soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

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trends in analytical chemistry, vol. 14, no. 2, 1995 83 Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities SE. Blondelle, E. Perez-Pay& C.T. Dooley, C. Pinilla, R.A. Houghten * San Diego, CA, USA Molecular diversity provides a source of novel lead compounds for drug discovery. In particular, soluble combinatorial libraries composed of millions of peptides, peptido- mimetics and organic compounds have lead to the successful identification of new phar- macophores. The current development of structurally defined combinatorial libraries will broaden the applicability of combinato- rial chemistry. 1. Introduction While the search for novel pharmacophores from natural sources continues to be an important source for therapeutically interesting lead compounds, a more direct approach has recently been developed which involves the generation and systematic screening of immense molecular diversities (i.e., tens to hundreds of millions). A number of differ- ent strategies have been developed using the prin- ciple of solid-phase synthesis to synthesize these diversities in a manner which permits their use for biological screening. The generation of such ‘chemical libraries’ was originally focused on pep- tides and nucleotides, for which synthetic proce- dures were straightforward and well established. Peptides have long been used as starting com- pounds for the development of novel drugs, even though they are limited as potential therapeutic agents due to their typical lack of oral activity, susceptibility to proteolytic breakdown, and ina- bility to pass through the blood brain barrier (although it should be noted that therapeutic agents such as calcitonin and cyclosporin A, which are medically and economically important drugs, are peptides). Advances in synthetic procedures over the last 100 years now enable scientists to generate large * Corresponding author. 0 1995 Elsevier Science B.V. All rights reserved collections of individual compounds for screening and research purposes. Thousands of peptides, which are key components in all mammalian bio- chemical systems, can now be prepared quickly and inexpensively by a modestly equipped labo- ratory, using either standard manual procedures [ l-61 or automated synthesizers [ 7,8]. A novel, light-directed approach for the synthesis of thousands of peptides on silica surfaces has also been reported [9]. This article briefly reviews a number of techniques used to generate combina- torial libraries of peptides, peptidomimetics and organic compounds, with an emphasis on the approaches carried out in this laboratory. 2. Chemical library preparation 2.1. Library types The term ‘library’ is now commonly used to describe virtually any collection of compounds ranging in number from less than 10 [ 9-121 to literally trillions. The analoging systems which enable large numbers of compounds to be prepared separately using parallel synthesis methods include Geysen’s pin approach [ 21 and Houghten’s T-bag method [ 31, along with a number of later variations [ 121. While such collections, or series of analogs, are now also called libraries, the original meaning of the library concept referred to vast numbers (millions) of compounds prepared in a highly sys- tematic manner, which consisted of all combina- tions of a class of compounds [ 2,3]. The premise of such libraries is that they enable completely novel, biologically active compounds to be iden- tified through screening without any prior struc- tural or sequence knowledge. A wide range of combinatorial libraries made up of the requisite millions of compounds typically required for de novo discovery have now been described [ 13,141. The two main approaches gen- erally used involve: 0 libraries comprised of large collections of individual compounds, each physically sepa- rated from one another by being attached to separate beads, phage, etc.; and 016S-9936/95/$09.50

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Page 1: Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

trends in analytical chemistry, vol. 14, no. 2, 1995 83

Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

SE. Blondelle, E. Perez-Pay& C.T. Dooley, C. Pinilla, R.A. Houghten * San Diego, CA, USA

Molecular diversity provides a source of novel lead compounds for drug discovery. In particular, soluble combinatorial libraries composed of millions of peptides, peptido- mimetics and organic compounds have lead to the successful identification of new phar- macophores. The current development of structurally defined combinatorial libraries will broaden the applicability of combinato- rial chemistry.

1. Introduction

While the search for novel pharmacophores from natural sources continues to be an important source for therapeutically interesting lead compounds, a more direct approach has recently been developed which involves the generation and systematic screening of immense molecular diversities (i.e., tens to hundreds of millions). A number of differ- ent strategies have been developed using the prin- ciple of solid-phase synthesis to synthesize these diversities in a manner which permits their use for biological screening. The generation of such ‘chemical libraries’ was originally focused on pep- tides and nucleotides, for which synthetic proce- dures were straightforward and well established. Peptides have long been used as starting com- pounds for the development of novel drugs, even though they are limited as potential therapeutic agents due to their typical lack of oral activity, susceptibility to proteolytic breakdown, and ina- bility to pass through the blood brain barrier (although it should be noted that therapeutic agents such as calcitonin and cyclosporin A, which are medically and economically important drugs, are peptides).

Advances in synthetic procedures over the last 100 years now enable scientists to generate large

* Corresponding author.

0 1995 Elsevier Science B.V. All rights reserved

collections of individual compounds for screening and research purposes. Thousands of peptides, which are key components in all mammalian bio- chemical systems, can now be prepared quickly and inexpensively by a modestly equipped labo- ratory, using either standard manual procedures [ l-61 or automated synthesizers [ 7,8]. A novel, light-directed approach for the synthesis of thousands of peptides on silica surfaces has also been reported [9]. This article briefly reviews a number of techniques used to generate combina- torial libraries of peptides, peptidomimetics and organic compounds, with an emphasis on the approaches carried out in this laboratory.

2. Chemical library preparation

2.1. Library types

The term ‘library’ is now commonly used to describe virtually any collection of compounds ranging in number from less than 10 [ 9-121 to literally trillions. The analoging systems which enable large numbers of compounds to be prepared separately using parallel synthesis methods include Geysen’s pin approach [ 21 and Houghten’s T-bag method [ 31, along with a number of later variations [ 121. While such collections, or series of analogs, are now also called libraries, the original meaning of the library concept referred to vast numbers (millions) of compounds prepared in a highly sys- tematic manner, which consisted of all combina- tions of a class of compounds [ 2,3]. The premise of such libraries is that they enable completely novel, biologically active compounds to be iden- tified through screening without any prior struc- tural or sequence knowledge.

A wide range of combinatorial libraries made up of the requisite millions of compounds typically required for de novo discovery have now been described [ 13,141. The two main approaches gen- erally used involve: 0 libraries comprised of large collections of

individual compounds, each physically sepa- rated from one another by being attached to separate beads, phage, etc.; and

016S-9936/95/$09.50

Page 2: Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

84 trends in analytical chemistry, vol. 14, no. 2, 1995

A Physical Preparation

Combine and Mix Resin

1

B Chemical Preparation

Coupling (mixture of

1

amino acids)

Boc-Ala Boc-Asp(l3zI) Boc-Glu(Bzl) . . .

recombine process or (B) a possible combinations of the

Fig. 1. Generation of equimolar resin mixture using (A) the divide, couple, and chemical ratio of amino acids. (A) For the preparation of peptides composed of all 20 L-amino acids, the resins are divided and compartmentalized into 20 separate polypropylene packets at each coupling step, then mixed prior to the wash, deprotection and neutralization steps [31]. (B) A predetermined ratio of amino acids [20] is used at each coupling step.

0 combinatorial libraries comprised of pools or mixtures of compounds.

The use of combinatorial libraries is a ‘numbers game’ in which literally tens to hundreds of mil- lions of compounds can now be screened for activ- ity as rapidly as overnight. In the discovery of novel highly active compounds, it is advantageous if all possible combinations of amino acids or building blocks are present. The differences between the two types of libraries result from the synthesis method used for their generation. In the first case, each individual compound is prepared on separate solid supports (i.e., plastic pins, resin packet-‘T bags’, resin beads, etc.) or on spatially separated regions of the same support during the synthesis process (i.e., silica surface, cellulose surface, etc.). This can be easily performed using the different simul- taneous multiple synthesis techniques. In the case of pools of compounds, either the growing peptide chains are prepared by mixing of the resins follow- ing each coupling step [i.e., the divide, couple and recombine (DCR), or ‘split’ resin method, see below; Fig. 1 A] or by using a mixture of building blocks that are incorporated simultaneously during the coupling procedures (Fig. 1B).

2.2. Preparation of combinatorial libraries

Two major approaches have been used for the generation of combinatorial libraries. The first one involves the DCR process (also known as the ‘split’ resin method, and the one bead/one peptide approach) which has been independently reported for the synthesis of peptide combinatorial libraries by this laboratory and two other groups [ 1% 171. For example, when one is synthesizing libraries of peptides made up of all the combinations of the 20 naturally occurring L-amino acids, the coupled resin is divided into 20 equal portions, then each portion is separately coupled to a single amino acid. The 20 equal portions are then mixed together for the required deprotection and neutralization steps. This resin mixture is then redivided again into 20 equal portions for the next individual coupling steps. This process is repeated until the synthesis is complete (Fig. 1 A). The essence of this approach is that highly complex mixtures of com- pounds can be prepared, in which each is on a separate bead (i.e., millions of beads/millions of peptides [ 161)) or if the final pooled mixtures of resin-bound compounds are cleaved from the resin

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trends in analytical chemistry, vol. 14, no. 2, 1995 a5

support, then virtually equal amounts of hundreds of thousands to billions of compounds can be pre- pared in soluble form [ 151. The DCR process, by its very nature (i.e., the exactitude of the physical process of weighing the separate resins, mixing them, subdividing and weighing them again), assures the generation of equimolar or very close to equimolar compound-bound resins.

In a second approach, mixtures of all of the desired protected amino acids or building blocks are used simultaneously in the coupling step during solid-phase synthesis (Fig. 1 B) [ 18,193. If one is using the 20 naturally occurring L-amino acids, then six couplings of such a mixture will result in 64. lo6 hexamers. One perceived disadvantage of this method is the potential non-equimolarity of the resultant mixtures due to the inherent variations in coupling rates between amino acids or building blocks. In the case of peptide libraries, kinetic stud- ies of the relative coupling rates of incoming amino acids performed in this laboratory have lead to the determination of amino acid ratios necessary to ensure close to equimolarity peptide mixtures within a library [20]. The success of this second approach has been repeatedly proven by compar- ative biological studies using peptide libraries rep- resenting the same peptide diversity but prepared by either method [ 15,18,19,2 11. Thus, identical sequences recognized by a monoclonal antibody raised against a peptide [ 15,181, as well as peptide sequences having binding affinity to opioid recep- tors [ 19,211, have been identified through the screening of two libraries prepared by the two approaches. The advantages of this method over the DCR method are the ease of the synthesis proc- ess, as well as the ability to prepare complete librar- ies with more than three or four mixture positions. Furthermore, the defined positions in the library can be readily located in positions other than the N-terminal.

3. Library screening approaches

3.1. Molecular biology approach: pep tides displayed on phage

Large collections of peptides have been created by the molecular cloning and expression of random mixtures of oligonucleotides. Two of the central features of this approach are the ability to determine which of the peptides are responsible for the spe- cific binding of the peptide-phage to an immobi-

lized soluble receptor, and the ability to generate libraries of long peptide or protein sequences. Sev- eral approaches for the generation of such libraries have been developed in the past few years using the fusion of peptides to a cell surface protein to obtain millions of random peptide sequences (reviewed in Ref. [22] ). The active peptide- expressing phage particles can be enriched in a selection process termed ‘biopanning’, until a spe- cific active sequence can be determined by sequencing the protein-encoding region of the phage DNA. Such libraries have been successfully used to study binding to antibodies and receptors (reviewed in Ref. [ 221) .

3.2. Chemical approach: support-bound peptides

The first report of this approach involved the synthesis of mixtures of tens to hundreds of thousands of octapeptides on plastic pins [ 231. These libraries, combined with an iterative process, were purported to enable the identification of indi- vidual sequences, termed ‘mimotopes’ (i.e., mim- ics of discontinuous epitopes), which bound to a monoclonal antibody [ 23,241. This approach has met with limited success.

A second immobilized method, referred to as the ‘one bead/one peptide’ approach, has been pre- sented in which one to three million individual peptides, each attached to a separate resin bead, are generated and assayed for binding to antibodies or receptors [ 161. This approach uses calorimetric methods to visualize the bead to which the protein is attached. The peptide associated with the specific protein-binding bead is then microsequenced to identify the peptide. The active sequences in such libraries can also be identified by the determination of an attached polynucleotide [ 2.51, polypeptide [ 26,271, or halogenated aromatic tag [ 281.

Another approach for the generation of support- bound libraries involves the preparation of peptide mixtures by standard solid-phase chemical synthe- sis methods on cotton supports [29]. When cleaved in situ, such libraries have been used to identify new trypsin inhibitors [ 291.

3.3. Chemical approach: soluble combinatorial libraries

As first presented by this laboratory, large diver- sities of compounds can be prepared which are not attached to any solid support, and which can there- fore be used in solution in virtually any assay sys-

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86 trends in analytical chemistry, vol. 14, no. 2, 7995

steps Procow sequence

1. screening 00XxXx SelectIon RRXXXX

2. SyllthWlS RROXXX

3. Scmenlng RROXXX Selectton RRWXXX

4. DyIlthCAs RRWOXX

5. susenklg RRWJXX S&dOIl RRWCXX

6. synlhe*rlr RRWCOX

7. SCWWllllg RRWCOX SElW6OIl RRWCKX

a. Syntherlr RRWKO

9. Screening RRWKO Selectbn RRWCKR

Groups

400 x 1 x

20 x

20 x 1 x

20 x

20 x 1 x

20 x

20 x 1 x

20 X

20 x 1 x

ff peptldaslgmup Total # of peptides

160,000 64.000.000 160,000 160,000

6,000 160,000

160,000 6,000

6.000 6,000

400

400 400

20

20 20

1

1

1

6.000

6,000 400

400

400 20

20

20 1

Fig. 2. iterative process of selection and synthesis. O=defined position with one of the 20 naturally occurring L-amino acids; X = close to equimolar mix- ture of the 20 naturally occurring L-amino acids.

tern [ 15,181. The libraries are prepared using the synthetic diversity approach called simultaneous multiple peptide synthesis method (SMPS), also known as the ‘T-bag’ approach [ 3,301, which by its nature enables the rapid generation of cleavable peptide mixtures. The synthesis process enables the peptide mixtures within a library to be prepared in separate groups, each being characterized by a sin- gle or multiple defined residue at a given posi- tion( s). Thus, this simple self ‘coding’ of each peptide mixture group enables the rapid identifi- cation of the active sequence(s) .

We have devised two separate strategies for the identification of active sequences from mixtures of millions of compounds present in a combinatorial library (i.e., the ‘deconvolution’ process). The first strategy involves an iterative process of selection and synthesis, which results in the identification of one residue or building block per assay and syn- thesis cycle [ 151. An example which illustrates this process for a hexapeptide library is shown in Fig.

steps Process sequsnce Groups X peptldeslgmup Total I of peptides

1. scresning 0xXxXx 20 x 3,200,OOO 64,000,OOO Screening x0xXxX 2c x 3.200,OOO 64,000,000 Screening XXOXXX 20 x 3,200.OOO 64.000.000 scresning XXXOXX 20 x 3.200.000 64.000.000 SCW3lllllg XXXXOX 20 x 3.200,OOO 64.000.000 sueaIling XXXXXO 20 x 3.200.000 64,000.000

SelectIon RXXXXX 1 x 3.200,OOO 3.200.000 Selwtlon XRXXXX 1 x 3.200.000 3.200.000 Selecuon XXwXX% 1 x 3.200.000 3.200.000 Selecuon XXXCXX 1 x 3.200.000 3.200.000 Selealon XXXXKX 1 x 3.,!00,000 3.200.000 Selectton XXXXXR 1 x 3.200.000 3.200.000

2. Synthasls RRWCKR 1 x 1 1

Fig. 3. Positional scanning process. 0 and X are defined as described in Fig. 2.

2. Four repetitive steps of selection and synthesis are required to identify the individual active com- pounds. One or more peptide mixtures can be cho- sen at each selection step. The selection criteria are based on a fully empirical approach of identifying the highest activities of interest and/or variation in the chemical nature of the amino acids or building blocks between the most active mixtures.

A second deconvolution strategy, termed the positional scanning approach, enables active sequences to be identified in a single screening assay [ 18,19,2 13. A positional scanning combi- natorial library is divided into separate positional libraries, each of which contains only a single defined residue or building block (Fig. 3, for a hexapeptide library). It should be noted that each separate positional library (e.g., first-position library AC-OXXXXX-NH,, etc.) consists of the same number of compounds. They differ from one another only by the defined position of these com- pounds within the different groups (e.g., group AC-AXXXXX-NH,, etc.). When used in concert, the data derived from each single-position library yield information about the most important resi- due(s) or building block(s) for every position in a sequence. The information is then used to syn- thesize individual compounds representing all pos- sible combinations of the most active building block(s) identified at each position. This confirms the information obtained from the initial screening of the separate positional libraries, and enables the identification of the most active compound(s). Both strategies have been successfully used for the identification of antigenic determinants [ 15,18,21,3 l-331, compounds binding to opioid receptors [ 19,21,34], new antimicrobials [ 15,3 1,35-371, and new inhibitors of enzymes [ 2 1,291 or of lytic compounds [ 2 1,351.

4. Soluble synthetic combinatorial libraries (SCLs)

4.1. Peptide SCLs

A number of peptide SCLs have been prepared and screened for biological activity in this labora- tory using competitive ELISA, radio-receptor assays, microdilution assays, etc.. The first of these libraries to be synthesized was composed of mixtures of hexapeptides, in which the first two positions were defined with one of the 20 naturally occurring L-amino acids, with the remaining four

Page 5: Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

AC- RROXXX- NH,

3I

0 I x A CDE FFH I KLMNPQRSTVWY

AC- RRWOXX- NH,

1

X A CDE FGH I KLMNPQRSTVWY

AC-RRWCOX-NH, AC- RRWWOX- NH, , 10,

87

X A CDE FGH I KLMNPQRSTVWY X A CDE FGH I KLMNPQRSTVWY

AC-RRWCKO-NH, AC- RRWWRO - NH, ^^.

x ACDE FFH I KLMNPQRSTVWY X A CDE FGH 1 KLMNPQRSTVWY

AC-RRWCKR-NH, IC,,=67pg/ml AC-RRWWRR-NH, IC,=26pg/ml

Fig. 4. identification of antifungal hexapeptides. The activity was determined against 1 O5 CFU/ml Candidaalbicans following a 48 h incubation at 30°C as described elsewhere [37]. The IC,, values (in pg/ml, peptide mixture concentration necessary to inhibit 50% cell growth) were determined from two-fold serial dilutions of the peptide mixtures, and calculated using a sigmoidal curve fitting method. Each graph represents a screening step carried out during the iterative process from the peptide mixture AC-RRXXXX-NH,. Each bar represents the reciprocal of the IC,, values, and is labeled on the xaxis by the amino acid used to define the ‘0’ position. X represents the peptide mixture from the previous iterative step.

positions as mixtures of the same amino acids [ 151. An example of the use of this SCL is illustrated here for the identification of new antifungal hexa- peptides against Candida albicans (Fig. 4). A number of hexapeptide sequences have been iden- tified in a similar manner which have potent activ- ity against gram-positive bacteria such as Staphylococcus aureus and Streptococcus sanguis

[ 15,3 11, and/or against gram-negative bacteria such as Escherichia coli and Pseudomonas aeru- ginosa [36]. A non-acetylated version of this library in the positional scanning format, as well as non-acetylated and acetylated hexapeptide librar- ies made up entirely of D-amino acids, have been prepared and tested in various in vitro assays [ 381, and even in an in vivo assay to identify peptides

Page 6: Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

trends in analyticalchemistry, vol. 14, no. 2, 1995

A Standard SPCL

AC-OOXXXX-NH,

400 x 160,000 = 64.000,000 hexapeptides

B Disulfide Labrary

1 Non-axidrzed

AC-OCXX-NH, (L-amino acid) Ac-aca-NH, (D-amino sad)

20 x 400 = 6,000 tetrapeptides 20 x 400 = 6,000 tetrapeptides

2 Oxidized

AoOCXX-NH, A&a-NH,

400 x 160,000 = 64.000.000 cctapeptides

Fig. 5. Disulfide libraries.

which regulate blood pressure and/or heart rate

c391. While L-amino acid peptides are expected to be

susceptible to proteolytic breakdown, and, in turn, may be limited to topical therapeutic use, the inser- tion of D- or unnatural amino acids into the sequence is anticipated to increase the duration of activity and applicability of such compounds. Indi- vidual tetrapeptides were thus identified through the screening of a tetrapeptide library made up of L-, D-, and unnatural amino acids that show potent antimicrobial activities against different microor- ganisms [ 35,371.

4.2. Disulfide-linked dimer SCLs

The identification of discontinuous determinants has not generally been successful using any of the existing library approaches. If a library occupying a broader conformational space of interactive res- idues can be generated, then it should be possible for discontinuous determinants of proteins to be identified. One way to accomplish this is through the use of disulfide-linked peptide libraries. The oxidation of peptides in a monomeric cysteine-con- taining peptide mixture with a different monomeric cysteine mixture results in a cysteine dimer library. The number of newly generated disulfide-linked compounds in this cysteine library is equal to the number of compounds in the first set multiplied by the number in the second set. This enables, for example, a diversity of 64. lo6 different dimers to be generated from two separate cysteine monomer mixtures of 8000 cysteine peptides (i.e., OCXX made up of L-amino acids and ocxx made up of D- amino acids). Thus, in the simple example illus- trated in Fig. 5, each peptide in mixture A can randomly associate with itself or with any of the

peptides in mixture B. In our initial studies this oxidation was carried out by direct treatment of the cysteine mixtures with H202. One finds that the oxidation of an individual cysteine-containing pep- tide ‘A’ and a second individual cysteine-contain- ing peptide ‘B’ will yield a mixture of cysteine dimers: A-A, A-B = B-A, B-B in the ratios of 1:2: 1, since A-B = B-A. In an RP-HPLC confor- mational study using two model peptides, Ac- DVPDYAC-NH2 (peptide A) and Ac- PYPLNSC-NH2 (peptide B), the oxidation of these two peptides by H202 yielded a ratio of 1.2: 1.9:0.9 (AA:AB/BA:BB) .

When one forms a self disulfide set from all combinations of the 20.1 92 = 7220 peptides con- tained within the OCXX-NH2 monomer set, there are not (20 - 192) 2 = 52 128 400 combinations in the resulting disulfide library, as would be the case if two completely different monomeric sets were brought together (i.e., one containing only L-amino acid peptides and the other only D-amino acids peptides). The actual number of unique com- pounds is 26 067 810. This number is calculated from the number of unique mixtures and the num- ber of unique compounds in each library. The num- ber of unique sub-libraries is CN, where N is the number of amino acids in the 0 position, which then becomes CN=N2- [(N2-N)/2]. The 210 unique sub-libraries consist of 20 homodimeric sub-libraries (where 0 in each set is the same amino acid) and 190 heterodimeric sub-libraries (where 0 in each set is a different amino acid). Each of the 20 homodimeric sub-libraries contain C192=65 341 unique compounds, while each of the 190 heterodimeric sub-libraries contain C( 1 92 + 192) = 261 003 unique compounds at l/2 the concentration of the compounds found in the homodimeric sub-libraries. In addition, due to the formation of the A-A:A-B = B-A:B-B com- binations, each heterodimeric sub-library is also contained in the two possible homodimeric mixtures, each consisting of C 192 = 65 341 com- pounds, at the same concentration as the heterodi- merit compounds. In a preliminary study, we have examined the oxidation of AC-OCXX-NH, (made up of L-amino acids but with cysteine excluded for the X positions). This results in 210 discrete mixtures, each made up of 65 341 dimers (this library contains a total of 13 72 1 6 10 dimers) . This disulfide-linked SPCL was screened in an opioid receptor binding assay [ 191. The most effective mixture was found to be di [ AC-RCXX-NH,/Ac- WCXX-NHJ with an I& of 82 14 nA4. The activ-

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trends in analytical chemistry, vol. 14, no. 2, 7995 89

ity of this heterodimer is potentially twice as high as found, since the oxidation process yields a mix- ture of both the desired heterodimer as well as the homodimers, neither of which were active. The iterative process is being carried out in order to define the next positions of these peptide mixtures. It should be noted that from the 400 linear mixtures of the standard SPCL, (i.e., AC-OOXXXX-NH2), the most active individual mixture was found to be AC-RWXXXX-NH2, with an I& of 2128 ruJ4 [ 341. Since the final activities in the linear case were in the 4-10 ti range, it is anticipated that substantial improvement will result as the iterative process is advanced in this disulfide case.

4.3. Peptidomimetic SCLs

In order to enhance the physico-chemical prop- erties of peptides to broaden their range of utility (i.e., enhanced resistance to proteolytic enzymes, high water solubility, favorable aqueous-organic partitioning characteristics, etc.), interest has been high in the development of soluble peptidomimetic libraries. Advances in synthesis technologies and chemistries have lead to the generation of pepti- domimetic and non-peptide compounds (summa- rized in Ref. [ 401). For instance, the preparation of libraries of oligomeric N-substituted glycines, termed peptoids, has been reported [ 4 I].

In this laboratory, we have developed an effi- cient method for the generation of peptidomimetic and chemical combinatorial libraries by chemically transforming existing peptide SCLs. For example, the permethylation of the amide nitrogens of pep- tide SCLs can be carried out while they remain attached to the solid support [42]. This approach capitalizes on well-established solid-phase synthe- sis methods and enables a wide range of useful chemical diversities to be envisioned. The defining requirement of this approach is its ability to effec- tively transform a pool of diverse chemical moie- ties in a clearly understandable manner (it should be noted that while quantitative transformations are not required, reproducibility is essential). This has been successfully accomplished by the preparation of a library resulting from permethylating the amide nitrogens of an existing hexapeptide posi- tional scanning SCL (Fig. 6) [42]. This library has been screened for antimicrobial activity against different microorganisms in a manner similar to the original peptide SCLs. A series of permethylated compounds were identified having potent antimi- crobial activity against gram-positive bacteria

R, 0 R, 0 R, 0 R, 0 y, : ff+ f I c~.,.N~-~-N_~_~_N_~_e_N_~_~_~_~_~_~_~_~_~_~

I

a3 &I3 &-I, a? &-I3 Fig. 6. N-permethylated positional scanning SCLs. One R group of R,, R,, R,, Rq, R, or R, represents the side chain groups of one of the 20 naturally occurring L-amino acids. The remaining R groups represent a close to equimolar mixture of the side chain groups of the 20 naturally occurring L-amino acids. The func- tionalities of the side chain groups have also been methylated for C, D, E, H, K, N, Q, R, W and Y.

(e.g., pm [ LFIFFF] , MIC = 8-l 0 pg/ml against a methicillin-resistant S. aUYeUS strain). We have successfully extended this method for the prepa- ration of peralkylated libraries derived from related chemical modifications using ethyl bromide, ben- zyl bromide, ally1 bromide, etc.

4.4. Organic chemical libraries

Combinatorial libraries of organic compounds can be expected to provide new candidates for the rapid screening of traditional ‘drug-like’ chemical structures. The synthesis of collections of benzo- diazepine-derivatives on plastic pins has recently been reported [ 11,431. A related approach, termed diversomers, has been developed for the synthesis of a collection of benzodiazepines and hydantoins using glass-fitted chambers [ 121. These are vari- ations on the multiple synthesis methods described earlier [ 2,3]. While such approaches lead to the preparation of small collections of individual organic compounds (i.e., tens to hundreds), they as yet do not involve combinatorial chemistry. This is in contrast to pools of millions of peptidomi- metics generated as described above for permethy- lation of peptide SCLs.

4.5. Future directions - fully synthetic receptors and enzymes

The use of synthetic combinatorial libraries to generate immensely diverse planer or topographi- cal landscapes is now possible. Thus, one can read- ily envision the design of fully synthetic ‘receptors’ which are custom designed to bind to specific ligands of virtually any type, as well as the custom design of ‘enzymes’ (i.e., complexes having cata- lytic properties) able to cleave or combine a wide variety of building blocks. Such libraries must fulfil the minimum structural requirement necessary to

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90

mimic the natural ‘enzymes’ or ‘receptors’. We have successfully designed conformationally defined libraries consisting of combinatorial build- ing blocks inserted in a structurally defined peptide sequence. This new structural approach has been successfully used to identify peptides that catalyze hydrolysis and decarboxylation reactions. The ini- tial catalytic reaction studied was the decarboxyl- ation of oxaloacetate, which is involved in the industrial synthesis of phenylalanine [ 441. Using a conformationally defined SCL derived from an amphipathic a-helical 1%mer peptide [ 451, we have identified a set of individual peptides which have substantially higher catalytic activity than the recently reported catalytic peptide oxaldie- 1 [ 461. Thus, YKLLKELLAKLKWLLRKL-NH2 was found to catalyze the decarboxylation of oxaloac- etate with a catalytic constant kcat = 1.5 . lo-’ s- ‘. This reaction proceeds at a rate lo3 to 104-fold faster than the simple amine catalysts.

Such enzymes and/or receptors can be envi- sioned to fulfil a wide range of basic research, ther- apeutic and industrial needs. Such breadth of use must be considered not an ‘if’ or ‘perhaps’ sce- nario, but rather a ‘when’ scenario, since these tech- niques cut across and are already used in a wide range of disciplines, including molecular biology and chemistry. Thus, certainly before the year 2000, artificial receptors, enzymes, templates, and self-assembling proteins will be available for a broad cross-section of applications.

5. Conclusion

The generation of soluble SCLs comprised of tens to hundreds of millions of peptides, peptido- mimetics and organic compounds can be now car- ried out with a high degree of confidence and exac- titude [ 15,18,47,48]. Such libraries have proven to be widely useful for the identification of a variety of biologically active compounds. Since the major- ity of pharmacologically relevant assays involve receptors that are membrane-bound (e.g., bacterial and viral cell membranes, etc.), solubility is a key factor to consider for drug screening. Future com- binatorial studies can be expected to be directed toward the preparation of large chemical combi- natorial libraries. The currently available libraries, as well as the development of libraries of organic compounds, will play an increasingly important

trends in analytical chemistv, vol. 14, no. 2, 1995

role in the search for novel pharmacophores and for the development of new methods useful in all areas of basic research.

Acknowledgements

This work was funded in part by Houghten Phar- maceuticals, Inc., San Diego, CA, USA and a NATO Postdoctoral Fellowship to E.P.P.

References

[II

(21

13

[4

[71

[91

[lOI

[Ill

1121

[I31

R.B. Merrifield, J. Am. Chem. Sot., 85 (1963) 2149-2154. H.M. Geysen, R.H. Meloen and S.J. Barteling, Proc. Natl. Acad. Sci. USA, 81 ( 1984) 3998- 4002. R.A. Houghten, Proc. Natl. Acad. Sci. USA, 82 (1985) 5131-5135. R. Frank and R. Doring, Tetrahedron, 44 (1988) 6031-6040. J. Eichler, M. Bienert, N.F. Sepetov, P. Stolba, V. Krchn& 0. Smekal, V. Gut and M. Lebl, in R. Epton (Editor), Innovation and Perspective in Solid Phase Synthesis, Solid Phase Conference Coordination, Birmingham, 1990, p. 337. E. Atherton, W. Hiibscher, R.C. Sheppard and V. Woolley, Hoppe-Seylers Z. Physiol. Chem., 362 (1989) 833-839. G. Schnorrenberg and H. Gerhardt, Tetrahedron, 45 (1989) 7759-7764. H. Gausephol, M. Kraft, C. Boulin and R.W. Frank, in J.E. Rivier and G.R. Marshall (Editors), Proceedings of the 1 lth American Peptide Symposium, Escom, Leiden, 1990, p. 1003. S.P.A. Fodor, J.L. Read, M.C. Pit-rung, L. Stryer, A.T. Lu and D. Solas, Science, 25 1 ( 1991) 767- 773. Y.C.Cho,E.J.Moran,S.R.Cherry, J.C. Stephans, S.P.A. Fodor, C.L. Adams, A. Sundaram, J.W. Jacobs and P.G. Schultz, Science, 261 (1993) 1303-l 305. B.A. Bunin, M.J. Plunkett and J.A. Ellman, Proc. Natl. Acad. Sci. USA, 91 ( 1994) 470847 12. S.H. Dewitt, J.S. Kiely, C.J. Stankovic, M.C. Schroeder, D.M.R. Cody and M.R. Pavia, Proc. Natl. Acad. Sci. USA, 90 (1993) 6909-6913. R.A. Houghten, Trends Genet., 9 (1993) 235- 239.

[ 141 R.A. Houghten, Current Biology, 4 ( 1994) 564- 567.

[ 151 R.A. Houghten, C. Pinilla, S.E. Blondelle, J.R. Appel, C.T. Dooley and J.H. Cuervo, Nature, 354 ( 1991) 84-86.

Page 9: Soluble combinatorial libraries of organic, peptidomimetic and peptide diversities

trends in analytical chemistry, vol. 14, no. 2, 1995 91

[ 161 K.S. Lam, SE. Salmon, E.M. Hersh, V.J. Hruby, W.M. Kazmierski and R.J. Knapp, Nature, 354 ( 1991) 82-84.

[ 171 A. Furka, F. Sebestyen, M. Asgedom and G. Dibo, Int. J. Pept. Protein Res., 37 (1991) 487-493.

[ 1 S] C. Pinilla, J.R. Appel, P. Blanc and R.A. Houghten, Biotechniques, 13 ( 1992) 901-905.

[ 191 C.T. Dooley and R.A. Houghten, Life Sci., 52 (1993) 1509-1517.

[20] J.M. Ostresh, J.H. Winkle, V.T. Hamashin and R.A. Houghten, Biopolymers, 34 (1994) 1681- 1689.

[ 211 C. Pinilla, J.R. Appel, S.E. Blondelle, C.T. Dooley, J. Eichler, J.M. Ostresh and R.A. Houghten, Drug Dev. Res., 33 ( 1994) 133-145.

[ 221 J.K. Scott and L. Craig, Curr. Opin. Biotechnol., 5 ( 1994) 40-48.

[ 231 H.M. Geysen, S.J. Rodda and T.J. Mason, Mol. Immunol., 23 ( 1986) 709-715.

[24] H.M. Geysen and T.J. Mason, Biomed. Chem.

[251

1261

v71

[281

u91

[ 301

[311

[321

[331

[341

[351

[361

Lett., 3 ( 1993) 397-404. M.C. Needels, D.G. Jones, E.H. Tate, G.L. Heinkel, L.M. Kochersperger, W.J. Dower, R.W. Barrett and M.A. Gallop, Proc. Natl. Acad. Sci. USA, 90 (1993) 10700-10704. J.M. Kerr, S.C. Banville and R.N. Zuckermann, J. Am. Chem. Sot., 115 ( 1993) 2529-2531. V. Nikolaiev, A. Stierandovh, V. Krchn& B. Seligmann, K.S. Lam, S.E. Salmon and M. Lebl, Peptide Res., 6 (1993) 161-170. M.H.J. Ohlmeyer, R.N. Swanson, L.W. Dillard, J.C. Reader, G. Asouline, R. Kobayashi, M. Wigler and W.C. Still, Proc. Natl. Acad. Sci. USA, 90(1993) 10922-10926. J. Eichler and R.A. Houghten, Biochemistry, 32 (1993) 11035-l 1041. R.A. Houghten, M.K. Bray, S.T. De Graw and C.J. Kirby, Int. J. Pept. Protein Res., 27 ( 1986) 673-678. R.A. Houghten, J.R. Appel, S.E. Blondelle, J.H. Cuervo, C.T. Dooley and C. Pinilla, Biotechniques, 13 ( 1992) 4 12-42 1. J.R. Appel, C. Pinilla and R.A. Houghten, Immunomethods, 1 ( 1992) 17-23. C. Pinilla, J.R. Appel and R.A. Houghten, Gene, 128 (1993) 71-76. C.T. Dooley, N.N. Chung, P.W. Schiller and R.A. Houghten, Proc. Natl. Acad. Sci. USA, 90 ( 1993) 10811-10815. S.E. Blondelle and R.A. Houghten, in John W. Crabb (Editor), Techniques in Protein Chemistry V, Academic Press, Orlando, CA, 1994, p. 509. R.A. Houghten, K.T. Dhin, D.E. Burcin and S.E. Blondelle, in R.H. Angeletti (Editor), Techniques in Protein Chemistry IV, Academic Press, Orlando, CA, 1993, p. 249.

[371

[381

[391

[401

[41 I

~421

[431

[441 [451

[461

I471

[481

SE. Blondelle, E. Takahashi, P.A. Weber and R.A. Houghten, Antimicrob. Agents Chemother., 38 ( 1994) 2280-2286. C.T. Dooley and R.A. Houghten, in R.S. Hodges and J.A. Smith (Editors), Peptides: Chemistry, Structure and Biology. Proceedings of the 13th American Peptide Symposium, Escom, Leiden, 1994, p. 984. R.A. Houghten, in B. Gutte (Editor), Peptides: Synthesis and Applications, Academic Press, San Diego, CA, 1994, in press. R.N. Zuckermann, Curr. Opin. Struct. Biol., 3 ( 1993) 580-584. R.J. Simon, R.S. Kania, R.N. Zuckermann, V.D. Huebner, D.A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C.K. Marlowe, D.C. Spellmeyer, R. Tan, A.D. Frankel, D.V. Santi, F.E. Cohen and P.A. Bartlett, Proc. Nut/. Acad. Sci. USA, 89 ( 1992) 9367-937 1. J.M. Ostresh, G.M. Husar, S.E. Blondelle, B. Dijrner, P.A. Weber and R.A. Houghten, Proc. N&l. Acad. Sci. USA, 91 ( 1994) 11138-l 1142. B.A. Bunin and J.A. Ellman, J. Am. Chem. Sot., 114(1992) 10997-10998. J.D. Rozzell, Jr., Enzyme, 136 ( 1987) 479-503. S.E. Blondelle and R.A. Houghten, Biochemistry, 31 (1992) 12688-12694. K. Johnsson, R.K. Allemann, H. Widmer and S.A. Benner, Nature, 365 ( 1993) 530-532. A. Wallace, S. Altamura, C. Toniatti, A. Vitelli, E. Bianchi, P. Delmastro, G. Ciliberto and A. Pessi, Pept. Res., 7 ( 1994) 27-31. S.E. Salmon, K.S. Lam, M. Lebl, A. Kandola, P.S. Khattri, S. Wade, M. P&tek, P. Kocis, V. Krchn&, D. Thorpe and S. Felder, Proc. Nutl. Acad. Sci. USA,90(1993) 11708-11712.

R.A. Houghten, Ph.D., is the President of Torrey Pines lnsiitute for Molecular Studies (3550 Genera/ A tomics Ct., San Diego, CA, USA) and the Founder and Chief Scientific Officer of Houghton Parmaceuticals Inc. (San Diego, CA, USA). His research interests include all aspects of peptide synthesis, especially those involving solid-phase methodology, and the development and use of synthetic combinatorial libraries. Sylvie E. Blonde//e, Ph.D., is an Assistant Member at Torrey Pines Institute for Molecular Studies. Her research is focused on the induced conformations of biologically active peptides, which is current/y funded by the National Institutes of Health, and the development of novel antimicrobial compounds derived from synthetic combinatorial libraries. Enrique P&fez-Pay& Ph.D., is a doctoral research fellow at Torrey Pines Institute for Molecular

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92 trends in analytical chemistry. vol. 14, no. 2, 7995

Studies and the recipient of a NATO Postdoctoral Fellowship. Colette T. Dooley, M.Sc., is a Senior Scientist at Torrey Pines Institute for Molecular Studies. Her

Clemencia Pinilla. Ph.D., is an Assistant Member at Torrey Pines Institute for Molecular Studies. She recently received funding from the U.S. Army Medical Research and Development Command to

current research is focused on the development of potent opioid compounds using synthetic combinatorial libraries.

develop improved methods for breast cancer detection using peptide libraries and monoclonal antibodies.

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