess the reliability of the assay over time.
.DELTA..DELTA.G for the two data sets (.about.100 cal mol.sup.-1)
may be taken as an estimate of the error in the turn energies
reported here. .sup.c .DELTA..DELTA.G may be compared to the value
of -0.52 .+-. 0.11 kcal mol.sup.-1 (277 K) recently reported by
Syud et al. (1999) supra.
[0166] Alternatively, substitution energies for the strand position
may be obtained by plotting the same data, grouped instead by the
residue X (not shown). The correlations are again excellent, and
the slopes yield the free energy changes (Table 4). The range of
energies is larger than that reported in Example 1 for peptide 1
(1.42 vs. 0.85 kcal mol.sup.-1). Much of the difference is traced
to those substitutions at the bottom of the stability scale
(particularly asp). The less stable of the gly-asn turn peptides
are not detectably structured, and C.sub.eff assays do not register
any difference between them. Thus, the data obtained in peptides
with the stronger turn sequences provide a more complete view of
the strand substitution energies. TABLE-US-00006 TABLE 4 Relative
Energetic Contributions from Strand Residue X residue X slope
.sup.a .DELTA..DELTA.G, kcal mol.sup.-1 b .DELTA..DELTA.G, GN turn
.sup.c trp 2.92 -0.62 -0.53 tyr 1.27 -0.14 -0.08 val 0.66 0.24 0.09
thr 0.45 0.46 0.30 asp 0.25 0.80 0.32 .sup.a C.sub.eff values were
plotted against those of leucine peptides 1-4. .sup.b
.DELTA..DELTA.G = -RT ln(slope), T = 293 K. .sup.c .DELTA..DELTA.G
for the gly-asn turn series (.DELTA..DELTA.G = -RT ln {C.sub.eff,
X/C.sub.eff, leu}), as described in Example 1.
[0167] In order to assess how the turn types affect the hairpin
structure, the tryptophan analogs of 2 and 3 were characterized by
NMR spectroscopy, and structures were calculated as described in
Example 1 for peptide 1. The comparison of minimized mean
structures in FIG. 7 reveals that the backbone and side chain
conformations are very similar for the non-turn residues
(RMSD.about.0.3 .ANG.) regardless of the type of turn present.
Thus, consistent with the linear correlations in C.sub.eff (FIG.
6), these three turns do not exert any structural influence on the
adjacent strands.
[0168] The importance of the turn sequence and good cross-strand
pairing to hairpin structure has been addressed in many model
studies. However, there is little quantitative data or systematic
evaluation of residue substitutions. Our data show that, for these
simple cyclic peptides, substitutions in a strand site and in the
turn conform to simple linear free-energy relationships and have
independent and additive effects on hairpin stability. This is
unexpected, given their proximity in the structure (FIGS. 3 and 7)
and the reported sensitivity of calculated turn energies to
features of the protein anchorage Mattos et al. (1994) J. Mol.
Biol. 238:733. Nonetheless, it would appear that coupling between
these turns and the strands is negliglible compared to the large
influence each exerts alone. This suggests that .beta.-hairpin
stability may be understood by separate analysis of these
components.
Example 4
Quantification of Energetic Contributions from Cross-Strand
Residues
[0169] The results of above Examples revealed tryptophan to be
quite stabilizing in the non hydrogen-bonded (NHB) strand site X of
peptide 1, when paired with a cross-strand leucine. The tryptophan
peptide (bhpW) was highly structured in water, adopting the
intended hairpin conformation (FIG. 3). Here we investigate the
relationship between the NHB cross-strand residues. Remarkably, we
find that residue preferences for the two structurally inequivalent
sites are the same, and that specific pair interactions produce
only minor deviations from the single site contributions.
Accordingly, a tryptophan-tryptophan cross-strand pair appears to
be optimal for hairpin stability.
[0170] Our observation of a stabilizing contribution from
tryptophan prompted us to question how general the effect might be.
The tryptophan in peptide bhpW (FIG. 3) is spatially near both the
cross-strand leucine and the side chains of residues in the type
II' turn. Therefore, it seemed possible that the effect of
tryptophan might depend on stabilizing contacts with these other
residues. In order to address this question, we reversed the
hydrophobic pairs (peptide 5), varying the amino acid at position 8
(nearest the disulfide, FIG. 3) with leucine fixed at position 3.
Effective concentrations (C.sub.eff) of the cysteine thiols were
determined as in our previous studies. TABLE-US-00007 CTXEGNKLTC 1
SEQ ID NO:20 CTLEGNKXTC 5 SEQ ID NO:21 CTXEGNKWTC 6 SEQ ID NO:22
CTWEGNKXTC 7 SEQ ID NO:23 X = W, Y, F, L, M, I, V or A.
[0171] We find that tryptophan at position 8 is the most
stabilizing of those residues tested (FIG. 8A). Significantly, the
C.sub.eff values are quite close for the trp-leu and leu-trp pairs,
indicating that the two arrangements are about equivalent
energetically. This result appears to hold for other residue pairs
with leucine: the rank order and numeric values of C.sub.eff are
similar, but not exact, in the two series (FIG. 8A).
[0172] To test whether the equivalence of reversed hydrophobic
pairs might be more general, we examined peptide series 6 and 7, in
which residues are instead paired with a cross-strand tryptophan
(FIG. 8B). As with leucine pairs, a close correspondence is seen
between the two tryptophan series, both in rank order and value of
C.sub.eff. We conclude that the two cross-strand sites are
essentially equivalent, despite the differences in side chain
position relative to the turn and disulfide.
[0173] The two leucine series (1 and 5) may be compared to the
tryptophan series (6 and 7). The trends in the two data sets are
remarkably similar (FIGS. 8A and B), suggesting that the
cross-strand residues contribute to stability in an independent
manner. To explore this idea, we calculated free energy differences
for substitutions in each of the peptide series relative to a
reference peptide in that series (.DELTA..DELTA.G=-RT ln
(C.sub.eff, X/C.sub.eff, ref)). Representative comparisons are
plotted in FIG. 9.
[0174] Linear free energy relationships exist among the four data
sets. This is seen in comparisons of particular cross-strand pairs
switched between NHB sites 3 and 8, and also for comparisons of trp
pairs with leu pairs in the same orientation (FIG. 3). (There is
more scatter in the latter correlations.) The slopes (.rho.) were
determined using .DELTA..DELTA.G values scaled to two different
reference peptides in each series (X=ala and trp). The pvalues
obtained were not greatly different (Table 5). TABLE-US-00008 TABLE
5 Slopes (.rho.) of Hammett plots for peptide series 1, 5-7 x-axis
data set .rho., X3 vs. X8 .rho., leu vs. trp W3X8.sup.a 1.15
(1.11).sup.b 0.47 (0.43).sup.b L3X8 0.98 (0.86).sup.b -- X3W8 --
0.43 (0.32).sup.b .sup.aPlots vs. W3X8 data are shown in FIG. 9.
Values in parentheses were obtained using the tryptophan peptide in
each series as internal reference (instead of the alanine
peptide).
[0175] Consistent with the idea that positions 3 and 8 are
equivalent, .rho. is near 1 for plots comparing these data. In
contrast, when leu pairs are compared to trp pairs, .rho. is about
0.4. This means that for a given pair of residues X, the expected
difference in hairpin stability is .about.2.5-fold larger with trp
as the cross-strand partner. Given these simple relationships,
.DELTA..DELTA.G could be calculated for any substitution relative
to a reference pair by multiplying a substituent energy
.sigma..sub.x by .rho. for the cross-strand partner (see below).
This is surprising, as these residues are within contact distance,
and it has important implications for .beta.-hairpin design.
[0176] Statistical analyses of HB and NHB cross-strand pairs in
.beta.-sheet proteins find many residue pairs to be positively or
negatively correlated with high confidence. Largely in accord with
the statistical preferences, protein mutagenesis studies have
identified interaction energies as large as 1 kcal mol.sup.-1
between HB pairs. It has been proposed that including cross-strand
pairs preferred in proteins might improve stability or fix strand
register in isolated .beta.-hairpins.
[0177] To look for these effects, we calculated pair interaction
energies (FIG. 10). C.sub.eff ratios yield .DELTA..DELTA.G for the
single or double substitutions. Typically, the difference between
.DELTA..DELTA.G for the double substitution and
.SIGMA..DELTA..DELTA.G for the single substitutions is taken as an
interaction energy. In the example shown, this would be -136 cal
mol.sup.-1 for the trp-tyr pair relative to a leu-leu reference
state. If the the single substitution energies are calculated
sequentially, scaling by .rho. in the second step, the discrepancy
is only +41 cal mol.sup.-1 (insignificant in these experiments).
For the phe-trp pair (compare FIG. 2, top and bottom), a similar
analysis yields .DELTA..DELTA..DELTA.G=-253 cal mol.sup.-1 when
.rho. is included. This discrepancy is significant, and it suggests
that there might be some specific structural advantage in pairing
phe with trp (beyond the general superiority of trp). It is
interesting that the discrepancy is small when compared to the
total range of energies seen for single site substitutions (FIG.
9); we believe that such pair-specific effects (and experimental
error) may explain the scatter in our correlations.
[0178] Our experiments show clearly that cross-strand tertiary
contacts enhance hairpin stability. Notably, introduction of the
trp-trp pair results in a large stabilization compared to our
original peptide bhpW (FIGS. 3 and 8), and we believe that, despite
its rarity in proteins, trp-trp is the optimal NHB pair for
isolated hairpins. In most cases, the pair interaction energies we
obtain through conventional double mutant analysis are adequately
explained by differences in .rho.. That is, these energies are not
specific to a single pair, but instead reflect greater or lesser
sensitivity to all residue substitutions opposite a given
cross-strand partner. Therefore, we conclude that the combined
single site preferences (.sigma. and .rho.) are most important in
predicting hairpin stability. Significantly, it should be possible
to make these predictions from a limited basis set of experimental
data.
Example 5
Construction of Phage-Displayed Libraries Based on the trp Peptide
Scaffold
[0179] Libraries of random peptides fused to the gene 8 protein of
the filamentous bacteriophage M13 were produced by Kunkel
mutagenesis of plasmid pS1302b, a derivative of pS349 (U.S. patent
application Nos. 60/103,514 and 60/134,870, incorporated herein by
reference). Plasmid pS1302b includes the tac promoter and malE
leader sequence of pS349. The hGH sequence and Gly/Ser-rich linker
sequence of pS349 were replaced by the sequence: TABLE-US-00009
5'-TAA-TAA-TAA-ATG-GCT-GAT-CCG-AAC- (SEQ ID NO:24)
CGT-TTC-CGC-GGT-AAA-GAT-CTG-GGT- GGC-GGT-ACT-CCA-AAC-GAC-CCG-CCA-
ACC-ACT-CCA-CCA-ACT-GAT-AGC-CCA- GGC-GGT-3'
[0180] The inserted sequence encodes three stop codons, the GD
epitope tag, and a linker selected for high-level display of hGH.
The plasmid also includes the lac repressor (laclq) and the
ampicillin resistance gene from pS349. The oligonucleotide used to
construct the library was: TABLE-US-00010
5'-TCC-GCC-TCG-GCT-TAT-GCA-NNS-TGC- (SEQ ID NO:25)
ACT-TGG-NNS-NNS-NNS-NNS-CTG-ACT-
TGT-NNS-ATG-GCT-GAT-CCG-AAC-CGT-3'
[0181] The form of the random peptides was therefore XCTWX4LTCX. A
library of 10.sup.9 to 10.sup.10 individual transformants was
prepared by previously described methods (U.S. patent application
Nos. 60/103,514 and 60/134,870). Approximately one-third of
individual clones encoded a functional peptide sequence. The
remainder were starting template, contained stop codons, or
contained single nucleotide deletions. The library size is thus
adequate to include several copies of each possible random
sequence.
Example 6
Selection of Binding Peptides from the Structurally-Biased
Library
[0182] Nunc MaxiSorp plates were coated overnight with 2 .mu.pg/mL
rhuFc-epsilon-RI-IgG fusion in PBS. Plates were then blocked for
one hour at room temperature with 0.5% BSA (Sigma A-7638) in PBS.
Negative wells were prepared by coating only with 0.5% BSA. Phage
(10.sup.11 ifu per well) were added to ten each positive and
negative wells and incubated with shaking for 20 h at room
temperature. After extensive washing to remove
nonspecifically-bound phage, binders were eluted by treatment with
0.2 M glycine, pH 2 for five minutes. The eluted phage were then
neutralized by addition of TRIS base and used to infect a culture
of E. coli (XL1-blue, Stratagene). Several cycles of binding,
elution, and amplification (3-5 total) were conducted under similar
conditions. 192 individual clones were screened for binding to the
target receptor by incubation of phage supernatant with plates
prepared as described for phage sorting. After washing, wells were
treated with alpha-M13-HRP conjugate (Pharmacia Biotech
27-9421-01), and bound antibody was detected with OPD substrate
(Sigma P-9187). Plate absorbance (A.sub.492-A.sub.405) was compared
between positive and negative plates to identify those clones
positive for receptor binding. Twelve such clones were
identified.
[0183] The sequence of positive clones is identified by sequencing
the encoding DNA. Peptides corresponding to the displayed sequences
(i.e., 12-mers) are synthesized using standard solid-phase methods.
The peptides are then assayed using an appropriate biological or
binding assay to determine their potency. Peptides can be evaluated
for hairpin structure using any of the known techniques outlined
above: circular dichroism, NMR, or disulfide equilibrium.
Substitutions may then be made in the peptides to determine the
relative contributions of the selected turn residues to binding.
Ideally, these substitutions will not disrupt the scaffold
structure. Once the nature of the binding motif is understood, the
turn sequence can then be transferred onto a suitable organic
scaffold for further optimization.
Example 7
Stability of the Decamer bhp Scaffold with Specific HB Residues
[0184] The present example investigates substitutions at the
hydrogen-bonded (HB) strand positions 2 (i.e., A1) and 9 (i.e., A5)
of the bhp scaffold. The results show that the stability
determinants at these sites are different than those at the
non-hydrogen-bonded (NHB) positions 3 (i.e., A2) and 8 (i.e., A4).
In marked contrast to the observed equivalence of positions 3 and
8, positions 2 and 9 have very different residue preferences from
each other. In particular, a very large stabilization to the cyclic
hairpin fold from a valine residue at position 9; a much smaller
stabilization occurs in Val2 variants. This effect is evident from
C.sub.eff-derived free energy differences and from NMR structural
analysis of Val9 and Val2 bhp analogues. Further 10 evidences
suggest that the strong stabilizing effect of Val9 in the bhp
peptides is a consequence of its proximity to the cross-strand
disulfide. Intriguingly, Val9 is often present in
disulfide-cyclized .beta.-hairpins selected from phage-displayed
peptide libraries, suggesting that its stabilizing effect is
general for this fold.
[0185] In order to evaluate the residue preferences at
hydrogen-bonded strand positions of .beta.-hairpins, the previously
described cyclic peptide bhpW was used as a host. This peptide
adopts a well-defined hairpin conformation, and its NMR structure
is illustrated in FIG. 3. The disulfide of bhpW helps to stabilize
the hairpin conformation and simultaneously provides an assay for
structure. Changes in hairpin stability can be quantified by
changes in the position of the thiol-disulfide equilibrium.
Residues 2 and 9 of bhpW are cross-strand from one another and form
backbone hydrogen bonds. Several substitutions were then introduced
at positions 2 and 9 in bhpW: TABLE-US-00011 bhpW CTWEGNKLTC SEQ ID
NO:2 X2 series CXWEGNKLTC SEQ ID NO:26 X9 series CTWEGNKLXC SEQ ID
NO:27 KGNE turn CTWKGNELTC SEQ ID NO:28 V2H9 pair ("VH) CVWEGNKLHC
SEQ ID NO:29 H2V9 pair ("HV") CHWEGNKLVC SEQ ID NO:30 HV, X3 series
CHXEGNKLVC SEQ ID NO:31
[0186] The relative stabilities of the bhpW variants were
determined from the effective concentrations (C.sub.eff) of the
cysteine thiols (see the experimental section of Example 1) and are
shown in FIG. 11. Substitutions at both positions have large
effects on hairpin stability. The range of stabilities is 1 kcal
mol.sup.-1 for substitutions at position 2 (FIG. 11A) and 0.7 kcal
mol.sup.-1 for the same substitutions at position 9 (FIG. 11B). The
rank order of residue preferences at each of these positions is
rather different than what were observed previously for the NHB
positions 3 and 8 (see Example 4). Significantly, the pattern of
stability changes for position 2 is completely different from that
of position 9. In contrast, residue preferences are the same for
positions 3 and 8. Therefore, in addition to the influence of a
cross-strand hydrogen bond between residues 2 and 9, the bhp
hairpins exhibit a localized asymmetry that analysis of residue
preferences in these sites.
[0187] It is possible that the asymmetry in residue preferences at
positions 2 and 9 reflects differences in contacts with nearby side
chains. Peptides were prepared in which a valine residue was paired
with a cross-strand histidine instead of a threonine. We also
swapped residues 4 and 7, whose side chains are on the same face of
the hairpin as residues 2 and 9. In another hairpin model system,
"diagonal" interactions between side chains of NHB residues, placed
in proximity by the interstrand twist as are HB residues 4 and 9
here, have been proposed to influence hairpin stability. Syud et
al. (2001) J. Am. Chem. Soc. 123:8667-8677. In both cases, the
strong stabilizing effect of valine at position 9 remains (FIG.
12). It would seem therefore, that some structural feature other
than particular side chain-side chain contacts is responsible for
this effect.
[0188] Tryptophan residues at NHB strand positions have been
associated with a distinct twist in .beta.-hairpin structures; it
is possible that this creates a local conformational distortion,
influencing residue preferences at adjacent positions. To
investigate this, we fixed residues 2 and 9 in the more stabilizing
"HV" combination, then varied position 3. The relative stabilities
of these peptides are compared to those of the analogous T2T9
peptides in FIG. 13. The HV peptides are uniformly more stable than
their TT counterparts, as indicated by their higher C.sub.eff
values. Significantly, the substitution free energy differences
within each series are linearly correlated. This demonstrates that
the large stabilization imparted by the valine 9 substitution does
not depend on the presence of a particular residue pair at the
neighboring NHB sites. Interestingly, the slope of the plot is not
1, but instead indicates that the HV host is more sensitive to
substitutions at position 3 by a factor of 2. Taken together, the
data in FIGS. 11-13 suggest that the strong, asymmetric
stabilization by valine at position 9 is a general property of
CX.sub.8C disulfide-constrained hairpins.
[0189] In order to assess the effect that the substitutions at the
HB site had on hairpin conformation, structures were determined for
HV and VH on the basis of .sup.1H NMR data. The NMR data for HV are
in full agreement with this being a highly structured
.beta.-hairpin: the .sup.1H NMR secondary chemical shifts (the
differences between observed and random coil shifts) are much
larger than for bhpW, and .sup.3.sup.JH.alpha.-H.beta. coupling
constant values indicate defined side chain rotamers for three
pairs of strand residues (Cys1-Cys10, His2-Val9, Trp3-Leu8; Table
6). In particular, His2 and Val9 can be unambiguously assigned to
adopt X.sub.1 rotamers of +60.degree. and 180.degree.,
respectively. For peptide VH, the restraints generated from the NMR
data clearly define a conformation, but the H.sup..alpha. secondary
chemical shifts are less extreme than for HV. Furthermore,
.sup.3.sup.JH.alpha.-H.beta. coupling constants indicate motional
averaging for the Val2, His9, Trp3 and Leu8 side chains (Table 6).
All of these features indicate reduced stability for VH compared to
HV, consistent with the C.sub.eff measurements. The relative
structural stability of HV and VH does not depend on the presence
of histidine; very similar NMR parameters are also observed for
peptides in which the histidine is replaced by threonine (Table 6).
That is, TV has well-defined side chain conformations, whereas VT
exhibits conformational averaging at the side chain level.
TABLE-US-00012 TABLE 6 .sup.3J.sub.H.alpha.-H.beta. for Hydrogen
Bonded Cross-Strand Residues position 2 position 9 peptide
.sup.3J.sub.H.alpha.-H.beta., Hz .chi..sub.1
.sup.3J.sub.H.alpha.-H.beta., Hz .chi..sub.1 C.sub.eff, mM bhpW
(TT) 7.9 n.d..sup.c 6.8 n.d. 210 HV 3.8, 3.8.sup.b +60.cndot. 9.8
180.cndot. 1181 VH 5.6 n.d. 7.4, 7.6 n.d. 293 TV 4.2 +60.cndot. 9.7
180.cndot. 677 VT 5.9 n.d. 6.5 n.d. 305 .sup.bValues representing
non-averaging X.sub.1 torsion angles are indicated by bold
typeface. .sup.cnot defined. .sup.dnot applicable.
[0190] The structures determined for HV and VH are quite similar at
the backbone level (RMS difference of 0.37 .ANG. between the mean
structures): both peptides adopt a type II' turn and have a
considerable right-handed twist of the strands, as was seen
previously for bhpW. Calculation of the interstrand twist angle
indicates that for the cyclized hairpins, the twist is not uniform,
with less twisting adjacent to the disulfide (.THETA.<20.degree.
between the Cys1, Cys10 pair and the 2, 9 pair) and more twisting
before and after the NHB pair Trp3, Leu8
(.THETA..about.30.degree.). Since these measurements involve atoms
from both strands, the degree of twist present in each individual
strand is not apparent from .THETA.. Alternatively, the sum of
.psi. from one residue and .phi. from the succeeding residue
(.psi..sub.i+.phi..sub.i+1) gives an indication of the twist
between consecutive .alpha.-carbons in the same strand, with
non-twisted strands having a value of zero and strands of
right-handed twist having a positive value. The twist data for the
HV and VH ensembles are compared to bhpW in FIG. 14. These data
clearly indicate that the majority of the twist contributing to the
large interstrand .THETA. values occurs on the N-terminal side of
the NHB hydrophobic residues (45-65.degree. between His/Val/Thr2
and Trp3, and between Lys7 and Leu8, compared to <20.degree.
between Trp3 and Glu4 and between Leu8 and Val/His/Thr9). Although
the ranges of .psi..sub.i+.phi..sub.i+1 within the three ensembles
overlap, the data in FIG. 15 indicate that compared to VH, HV has a
slightly more pronounced twist preceding the NHB sites and a less
pronounced twist around Cys1 (assuming a normal distribution of
dihedral angles within each ensemble, an independent samples t-test
indicates that the mean .psi..sub.i+.phi..sub.i+1 for HV and VH are
different for residues 1-2, 2-3, and 7-8 with a p-value of less
than 0.01). This suggests that swapping the location of His and Val
side chains does have a subtle influence on backbone geometry.
[0191] The main difference between the HV and VH ensembles is the
degree of side chain order. Although Cys1, Trp3, Leu8 and Cys10
adopt the same X.sub.1 in HV and VH (-60.degree. for Cys1, Trp3 and
Cys10; 180.degree. for Leu8), the magnitudes of the
.sup.3J.sub.H.alpha.-.sub.H.beta. are less extreme in the latter,
suggesting that the side chains of VH are transiently sampling
alternate conformations. More importantly, in HV the X.sub.1
rotamers of His2 and Val9 are fixed (+60.degree. and 180.degree.,
respectively) bringing these two side chains into van der Waals
contact above the cross-strand hydrogen bonds. Due to the twisting
of the strands, the .gamma.2 methyl group of Val9 is also brought
into van der Waals contact with the side chain methylene groups of
both Glu4 and Lys7. However, in VH, Val2 populates both the
-60.degree. and 180.degree. rotamer wells, and most of the
structures in the ensemble have a X.sub.1 of -60 for His9,
orienting the side chain in the direction of the turn. Moreover,
the twisting of the strands directs the Val2 side chain towards the
termini of the peptide. Thus, there is little or no contact between
these side chains, and the backbone hydrogen bond between these
residues is more exposed to solvent.
[0192] In order to assess whether the asymmetric stabilization seen
for the HV peptide was related to differences in interstand twist
imposed by the disulfide bond, peptides HV and VH were examined by
NMR after reduction. Surprisingly, reduced HV has observable
residual structure: 4 .sup.3.sup.JHN-H.alpha. coupling constants
exceed 8 Hz, Leu8 methyl chemical shifts are 0.41 ppm and 0.33 ppm
upfield compared to random coil, and 3 backbone cross-strand NOEs
are observed (Cys1 H.sup..alpha. to Cys10 H.sup..alpha., His2
H.sup.N to Val9 H.sup.N and Glu4 H to Lys7 H.sup.N). In contrast,
there are no indications that reduced VH has a preferred
conformation. Presumably, Val9 can adopt a similar conformation in
the reduced peptide as it does in the oxidized form, while the
strong right-handed twist of the strands preceding position 3 (see
above) would not allow the Val2 side chain of either form to
interact with the C-terminal strand.
[0193] Taken together, the date shows that disulfide-cyclized
.beta.-hairpins can be markedly stabilized by a valine residue
immediately preceding the C-terminal cysteine, with isoleucine
appearing to be nearly as stabilizing. This effect may be due to a
favorable conformational relationship between Val9 and Cys10. For a
stable .beta.-turn phage display, the combination of a Trp residue
at position 3 or 8 and the very stabilizing His-Val 2,9-pair
reported here would appear especially useful. Compared to the least
stable combinations in which definite hairpin structure is
detectable, these substitutions stabilize the fold by more than 1.5
kcal mol.sup.-1. Inclusion of the optimal W3-W8 pair should
increase stability further, expanding the range of .beta.-turns
that might be structured. The data further suggests that libraries
with fixed CX.sub.nWVC motifs might also be useful in generating
structured hairpin ligands.
Example 8
Structure and Activity of the bhpBR3 Peptide
[0194] This example describes use of the bhp scaffold to generate a
turn peptide that mimics binding of the BR3 receptor to its ligand,
BAFF. BAFF (also known as BLyS, TALL-1, zTNF4, THANK and TNFS 13B),
a recently defined member of the TNF family, is a homotrimeric type
transmembrane protein expressed by macrophages, monocytes and
dendritic cells. Moore et al. (1999) Science 285:260-263; Schneider
et al. (1999) J. Exp. Med. 189:1747-1756. BAFF has been found
critical for the development and survival of peripheral B cells.
Gross et al. (2001) Immunity 15:289-302; Schiemann et al. (2001)
Science 293:2111-2114. Intriguingly, humans suffering from
autoimmune syndromes, including systemic lupus erythematosus (SLE),
rheumatoid arthritis, and Sjogren's syndrome where end organ damage
is primarily in the kidneys, joints and salivary/lacrymal glands,
respectively, have elevated levels of serum BAFF. Furthermore, BAFF
levels correlate with disease severity, consistent with a possible
role in the pathogenesis of these disabling maladies. Cheema et al.
(2001) Arthritis Rheum. 44:1313-1319; Groom et al. (2002) J. Clin.
Invest. 109:59-68; Zhang et al. (2001) J. Immunol. 166:6-10.
[0195] Of the three receptors for BAFF, only BR3 is specific; the
other two, TACI and BCMA, also bind the related ligand APRIL. Gross
et al. (2000) Nature 404:995-999; Thompson et al. (2001) Science
293:2108-2111; Yan et al. (2001 a) Curr. Biol. 11:1547-1552; Yan et
al. (2000) Nat. Immunol. 1:37-41. The extracellular domain of TACI
has a characteristic TNFR-like structure encompassing two
cysteine-rich domains (CRDs) that are the hallmark of the TNF
receptor family. These approximately 40 residue pseudorepeats have
a unique secondary structure, typically characterized by three
intrachain disulfides involving six highly conserved cysteines.
BCMA is unusual in that it contains only a single canonical CRD.
However, BR3 is even more divergent in that its extracellular
domain is composed of only a partial CRD, containing four cysteine
residues with spacing distinct from other TNFR modules
characterized previously. Bodmer et al. (2002) Trends Biochem. Sci.
27:19-26; Naismith and Sprang (1998) Trends Biochein. Sci 23:74-79.
Conventional members of the TNFR family utilize two CRDs for
binding ligand; contacts stem primarily from analogous loops from
each CRD interacting with two distinct surface patches on the
ligand (reviewed in Bodmer et al., 2002). Thus, how high-affinity
binding to BAFF is achieved by only a single, or partial, CRD like
that of BR3 is unclear. Recent crystal structures of BAFF reveal a
trimeric TNF-like fold with several distinguishing features
(Karpusas et al. (2002) J. Mol. Biol. 315:1145-1154; Liu et al.
(2002) Cell 108:383-394; Oren et al. (2002) Nat. Struct. Biol.
9:288-292), however no structure of any of the BAFF receptors has
been described.
Methods and Materials
Peptide Synthesis
[0196] MiniBR3 and bhpBR3 were synthesized as C-terminal amides on
a Pioneer peptide synthesizer (PE Biosystems) using standard Fmoc
chemistry. Their sequences are as follows (also shown in FIG. 15A):
TABLE-US-00013 Mini BR3: TPCVPAECFDLLVRHCVACGLLRTPR (SEQ ID NO:32)
bhpBR3: CHWDLLVRHWVC (SEQ ID NO:33)
[0197] For miniBR3, the side chain thiols of cysteines 19 and 32
were protected as the trifluoroacetic acid (TFA)-stable
acetamidomethyl (Acm) derivatives. Peptides were cleaved from resin
by treatment with 5% triisopropyl silane in TFA for 1.5-4 hr at
room temperature. After removal of TFA by rotary evaporation,
peptides were precipitated by addition of ethyl ether, then
purified by reversed-phase HPLC (acetonitrile/H.sub.2O/0.1% TFA).
Peptide identity was confirmed by electrospray mass spectrometry.
BhpBR3 was converted to the cyclic disulfide by dropwise addition
of a saturated solution of I.sub.2 (in acetic acid) to HPLC
fractions. After lyophilization, the oxidized peptide was purified
by HPLC. HPLC fractions containing reduced miniBR3 were adjusted to
a pH of .about.9 with NH.sub.4OH; the disulfide between cysteines
24 and 35 was then formed by addition of a small excess of
K.sub.3Fe(CN).sub.6, and the oxidized peptide purified by HPLC. Acm
groups were removed (with concomitant formation of the second
disulfide) by treatment of the HPLC eluate with a small excess of
I.sub.2 over .about.4 h. The progress of the oxidation was
monitored by analytical HPLC, and the final product was again
purified by HPLC. MiniBR3 was amino-terminally biotinylated while
on resin, then cleaved and purified exactly as described above for
the unmodified peptide.
NMR Spectroscopy
[0198] Two-dimensional (2D) NMR experiments were acquired and
analyzed as described in Starovasnik et al., (1996) Biochemistry
35:15558-155569, using a Bruker DRX-600 spectrometer at 293K on a
sample containing 2.9 mM bhpBR3, pH 4.5, with 0.1 mM DSS as a
chemical shift reference. Distance restraints were derived from 2D
NOESY spectra (.tau..sub.m 250 ms); dihedral angle restraints were
derived from analysis of a 2D DQF-COSY spectrum acquired in
92%H.sub.2O/8%D.sub.2O and a 2D COSY-35 spectrum acquired on a
sample dissolved in 100% D.sub.2O. Complete resonance assignments
and coupling constant values are included in the supplemental
data.
[0199] The three-dimensional structure of bhpBR3 was calculated
based on 78 NOE-derived (including 26 long-range) distance
restraints and 17 dihedral angle restraints. 100 initial structures
were calculated using DGII; 80 of these were further refined by
restrained molecular dynamics using DISCOVER as described
(Starovasnik et al., (1996) supra). Twenty structures having the
lowest restraint violation energy and good geometry represent the
solution conformation of bhpBR3. The model with the lowest rms
deviation (RMSD) to the average coordinates of the ensemble was
chosen as the representative structure (model 1 in the PDB file).
The final ensemble of twenty models satisfies the input data well,
having no distance or dihedral angle restraint violations greater
than 0.1 .ANG. or 1.degree., respectively. The structures are well
defined, with an average backbone RMSD to the mean coordinates of
0.42.+-.0.07 .ANG., and have good covalent geometry as judged by
PROCHECK (87% of the residues in the most favored, 9% in the
allowed, and 4% in the generously allowed regions of .phi..psi.
space). Laskowski et al., (1993) J. Appl. Crystallogr. 26:283-291.
The structure of bhpBR3 will be available from the RCSB Protein
Data Bank (ID code xxxx).
Competitive Displacement ELISA
[0200] Nunc Maxisorp 96-well plates were coated overnight at
4.degree. C. with 100 .mu.l of a 2 .mu.g/ml solution of BAFF in
carbonate buffer, pH 9.6. The plate was washed with PBS and blocked
with 1% skim milk in PBS. Serial dilutions of BR3 variants were
prepared in PBS/0.05% Tween 20 containing 3 ng/ml biotinylated
miniBR3. After washing with PBS/Tween, 100 .mu.l/well of each
dilution was transferred and incubated for 1 h at room temperature.
The plate was washed with PBS/Tween and incubated 15 min with 100
.mu.l/well of 0.1 U/ml Streptavidin-POD (Boehringer Mannheim) in
PBS/Tween. After washing with PBS/Tween followed by PBS, the plate
was incubated 5 min with 100 .mu.l PBS substrate solution
containing 0.8 mg/ml OPD (Sigma) and 0.01% H.sub.2O.sub.2. The
reaction was quenched with 100 .mu.l/well 1M H.sub.3PO.sub.4 and
the plate read at 490 nm. IC.sub.50 values were determined by a
four-parameter fit of the competitive displacement ELISA signal.
The concentrations of initial stock solutions of bhpBR3 were
determined spectrophotometrically as described (Gill and von
Hippel, (1989) Anal. Biochem. 182:319-326), while those of miniBR3
and BR3 extracellular domain were determined by quantitative amino
acid analysis.
The Turn Peptide bhpBR3 Competitively Binds to BAFF
[0201] As a first step towards developing agents to disrupt the
BAFF-BR3 interaction, an NMR analysis of the extracellular
ligand-binding domain of BR3 was performed. Intriguingly, only the
central one-third of the protein adopts a stable structure in
solution; this core is stabilized by two disulfide bonds connecting
Cys19/Cys32 and Cys24/Cys35. Consequently, a 26-residue miniBR3
peptide was synthesized, incorporating the 1:3, 2:4
disulfide-bonding pattern, and characterized structurally (FIG.
15A). Indeed, NMR spectra of miniBR3 indicated that this peptide
adopts essentially the same structure as in the context of the
full-length protein. Importantly, miniBR3 also binds with the same
affinity as full-length BR3 to BAFF (.about.70 nM IC.sub.50; FIG.
16A).
[0202] Given that high-affinity BAFF-binding was contained within a
26-residue core, attempts were made to further delineate the
BAFF-binding portion of BR3. In crystal structures of other
TNF-like ligand/receptor complexes, a receptor loop analogous to
BR3 residues .sup.26DLLVRH.sup.31 is involved in forming direct
contacts with the ligand. Bodmer et al. (2002) Trends Biochein. Sci
27:19-26. In the context of both full-length and miniBR3, NMR
analysis suggested that the sequence .sup.26DLLVRH.sup.31 presents
a type I .beta.-turn with backbone hydrogen-bonding between Asp26
and His31. Thus, a 12-residue peptide was synthesized in which the
six residues from BR3 were embedded within a disulfide-bonded
.beta.-hairpin (bhp) scaffold (FIG. 15A). As described previously,
the strong strand-strand interactions in these scaffolds can
structure a variety of .beta.-turns.
[0203] The peptide bhpBR3 adopts a remarkably stable conformation
in solution as indicated by a high degree of chemical shift
dispersion, extreme values for many of the backbone and side chain
coupling constants, and a large number of long-range NOEs present
in its NMR spectra. The three-dimensional structure of bhpBR3
consists of a .beta.-hairpin in which the BR3 turn sequence adopts
the type I .beta.-turn structure, as expected, with Arg30 adopting
a positive .phi. angle and the side chains of Asp26, Leu28, Val29,
and His31 projecting on one face of the .beta.-turn (FIGS. 15B and
15C). If BR3 binds BAFF using interactions homologous to those
observed for TNFR and DR5 (Hymowitz et al., (1999) Mol. Cell
4:563-571, then one would expect this face (the "bottom" face of
the turn shown in FIG. 15C) to contact BAFF.
[0204] Because bhpBR3 mimics structurally the BR3 turn, its ability
to compete with miniBR3 for binding to BAFF was tested in a
competitive displacement ELISA (FIG. 16A). Remarkably, the
12-residue peptide blocked binding of the larger core domain
(IC.sub.50=15 .mu.M), indicating that the critical binding
determinants do indeed reside in the six-residue turn shown in FIG.
15C. Further, the activity of bhpBR3 was confirmed in a bioassay:
bhpBR3, but not a control hairpin peptide, blocked BAFF mediated
NF-.kappa.B2/p52 induction in primary Bcells (FIG. 16B).
[0205] The finding that a .beta.-turn structure from BR3 has
significant affinity for BAFF has implications for recognition of
BAFF by its other receptors. TACI and BCMA share homologous
sequences in this loop region that would be expected to adopt a
similar turn conformation to that in BR3. Therefore, the
interactions of this turn with ligand will likely be a conserved
feature of all BAFF/receptor complexes. The surprising
identification of such a focused recognition epitope will provide
the framework for developing small-molecule peptidomimetic
inhibitors of the BAFF-BR3 interaction; these inhibitors may have
therapeutic potential in the treatment of autoimmune diseases such
as lupus.
[0206] While the invention has necessarily been described in
conjunction with preferred embodiments, one of ordinary skill,
after reading the foregoing specification, will be able to effect
various changes, substitutions of equivalents, and alterations to
the subject matter set forth herein, without departing from the
spirit and scope thereof. Hence, the invention can be practiced in
ways other than those specifically described herein.
Sequence CWU 1
1
60 1 18 PRT Artificial Sequence Turn Peptide 1 Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Cys 2 10
PRT Artificial Sequence Turn Peptide 2 Cys Thr Trp Glu Gly Asn Lys
Leu Thr Cys 1 5 10 3 12 PRT Artificial Sequence Turn Peptide 3 Ser
Cys Thr Trp Glu Gly Asn Lys Leu Thr Cys Lys 1 5 10 4 10 PRT
Artificial Sequence Turn Peptide 4 Cys Gly Asn Gln Gly Ser Phe Leu
Thr Cys 1 5 10 5 10 PRT Artificial Sequence Turn Peptide 5 Cys Thr
Trp Gln Gly Ser Phe Leu Thr Cys 1 5 10 6 12 PRT Artificial Sequence
Turn Peptide 6 Ser Cys Gly Asn Gln Gly Ser Phe Leu Thr Cys Lys 1 5
10 7 12 PRT Artificial Sequence Turn Peptide 7 Ser Cys Thr Asn Gln
Gly Ser Phe Leu Thr Cys Lys 1 5 10 8 12 PRT Artificial Sequence
Turn Peptide 8 Ser Cys Gly Trp Gln Gly Ser Phe Leu Thr Cys Lys 1 5
10 9 12 PRT Artificial Sequence Turn Peptide 9 Ser Cys Thr Trp Gln
Gly Ser Phe Leu Thr Cys Lys 1 5 10 10 12 PRT Artificial Sequence
Turn Peptide 10 Ser Cys Gly Asn Gln Gly Ser Phe Leu Thr Cys Lys 1 5
10 11 12 PRT Artificial Sequence Turn Peptide 11 Ser Cys Thr Trp
Gln Gly Ser Phe Leu Thr Cys Lys 1 5 10 12 10 PRT Artificial
Sequence Turn Peptide 12 Cys Thr Lys Val Trp Gln Leu Trp Thr Cys 1
5 10 13 12 PRT Artificial Sequence Turn Peptide 13 Ser Cys Thr Trp
Val Trp Gln Leu Leu Thr Cys Lys 1 5 10 14 12 PRT Artificial
Sequence Turn Peptide 14 Ser Cys His Phe Gly Pro Leu Thr Trp Val
Cys Lys 1 5 10 15 12 PRT Artificial Sequence Turn Peptide 15 Ser
Cys Thr Trp Gly Pro Leu Thr Leu Thr Cys Lys 1 5 10 16 10 PRT
Artificial Sequence Turn Peptide 16 Cys Thr Xaa Glu Gly Asn Lys Leu
Thr Cys 1 5 10 17 10 PRT Artificial Sequence Turn Peptide 17 Cys
Thr Xaa Glu Asn Gly Lys Leu Thr Cys 1 5 10 18 10 PRT Artificial
Sequence Turn Peptide 18 Cys Thr Xaa Glu Xaa Asn Lys Leu Thr Cys 1
5 10 19 10 PRT Artificial Sequence Turn Peptide 19 Cys Thr Xaa Glu
Xaa Gly Lys Leu Thr Cys 1 5 10 20 10 PRT Artificial Sequence Turn
Peptide 20 Cys Thr Xaa Glu Gly Asn Lys Leu Thr Cys 1 5 10 21 10 PRT
Artificial Sequence Turn Peptide 21 Cys Thr Leu Glu Gly Asn Lys Xaa
Thr Cys 1 5 10 22 10 PRT Artificial Sequence Turn Peptide 22 Cys
Thr Xaa Glu Gly Asn Lys Trp Thr Cys 1 5 10 23 10 PRT Artificial
Sequence Turn Peptide 23 Cys Thr Trp Glu Gly Asn Lys Xaa Thr Cys 1
5 10 24 102 DNA Artificial Sequence Synthesized Sequence 24
taataataaa tggctgatcc gaaccgtttc cgcggtaaag atctgggtgg cggtactcca
60 aacgacccgc caaccactcc accaactgat agcccaggcg gt 102 25 72 DNA
Artificial Sequence Oligonucleotide 25 tccgcctcgg cttatgcann
stgcacttgg nnsnnsnnsn nsctgacttg tnnsatggct 60 gatccgaacc gt 72 26
10 PRT Artificial Sequence Turn Peptide 26 Cys Xaa Trp Glu Gly Asn
Lys Leu Thr Cys 1 5 10 27 10 PRT Artificial Sequence Turn Peptide
27 Cys Thr Trp Glu Gly Asn Lys Leu Xaa Cys 1 5 10 28 10 PRT
Artificial Sequence Turn Peptide 28 Cys Thr Trp Lys Gly Asn Glu Leu
Thr Cys 1 5 10 29 10 PRT Artificial Sequence Turn Peptide 29 Cys
Val Trp Glu Gly Asn Lys Leu His Cys 1 5 10 30 10 PRT Artificial
Sequence Turn Peptide 30 Cys His Trp Glu Gly Asn Lys Leu Val Cys 1
5 10 31 10 PRT Artificial Sequence Turn Peptide 31 Cys His Xaa Glu
Gly Asn Lys Leu Val Cys 1 5 10 32 26 PRT Artificial Sequence
Peptide 32 Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg
His Cys 1 5 10 15 Val Ala Cys Gly Leu Leu Arg Thr Pro Arg 20 25 33
12 PRT Artificial Sequence Peptide 33 Cys His Trp Asp Leu Leu Val
Arg His Trp Val Cys 1 5 10 34 4 PRT Artificial Sequence Turn
Peptide 34 Glu Gly Asn Lys 1 35 4 PRT Artificial Sequence Turn
Peptide 35 Glu Asn Gly Lys 1 36 4 PRT Artificial Sequence Turn
Peptide 36 Gln Gly Ser Phe 1 37 4 PRT Artificial Sequence Turn
Peptide 37 Val Trp Gln Leu 1 38 4 PRT Artificial Sequence Turn
Peptide 38 Gly Pro Leu Thr 1 39 16 PRT Artificial Sequence Turn
Peptide 39 Met Gln Ile Gly Val Lys Asn Pro Asp Gly Thr Ile Thr Leu
Glu Val 1 5 10 15 40 16 PRT Artificial Sequence Turn Peptide 40 Met
Gln Ile Gly Val Lys Ser Pro Xaa Lys Thr Ile Thr Leu Lys Val 1 5 10
15 41 9 PRT Artificial Sequence Turn Peptide 41 Tyr Gln Asn Pro Asp
Gly Ser Gln Ala 1 5 42 10 PRT Artificial Sequence Turn Peptide 42
Ile Thr Ser Asn Ser Asp Gly Thr Trp Thr 1 5 10 43 10 PRT Artificial
Sequence Turn Peptide 43 Tyr Ile Thr Asn Ser Asp Gly Thr Trp Thr 1
5 10 44 12 PRT Artificial Sequence Turn Peptide 44 Arg Gly Ile Thr
Val Asn Gly Lys Thr Tyr Gly Arg 1 5 10 45 10 PRT Artificial
Sequence Turn Peptide 45 Ile Tyr Ser Asn Pro Asp Gly Thr Trp Thr 1
5 10 46 10 PRT Artificial Sequence Turn Peptide 46 Ile Tyr Ser Asn
Ser Asp Gly Thr Trp Thr 1 5 10 47 16 PRT Artificial Sequence Turn
Peptide 47 Lys Lys Tyr Thr Val Ser Ile Asn Gly Lys Lys Ile Thr Val
Ser Ile 1 5 10 15 48 16 PRT Artificial Sequence Turn Peptide 48 Gly
Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu 1 5 10
15 49 12 PRT Artificial Sequence Turn Peptide 49 Arg Tyr Val Glu
Val Xaa Gly Xaa Lys Ile Leu Gln 1 5 10 50 8 PRT Artificial Sequence
Turn Peptide 50 Xaa Cys Leu Xaa Ala Val Cys Xaa 1 5 51 8 PRT
Artificial Sequence Peptide 51 Ala Cys Ser Pro Gly His Cys Glu 1 5
52 6 PRT Artificial Sequence Turn Peptide 52 Cys Xaa Pro Gly Xaa
Cys 1 5 53 17 PRT Artificial Sequence Turn Peptide 53 Cys Glu Trp
Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Cys 1 5 10 15 Lys
54 16 PRT Artificial Sequence Peptide 54 Gly Cys Lys Pro Thr Phe
Arg Arg Leu Lys Trp Lys Tyr Lys Cys Gly 1 5 10 15 55 18 PRT
Artificial Sequence Cyclized Peptide 55 Cys Ala Gly Phe Met Arg Ile
Arg Gly Arg Ile His Pro Leu Cys Met 1 5 10 15 Arg Arg 56 10 PRT
Artificial Sequence Turn Peptide 56 Phe Cys Asn Gln Gly Ser Phe Leu
Cys Tyr 1 5 10 57 12 PRT Artificial Sequence Turn Peptide 57 Phe
Cys Tyr Ile Cys Glu Val Glu Asp Gln Cys Tyr 1 5 10 58 11 PRT
Artificial Sequence Turn Peptide 58 Cys Gly Val Ser Arg Gln Gly Lys
Pro Tyr Cys 1 5 10 59 12 PRT Artificial Sequence Turn Peptide 59
Xaa Cys Thr Trp Xaa Xaa Xaa Xaa Leu Thr Cys Xaa 1 5 10 60 6 PRT
Artificial Sequence Turn Peptide 60 Asp Leu Leu Val Arg His 1 5
* * * * *