peptide, peptidomimetic, and organic synthetic combinatorial libraries

Peptide, Peptidomimetic, and Organic Synthetic Combinatorial Libraries

Jutta Eichler, Jon R. Appel, Sylvie E. Blondelle, Colette T. Dooley, Barbara Dorner, John M. Ostresh, Enrique Perez-Paya, Clemencia Pinilla, and Richard A. Houghten,

Torrry Pines Iristittite for Molrcitlnr Studies, 3550 Grrirrnl Atomics Court, Snr7 Diego, Califorrzin 92121

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 11. Library Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. One Bead-One C B. Synthetic Combinatorial Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

1. Iterative Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 2. Positional Scanning SCLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

111. Utilization of SCLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Dual Positional SCLs . . . . . . . . . . . . . . . .

B. Opioid Receptor Ligands . . . . . . . . . . . . . . . . . . . .

D. Enzyme Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492 C. Antimicrobial Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

. . . . . . . . . . . . . . . . . 1V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

I. INTRODUCTION

Synthetic combinatorial libraries made up of up to tens of millions of peptides or peptidomimetics have emerged during the past 5 years as powerful tools for basic research and drug development. As first developed for peptides,1-3 the general concept of combinatorial libraries involves the generation of all possible sequence permutations for a peptide of a given length (i.e., 64 million for a hexapeptide composed of the 20 proteinogenic amino acids), in connection with a screening and selection process that enables the identification of unique, highly active peptides in the presence of millions of less active or inactive peptides. Since the individual synthesis of such large numbers of compounds is unrealistic, several approaches for the synthesis of mixtures composed of up to millions of peptides have recently been shown to be practical. Such libraries have been used to identify a variety of peptide ligands (e.g., antigenic peptides, receptor ligands, antimicrobial peptides, enzyme substrates, and inhibitors).4

Using recombinant DNA techniques, large numbers of peptides can be expressed randomly in a fusion phage vector ~ys t e rn .~ This method, however, remains restricted to the 20 proteinogenic amino acids as building blocks. Chemical approaches to the generation of peptide libraries, on the other hand,1-3,6 allow for the incorporation of nonpro- teinogenic7 and D-amino acids,8-10 as well as chemically modified amino acids” and other carboxylic acids.12.13

The generally poor oral bioavailability and rapid enzymatic breakdown of L-amino acid

“To whom correspondence should be addressed. Phone: 619-455-3803; fax: 619-455-3804

Medicinal Research Reviews, Vol. 15, No. 6, 481-496 (1995) 0 1995 John Wiley & Sons, Inc. CCC 0198-6325/95/060481-16

482 EICHLER ET AL.

peptides make them inferior drug candidates relative to other organic (i.e., nonpeptide) compounds. Therefore, the major research focus in the field of combinatorial chemistry is currently on the development of peptidomimetic'*,*'' and organic chemical libraries. 15

The majority of chemical methods used to synthesize combinatorial libraries utilize the concept of solid-phase synthesis,'6 which is based on the sequential assembly of compounds from a defined set of building blocks, in which the first building block is cova- lently attached to a polymeric solid support. This enables the excess of reagents to be removed by simple wash and filtration processes, and avoids the laborious isolation and purification of intermediates associated with the conventional synthesis in solution, thus greatly facilitating and accelerating the synthesis process.

The principal differences between the various methods of library synthesis are: (i) the solid support used, (ii) the means of incorporating mixtures of building blocks, (iii) the state (immobilized or in solution) in which the libraries are screened, and (iv) the manner in which the structures of the identified active compounds are determined.

11. LIBRARY SYNTHESIS

The majority of synthetic methods for the generation of combinatorial libraries were derived from various multiple peptide synthesis techniques developed during the past decade.17-22 The solid supports used for the synthesis of combinatorial libraries are either polymeric resins2Z3 or segmental carriers such as plastic pins,' glass slides,22 cotton,23 or cellulose membrane.24 When using a resin as the solid support, mixtures of compounds can be generated by coupling each building block to separate portions of the resin, followed by combining and mixing all of the resin portions before dividing them again prior to the next coupling step. This method, known as "portioning-mixing,"6 "divide-couple-recombine" (DCR),2 or "split synthesis,"3 is not feasible in conjunction with segmental solid supports. An alternative means to generate compound mixtures is the incorporation of mixtures of building blocks in a predetermined molar ratio, which compensates for the different coupling rates of the various building blocks.23,25,26 Li- braries generated by these methods are screened either immobilized (attached to the solid support) or in solution after cleavage from the solid support.

A. One Bead-One Compound Libraries

The majority of immobilized libraries are synthesized using a resin as the solid support and the "split synthesis" method, generating "one (resin) bead-one compound" libraries.3 These libraries are typically screened in a solid-phase assay, which is based on the physical selection of positively reacting resin beads, followed by structure determi- nation of the compounds attached to those active beads. One bead-one compound libraries have been used for the identification of antigenic peptides10,27,28 and compounds that bind to streptavidin,12,29,3" as well as for the mapping of endoprotease specificity.31 The multiple release of equimolar amounts of compounds from the resin

Richard A. Houghten is internationally recognized for his ~ i ~ o r k i n the areas of molecular diversity and combinatorial chemistry. Dr. Hougliten received his doctorate from the University of California, Berkeley, in 1975. He joined the molecular biology department of The Scripps Research lnstitute in 1981, and in 1989 founded Torrey Pines Zristitute for Molecular Studies (he maintains an adjunct membership at Scripps). He is also fhe founder of Multiple Peptide Systems (1986) and Houghten Pharinaceuticals, Inc. (1990).

SYNTHETIC COMBINATORIAL LIBRARIES 483

beads enables one bead-one compound libraries to also be screened in solution.32 For libraries composed of L-amino acid peptides, the structure of the compounds on the active beads can be readily determined by Edman degradation. One bead-one compound libraries made from building blocks other than the proteinogenic amino acids, however, require the presence of oligonucleotide,^^^^ peptide35.36 or other chemical tags37 to encode the structure of the library compound on each resin bead, which can then be decoded by sequencing or other analysis (e.g., gas chromatography, HPLC) of the coding tag. This requires an additional, independent chemistry for the assembly of the coding tag, so as not to interfere with the synthesis of the library.

B. Synthetic Combinatorial Libraries

Synthetic combinatorial libraries ( S C L S ) ~ , ~ ~ are composed of separate compound mixtures with one or more defined positions in the sequence. Since these mixtures are in solution (as opposed to immobilized libraries), SCLs can be screened in a variety of bio- assays, including those involving membrane-bound receptors or whole cells. Further- more, the systematic array (i.e., the use of defined and mixture positions) of SCLs eliminates the need for any kind of coding for building blocks other than the proteinogenic amino acids, since the identification of the structure of the active compounds of interest is inherent to the SCL approach (see below). SCLs are typically synthesized using the method of simultaneous multiple peptide synthesis, often referred to as the “tea bag method.”18 Briefly, the solid support resins for each peptide are separated from one another by containing them in sealed polypropylene mesh packets, thus enabling identical processes (i.e., removal of temporary protecting groups, wash steps, coupling of the same amino acid to several different peptide-resins) to be carried out in a common container, followed by unambiguous separation of the resin packets for individual coupling steps without cross-contamination.

1. lterative Process

The initial SCL was composed of 400 separate soluble hexapeptide mixtures, represented as O,O,XXXX, in which the first two positions (0, and 0,) were individually defined with one of the 20 proteinogenic amino acids, and the remaining four positions (X) were occupied by close to equimolar mixtures of 19 of the 20 proteinogenic amino acids (cysteine excluded). Thus, each peptide mixture is composed of 194 = 130,321 peptides, and the entire library represents 400 x 130,321 = 52,128,400 individual peptides.2

This type of SCL is typically synthesized using the DCR method mentioned above. Briefly, functionalized polystyrene resin crosslinked with divinylbenzene is contained in n ( n = number of building blocks for the mixture positions; 19 in the SCL described above) polypropylene mesh packets, and each building block is coupled to a separate resin packet. After washing off the excess of activated building blocks and removal of temporary protecting groups, the resins are taken out of the packets, combined, thor- oughly mixed, and then redivided into n packets for the next coupling step. After all mixture (X) positions have been incorporated, the resin is divided into m (m = number of peptide mixtures; 400 in the SCL described above) portions (packets), and the defined positions (0) are incorporated using standard methods of multiple solid-phase synthesis.

484 EICHLER ET AL.

In the initial screening of this type of SCL in a given bio-assay, the most active peptide mixtures are identified, followed by an iterative process of synthesis and screening, during which all positions of the active sequences are successively defined. This process involves ranking, selecting, and reducing the number of mixture positions (and thereby the number of peptides in each mixture), while defining one more position at each step. It is sometimes necessary to move forward with several peptide mixtures with similar activities in the initial screening. Those mixtures not pursued from the initial screening or during the iterative process, however, can be moved forward at a later date.

This SCL format has also been utilized for various other peptide and peptidomimetic libraries synthesized from D- and other nonproteinogenic amino acids, as well as carboxylic acids. A tetrapeptide library, represented as UZZZ, was prepared with the first position (U) defined with one of 58 amino acids (20 proteinogenic, 19 D- and 19 other nonproteinogenic amino acids), and the three remaining positions ( Z ) occupied by mixtures of the same set of amino acids, excluding D- and ~-cysteine.7 This library is made up of 58 separate peptide mixtures, each of which is composed of 563 = 175,616 individual peptides, and represents a total of 58 x 175,616 = 10,185,728 individual peptides.

Chemically modified SCLs can be generated through modification of the peptide bonds (e.g., N-alkylation and/or reduction) of existing peptide libraries, thus dramat- ically changing the physico-chemical character of the peptides, and greatly extending the range and repertoire of molecular diversity. The components of such transformed libraries, which have been termed “libraries from libraries,”14,39 are stable towards enzymatic degradation, since they lack the characteristic peptide bond -C&NH-. Two parent peptide libraries, OZZ and OZZZ, were synthesized using 52 amino acids as building blocks. The tripeptide library (OZZ) was permethylated, and the tetrapeptide library (OZZZ) perallylated and perbenzylated, thus generating three different N-alkyl- ated peptidomimetic libraries.40

The SCL approach has also been applied to the synthesis of conformationally defined libraries. The first of these libraries prepared was derived from melittin, a peptide known to adopt two a-helices linked by a hinge region. The ability of a peptide mixture to fold into an a-helical conformation was studied by replacing the five residues making up the hinge region with a single individually defined position and four mixture positions, thus generating 20 peptide mixtures, each composed of 130,321 individual peptides.41 The average folding propensity of each peptide mixture was determined by circular dichroism analysis.

2 . Positional Scanning SCLs

An alternative approach, termed a positional scanning SCL (PS-SCL),9,25,43 enables the identification of active individual compounds in a single assay, thus avoiding the iterative process of synthesis and screening associated with the original SCL format. A typical PS-SCL is composed of n positional SCLs (n = number of diversity positions). Accordingly, a hexapeptide PS-SCL is composed of six independent positional SCLs, and is represented as O,XXXXX, X02XXXX, XXO,XXX, XXX04XX, XXXXO,X, and XX- XXX06.43 As for the SCLs described above, 0 represents individually defined positions, whereas X stands for positions occupied by mixtures of building blocks. It should be noted that each positional SCL making up a PS-SCL, while addressing a specific position, represents the same collection of individual compounds. When used in concert, the screening of the n positional libraries making up a PS-SCL provides information about the most effective building blocks in each position for a given ligand-acceptor interaction,


as well as about the relative specificity of each position (i.e., the fewer building blocks found to be effective for a given position, the higher the specificity of that position). Synthesis of all possible combinations of the most effective building blocks at each position yields a range of individual compounds, which are then tested in order to establish their individual activities. Alternatively, each positional library can serve as a starting point for the iterative synthesis and screening process described above.9

The positional scanning format has also been used for the synthesis of a D-amino acid hexapeptide PS-SCL,9 as well as a decapeptide PS-SCL made up of 10 positional SCLs, which represent approximately 4 x 10'2 individual peptides.4 A multimeric peptide PS- SCL has also been presented.45 Recently, the positional scanning format has been applied to the preparation of peptidomimetic libraries. A hexapeptide PS-SCL was permethylated, thus generating a chemically transformed library ("libraries from libraries") of N-permethylated peptidomirneti~s.1~

A PS-SCL based on a cyclic peptide template was synthesized using 10 different carboxylic acids (Fig. 1) in addition to the 20 proteinogenic amino acids as building blocks, thus increasing the chemical diversity of this library. 13 This PS-SCL is composed of three positional SCLs, represented as cyclo[Lys(O,)-Lys(X)-Lys(X)-Glul-Gly-OH,

@J COOH

COOH

(I): 4-methoxy-2-quinolinewboxylic acid (2): I-adammtanecarboxylic acid

(3): piperonylic acid (4): 5-methyl-3-phenylisoxazole4-carboxylic acid

HzCOOH

0

(5): rhodanine-3-acetic acid (6): 2-norbornmeacetic acid

0

(7): nicotinic acid (8): 9-oxo-9H-thioxanthene-3~0xylic acid 10.1 0 dioxide

QCOOH 02N & k O O H

(9): 2-thiophenecarboxylic acid (10): 5-nitro-2-furoic acid

Figure 1. The 10 carboxylic acids used as building blocks for the cyclic peptide template PS-SCL.

486 EICHLER ET AL.

cyclo[Lys(X)-Lys(0,)-Lys(X)-Glul-Gly-OH and cyclo[Lys(X)-Lys(X)-Lys(0,)-Glul-Gly-OH. The cyclic template was generated prior to introducing the chemical diversity, thus avoiding the sequence-dependence associated with peptide cyclizations. The defined and mixture positions were incorporated by acylating the €-amino groups of the three lysine residues.

Since PS-SCLs are typically generated by coupling mixtures of building blocks to incorporate X positions, as opposed to the DCR method, which involves coupling of individual amino acids to separate portions of the solid support, PS-SCLs can be synthesized using resins as well as segmental carriers as the solid support. Immobilized peptide PS-SCLs have been prepared using cotton46 or cellulose membrane24 as the solid supports.

111. UTILIZATION OF SCLS

A. Antigenic Peptides

Peptide SCLs having varying lengths and formats have been used to identify the antigenic determinants recognized by various monoclonal antibodies (mAbs).2,9,38,43,44,47-53

The libraries were screened by competitive ELISA, which is based on the detection of the inhibition by peptide mixtures of antibody binding to a known antigen. Competitive ELISA permits the quantification of the inhibitory activity of each peptide mixture by determining its IC,, value, i.e., the concentration of peptide mixture necessary to inhibit 50% of the antibody binding to the known antigen.

1 . Dual Positional SCLs

An N-terminal acetylated, C-terminal amidated hexapeptide SCL (Ac-O,O,XXXX- NH,), composed of 400 peptide mixtures,, was screened against mAb 17/9, which was raised against a 36-residue fragment of the hemagglutinin of the influenza virus.54 The antigenic determinant recognized by this antibody (-DVPDYA-) had previously been identified and extensively characterized using omission and substitution analogs of a longer antigenic peptide (Ac-YPYDVPDYASLRS-NHJ,S, as well as by x-ray crystallogra- phy.56 Through the library screening, Ac-DVXXXX-NH, was found to be the most active peptide mixture (IC,,, = 58,000nM). The iterative process of synthesis and screening, during which all of the positions were successively defined, is illustrated in Figure 2. Twenty new peptide mixtures were synthesized, in which the third position of Ac- DVXXXX-NH, was defined (Ac-DVOXXX-NH,). The most effective inhibiting peptide mixture was Ac-DVPXXX-NH, (IC,,, = 15,000 nM). This process was repeated twice more in order to identify the most effective amino acids at the fourth (Ac-DVPDXX-NH,, IC,, = 1,100 nM), and at the fifth (Ac-DVPDYX-NH,, IC,, = 40 nM) positions, respectively. Upon defining the sixth and final position of the sequence, a set of 20 individual peptides (Ac-DVPDYO-NH,) was synthesized and assayed. Ac-DVPDYA-NH, (I&" =

2nM) was identified as the most active individual peptide. This sequence exactly matches the antigenic determinant found in earlier studies to be recognized by mAb 1 7/ 9.55

The same SCL was also screened against mAb 125-10F3, which was raised against a synthetic peptide representing a 31-residue fragment of the oncogene-related protein from v-fes. The iterative process was followed for the two most active peptide mixtures found (Ac-PYXXXX-NH, and Ac-YPXXXX-NH,).~~,~~ Individual peptides with nanomo- lar activities were identified; a number of these sequences represent analogs with substitutions at position six of the known antigenic determinant (-PYPNLS-).

Ac-DVOXXX-NH, 0 0 7 ,

Ac-DVPOXX-NH,

Ac- DVPDOX- NH,

Ac- DVPDYO- NH, -

Figure 2. Iterative process for Ac-DVXXXX-NH,. Each graph represents one step in the process (i.e., one more defined position). The bars within each graph represent the reciprocal IC,, values of the peptide mixtures defined at the 0 position with the amino acid specified on the x-axis. The first bar in each graph represents the most active peptide mixture from the previous step.

488 EICHLER ET AL.

In another study,50 the screening of the same SCL against an antipeptide antibody (mAb 222-35C8) resulted in the identification of the known antigenic determinant (--GRTTVF-), as well as completely unrelated sequences with similar or higher affinities, and single or double substitution analogs of the antigenic determinant.

A nonacetylated hexapeptide SCL with the same format (OOXXXX-NH,) was screened against mAb 3E7, which is known to recognize the N-terminus of P-endorphin. The peptide YGGFMT-NH,, which exactly matches the six N-terminal amino acid residues of P-endorphin, was identified through the screening of this library, along with an iterative synthesis and screening process.49

2. Positional Scanning SCLs

The screening of positional scanning SCLs (PS-SCLs),9.43,44,48,5*,52 in which every position is defined in a separate positional library (see above), enables the identification of the most effective amino acids or other building blocks in each position of the sequence for highly specific antigen-antibody interactions in a single assay. Table I illustrates five different antigen-antibody interactions that have been studied using N-acetylated (mAbs 17/9, 125-10F3, 12, and 134829), as well as nonacetylated (mAb 3E7) hexapeptide PS-SCLs. Although in most cases the library screening results provided sufficient information to locate the antigenic determinant within the immunogenic sequences, individual peptides representing all possible combinations of the most active amino acids found at each position were synthesized. The activities of those individual peptides were

TABLE I Identification of Antigenic Peptides from Hexapeptide PS-SCLs

# of Individ- Most Active Amino Acids ual 3 Most Active

Positions: Peptides Range of Individual Ref mAb 1 2 3 4 5 6 Synthesized IC,,, nM Sequences IC,,, (nM) #

17/9

125- 10F3

12

D I E D Y A V P S

Q P Y P N I L

P L R N

S S T P A H T S M M

134 829

3E7

M1

G D S T F E E Y H S

Y G A F L D G M N

P Q

D Y K A K A E E L L Q Q

12 2-33 Ac-DVPDYA-NH2 Ac-DIPDYA-NH, Ac-DVEDYA-NH,

12 13-33,000 Ac-PYPNLR-NH, Ac-PYPNIL-NH, Ac-PY PNLL-NH,

16 390-> 100,000 Ac-STTSMM-NH2 Ac-SSTSMM-NH, Ac-STTSAM-NH,

8 2-430 Ac-CESTFE-NH, Ac-GDSTYE-NH, Ac-GESTY E-NH,

2 2 2

13 18 21

390 873

2800 2 2 2

43 43 43 43 43 43

51 51 51 52 52 52

16 1-4 YGGFMP-NH2 YGGFMQ-NH, YGAFLP-NH,

16 3-14 DYKAKE-NH2 DYKEKL-NH2 DYKAKL-NH,

1 1 2

3 5 5

9 9 9

53 53 53


determined in order to confirm the screening results, as well as to determine the specificity of the antigen-antibody interactions. The N-acetylated PS-SCL had previously been screened against mAb 3E7. A free amino terminus is known to be essential for this interaction. Accordingly, no peptide mixture of the acetylated hexapeptide PS-SCL was found to bind to mAb 3E7.

A decapeptide PS-SPCL composed of more than 1012 sequences has been used for the identification of antigenic peptides for four different antibodies with up to 100-fold improved recognition compared to the original antigenic determinants.44~48

A hexapeptide PS-SCL bound to cotton carriers was screened against mAb 17/9 both immobilized by direct ELISA, in which the amount of antibody bound to the immobilized peptide mixtures was measured directly, as well as by competitive ELISA in solution, following cleavage of the peptide mixtures from the cotton carriers.46 The screening results are shown in Figure 3. Using direct ELISA, tyrosine was clearly identified as the most effective amino acid in position five. The two aspartic acid residues, however, which are known to be equally specific, could not be identified in positions one and four, neither could the less specific residues alanine, proline, and valine in positions six, three, and two, respectively. Conversely, competitive ELISA using the same peptide mixtures in solution yielded the correct amino acid for each position except for the second position, in which lysine was found to be more effective than the expected amino acids valine and isoleucine. These results indicate improved recognition of peptides free in solution by this antibody, as compared to peptides immobilized to a solid support.

As a result of these studies, it was found that mAbs exhibit various degrees of specificity, ranging from mAbs recognizing only conservative substitutions at one or two positions of the antigenic sequence, to mAbs that recognize sequences completely unrelated to the parent antigen while having comparable affinities. Utilizing the molecular diversity represented by SCLs, the extent of multiple binding specificities can be assessed in a systematic manner.

B. Opioid Receptor Ligands

In order to verify the utility of SCLs for the identification of receptor ligands, a nonacetylated dual positional peptide SCL (OIO,XXXX-NH,) was screened in an opioid receptor assay.38,57 This library was chosen since the natural opioid receptor ligands, the enkephalins (i.e., YGGFL and YGGFM), have a free amino terminus. The 400 peptide mixtures making up the library were tested for their ability to inhibit binding of [3H]- [D-Ala2,MePhe4,Gly-oI"]enkephalin (DAMGO, specific for the p,-opioid receptor) to membrane-bound receptors in crude rat brain homogenates. YGXXXX-NH, (IC50 = 3,452 nM) was found to be the most active peptide mixture. The four mixture positions were successively defined through the iterative synthesis and screening process described above. YGGFMA-NH, (I& = 28 nM) and YGGFLA-NH, (ICs0 = 59 nM) were identified as the most active individual peptides. The first five positions of these sequences exactly match the sequences of Met- and Leu-enkephalin, respectively. The sixth position was found to be relatively redundant, since the activities of the peptides did not change significantly upon truncation to pentapeptides when the sixth position was omitted.

A nonacetylated hexapeptide PS-SCL was screened for inhibition of either 3H- DAMGO (p,-receptor-specific),25 or 3H-DPDPE (6-receptor-specific).9 Individual peptides representing combinations of the most active amino acids found for each position in both

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assays (24 peptides for the preceptor and 32 peptides for the S-receptor) were synthesized and tested in the respective assays. While the most active peptide for the p.-receptor (YGGFMY-NH,, IC,, = 17 nM) contained the enkephalin sequence, in the S-receptor assay the best peptide (YGMFLV-NH,, IC,, = 45 nM) was approximately five times more active than the enkephalin containing sequences identified.

An N-acetylated sizing SCL (Ac-OXX-NH, through Ac-OXXXXXXX-NH,), ranging in length from three to eight positions, was used to determine the optimal length of possible N-acetylated opioid peptides. It should be noted that prior to this study, no acetylated opioid peptides were known. None of the mixtures five amino acids in length or shorter bound to the opioid receptor. Hexapeptide and longer peptide mixtures with arginine in the defined position were highly active.58 Consequently, the N-acetylated hexapeptide SCL (Ac-O,O,XXXX-NH,) was screened, resulting in the identification of Ac-RWXXXX-NH, and Ac-RFXXXX-NH, as the most active peptide mixtures. The iterative process for Ac-RFXXXX-NH, resulted in the identification of a new group of potent opioid receptor antagonists, termed acetalins (Ac-RFMWMO-NH,, 0 = K,T,R, IC,, = 5- 6 nM).S9

An N-acetylated hexapeptide SCL prepared entirely from D-amino acids (Ac-o,o,xxxx- NH,) was also screened in the opioid receptor assay, followed by the standard iterative process, which resulted in the identification of a potent all D-amino acid opioid peptide (Ac-rfwink-NH,, IC,, = 18 nM). This peptide was found to be a preceptor specific agonist and to produce analgesia in mice when administered intracerebroventricularly (i.c.v.) or intraperitonaelly (i.p.).8 These analgesic effects were antagonized by i.c.v. injection of naloxone, indicating that they were centrally mediated, and, consequently, that Ac-rfwink-NH, crosses the blood-brain barrier.

C. Antimicrobial Compounds

The increasing occurrence of bacterial strains which are resistant to commonly used antibiotics has led to a critical need for new antimicrobial agents. SCLs represent a powerful means to develop new antimicrobial leads for pharmaceutical research. Several microorganisms have been used in studies involving peptide SCLs: gram-positive bacteria such as Staphylococcus aureus and Staphylococcus sanguis, gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, and yeast such as Candida albicans.

A variety of potent antimicrobial peptides have been identified through the screening of L-amino acid peptide SCLs of different formats.2,38,6"-63 Since L-amino acid peptides are prone to proteolytic degradation, which limits their pharmaceutical utility to topical or intravenous application, compounds which are resistant to proteolytic degradation are expected to be of higher therapeutical value than L-amino acid peptides. Conse- quently, an N-permethylated peptidomimetic PS-SCL, the components of which have been shown to be resistant to enzymatic breakdown, was screened against S. a ~ r e u s . ~ ~ The screening data are shown in Figure 4. The following residues were chosen for the synthesis of individual permethylated peptides: phenylalanine, histidine, isoleucine, leucine, tryptophan', and tyrotine in position one; phenylalanine in position two; phenylalanine, isoleucine, and tryptophan in position three; phenylalanine and tryptophan in position four; phenylalanine and histidine in position five; and phenylalanine and histidine in position six. All possible sequence combinations of those amino acids generated a set of 6 x 1 x 3 x 2 x 2 x 2 = 144 individual permethylated peptides, which were synthesized and tested, resulting in the identification of the permethylated peptides pm[LFIFFF-NH,] and pm[FFIFFF-NH,] as the compounds with the strongest antibacterial

492 EICHLER ET AL.

Figure 4. Antimicrobial activity of the permethylated (pm-) PS-SCL against S. mireus. Each graph represents one positional SCL. The bars within each graph represent the reciprocal IC,,, values of the permethylated mixtures defined at the 0 position with the amino acids specified on the x-axis.

activity (IC50 = 6 pg/mL for both) against S. aureus. Similar activities were found against S. sanguis, whereas none of the permethylated compounds or mixtures were active against the gram-negative bacteria E . coli or yeast C. albicaiis (IC50 > 600 kg/mL).

In a separate study,7 a tetrapeptide library (UZZZ-NH,), which was synthesized from a set of 58 L-, D- and other nonproteinogenic amino acids (see above), was screened against S. aweus and E . coli. The peptide mixture with Na-Fmoc-E-lysine in the defined position (U) showed the highest activity in both assays (IC5" = 44 and 179 kg/mL, respectively). Following the iterative synthesis and screening process, (crFmoc4ys)WfR- NH, (MIC = 4-8 kg/mL against S. aweus ) and (aFmoc-E1ys)WKW (MIC = 16-31 Fg/mL against E. coli) were identified as the most active antibacterial compounds.

D. Enzyme Inhibitors

Enzyme inhibitors are of broad interest for biomedical research, as well as for their therapeutic potential. The possibility of identifying enzyme inhibitors through the screening of SCLs was studied using trypsin and chymotrypsin as model enzymes. Numerous polypeptide and protein inhibitors of these enzymes from plant and animal sources are known, which are highly specific, limited proteolysis substrates for their target enzymes.64 Their amino acid sequences contain at least one peptide bond, called the reactive site (-Pl-I','-), which specifically interacts with the active site of the cognate enzyme. The reactive sites of trypsin inhibitors have arginine or lysine at the P, position. A hexapeptide SCL was specifically designed to represent all possible reactive sites for trypsin inhibitors with the P, position (lysine or arginine) in every position of the sequence except the last. This library, which is composed of 10 sublibraries, represented as Ac-KOXXXX, Ac-ROXXXX, Ac-XKOXXX, Ac-XROXXX . . . through Ac- XXXXKO and Ac-XXXXRO, was screened in a trypsin inhibition assay, followed by an iterative synthesis and screening process. The most active trypsin inhibitor found was


Ac-AKIYRP-NH, (IC50 = 46 pM).23 This sequence was subsequently incorporated as a motif in a secondary library of 12-mer peptides, represented as Ac-XXXAKIYRPOXX. Upon iteratively defining all of the positions of the sequence, Ac-YYGAKIYRPDKM- NH, (IC50 = 10 pM) was found to have an approximately five-fold improved inhibitory activity compared to the hexapeptide from which it was derived.

Another hexapeptide SCL, represented as XXOOXX-NH,, was screened in a chymotrypsin inhibition assay, resulting in the identification of WFLYYC-NH, (ICso = 17 pM, unpublished) as the most active chymotrypsin inhibitor. This sequence contains four aromatic amino acid residues (tryptophan, phenylalanine, and two tyrosine residues), each of which may serve as the P, position of the reactive site, since aromatic amino acids are known to be specific for the P, position of chymotrypsin inhibitors.

Trypsin and chymotrypsin inhibitors were also identified through the screening of a hexapeptide PS-SCL synthesized entirely with D-amino acids. The most active inhibitors found were Ac-ryrpwp-NH, (IC50 = 62 pM) for trypsin9 and Ac-ygyyyr-NH, (IC50 = 36 pM) for chymotrypsin (unpublished). As expected, both peptides are completely stable towards proteolytic degradation.

Chymotrypsin inhibitors with no structural resemblance to any known inhibitors of this enzyme were identified through the screening of the cyclic peptide template PS- SCL described above.13 Upon screening of the three positional libraries making up this PS-SCL, the same carboxylic acids (i.e., piperonylic acid and thiophenecarboxylic acid) were found to be the most effective functionalities at each of the three positions (E-

amino groups of lysine). Eight individual compounds representing all possible sequence combinations of these "-substituted lysines were synthesized and tested. The structure of the most active inhibitor (ICsO = 51 pM) is shown in Figure 5. It should be noted that none of the compounds identified would have been found had only the proteinogenic amino acids been used as building blocks for the library.

Figure 5. Structure of the most active chymotrypsin inhibitor identified from the cyclic peptide template PS-SCL.

494 EICHLER ET AL.

E. Catalytic Peptides

Molecular recognition as the first step in the sequence of events of biological effects, such as biocatalysis, relies upon a conformationally defined environment. Based on this premise, a conformationally defined PS-SCL was designed and synthesized by replacing five positions of an amphipathic a-helical 18-mer peptide composed of alternating leucine and lysine residues,65 with one defined and four mixture positions, respectively.66 The initial reaction studied with this library was the decarboxylation of oxalacetate. A set of peptides which catalyze this reaction with substantially higher activities than a recently reported catalytic peptide (oxaldie-l)67 was identified. Among those peptides was YKLLKELLAKLKWLLRKL-NH,, which was found to catalyze the decarboxylation of oxalacetate with a catalytic constant of k,,, = 1.5 . l o -2. s-1. This reaction proceeds at a rate 103- to lO4-fold faster than the reaction catalyzed by simple amines, and was found to correlate well with the ability of this peptide to fold into an a-helical conformation.66

IV. CONCLUSIONS

The broad utility of SCLs relies upon the fact that they can be screened in virtually any bioassay. This is illustrated by the various levels of complexity of biological systems which can be studied using SCLs, from soluble receptors (i.e., antibodies, enzymes), to membrane-bound receptors (opioid receptors), to whole cells (bacteria, yeast). It should be noted, however, that SCLs are not limited to the soluble state, but can also be screened immobilized in assays where the solid-phase format is beneficial (e.g., direct binding assays involving soluble acceptors).

The versatility of the SCL concept is also reflected by the range of libraries with different formats and compositions. Starting with ”simple” L-amino acid peptide libraries, the SCL concept has since been used for the synthesis of peptide libraries composed of D- and other nonproteinogenic amino acids and carboxylic acids. Confor- mationally defined libraries have been prepared which enable the custom design of complex molecules with defined secondary structures that can perform specific biological functions. The concept of “libraries from libraries,” which is based on the post- synthetic chemical modification of parent peptide or nonpeptide libraries, opens the door to novel chemical diversities with great therapeutic potential.

The scope and versatility of synthetic combinatorial libraries can be expected to in- creasingly expand their significance for basic research and drug discovery.

We thank Eileen Silva for her assistance in preparing this manuscript. This work was funded by Houghten Pharmaceuticals, Inc., San Diego, California.

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