Solid-Phase Synthesis and Combinatorial Technologies

type 3

type 33

+

type 3 + 3

type 8

type 88

= gIII

+

= foreign insert

= displayed foreign peptides

Figure 10.6 Common display formats of filamentous phage libraries.

relevant target (vide infra). Selected phages are isolated, and further amplification/selection cycles (typically lasting one day) are performed until the required structural information and/or potency toward the target are obtained. The structure of displayed peptides is obtained from sequencing the DNA of the selected clones.

Amplification of a phage library by the biological machinery of the host is the major advantage of phage display in that large numbers of copies of peptides are obtained starting from cheap precursors (filamentous phage, E. coli cells, and oligonucleotide strands), and library copies and/or selected individuals can be replicated indefinitely. Among the few caveats to this technique is the fact that the abundance of each library member will necessarily be unbalanced because some of the 20 amino acids are coded only by one of the 64 DNA coding triplets, while others are coded by multiple codons and thus will be more represented in the peptide chains. A large redundancy must be used to represent all the library individuals in at least one copy. Display of large

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peptides in a type VIII phage may stress the E. coli machinery, forcing it to prepare too many copies of oligonucleotide and peptide oligomers, slowing down the life-cycle of the cell and again privileging the amino acids coded by multiple triplets in the library population.

When a library is prepared and displayed, it is screened and deconvoluted to find active structures from the library population. The phage capsid is stable enough to be purified from the biological medium while retaining its viability and infectivity to start a new selection/amplification cycle. The bonds between the inserted foreign peptides and the phage coat proteins, though, cannot usually be broken to release the peptides in solution. The fusion of foreign peptides onto pIII or pVIII coat proteins allows a large degree of conformational freedom for the displayed chains, making them solutionlike for the binding to any type of receptor. The screening therefore takes place “on-phage” and allows the recovery of viable phages to start a new amplification/selection cycle. It is flexible and sensitive, can spot even weakly binding activities for the selected receptor, and allows the fast, automated separation and isolation of target-bound phages from unbound phages. All of these properties are embedded in the concept of target-assisted screening (see Sections 7.2.2–7.2.4), providing that the selected target is bound onto a solid support to facilitate the separation of active phages from inactive library individuals. Suitable solid supports for large receptors include plastic surfaces, tubes, Petri dishes, and microtiter wells to which the target is adsorbed or bound nonspecifically to ensure a good percentage of adsorbed targets with accessible binding sites for the display library. If the target is a smaller entity, specific covalent linkages or interactions with supports (resin beads) or with supported molecules (target-specific antibodies) may be used to anchor it to the support. The phage library is incubated with the supported target and the desired, noncovalent interaction with phages displaying active peptide sequences takes place (step a, Fig. 10.7). The incubation medium containing unbound phages is separated and the solid support is washed thoroughly (step b); then the absorbed ligand is eluted from the support, thus recovering the first selected phage population (step c, Fig. 10.7). Either this population can be used for further rounds of amplification and selection if the observed activity is low or negligible or it can be structurally deconvoluted by DNA sequencing to determine the active polypeptide(s). Several rounds of selection and amplification are usually necessary to select peptides with the required levels of activity.

Two strictly related criteria are important for a selection protocol and must be carefully adjusted to obtain positive results from phage display. The first is the stringency of measurement, that is, the cutoff level at which a peptide sequence is recognized as active and the experimental conditions under which the activity is measured; the other is the yield, that is, the percentage of phage particles surviving the selection process. In the first selection round for a phage library, it is reasonable to look for sequences with weak activity so that a high loading (e.g., as found in a type VIII clone) and a loose stringency may produce starting points that can be optimized in further rounds of amplification and selection. These iterative cycles will progressively raise the cutoff level to allow the eventual isolation of populations containing a large number of phages bearing several highly active peptide

c

b

a:incubation of the phage library with the immobilized target; b: elimination of unbound phages;

c:elution and recovery of bound phages.

Figure 10.7 Selection of phages carrying binding sequences via target-assisted screening.

sequences. Further increase in affinity may come from using lower loading clones such as a type 33 or 3+3. Structural deconvolution by DNA sequencing may lead to the design of focused libraries where the DNA inserts are biased toward the positives obtained with the aim of optimizing the active structures from the primary phage library.

The next two sections describe several examples, providing both an overview of the main applications of phage display libraries and a brief bibliography. More detailed descriptions of the various aspects of a phage display can be found in several recent reviews (3, 4, 43–52).

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10.1.3 Phage Display Libraries: Looking for Small Peptide Recognition Motifs

The synthesis of peptide libraries to identify more or less refined ligand recognition motifs for the binding to a receptor is one of the main applications for phage display libraries. A recent example by Kraft et al. (53) reported the screening of a 7-mer (L1) and a 12-mer (L2) random commercial display library for binding to two different integrins (Fig. 10.8). The libraries were incubated on receptor-coated plates for 1 h, and the bound phages were eluted at acidic pH and amplified after removal of phages in solution. After three rounds of amplification and selection, the active phages were sequenced and a series of heptaand dodecapeptide structures were obtained (Fig. 10.9).

The known binding motif RGD was observed in many of the 12-meric bound sequences to αvβ6 integrin (51%, italic, Fig. 10.9), but a significant amount of peptides (27%, bold, Fig. 10.9) contained the unexpected X1X2DLX3X4LX5 motif. The X1–X5 positions showed marked preferences for specific amino acids (Fig. 10.9). The heptameric library L1 contained only 5% of the truncated DLXXL motifs, showing the importance of the full eight-residue recognition module found from L2 in defining specificity for the αvβ6 integrin. A specific clone (10.1, Fig. 10.10) was used to determine the biological properties of this class of binders. This clone 10.1 inhibited the RGD-dependent binding to αvβ6 integrin in vitro and in whole cells, and the importance of the eight residues was confirmed in a deletion study (Fig. 10.10). Many reports of binding motifs isolated from phage display libraries have appeared recently in the literature, and a few are referenced here for the interested reader (54–68).

Phage libraries have also been used to study the substrate specificity of enzymes by finding an improved artificial substrate. Coombs et al. (69) reported the detailed assessment of specificity for a serine protease belonging to the α-chymotrypsin family, the prostate specific antigen (PSA). They used both substrate optimization by singlepoint mutations and phage display libraries. The sequence of the 14-member substrate 10.2 (70) was used to start the iterative optimization process (Fig. 10.11) in which substitution or exchange of the P1, P2, or P2′ residues increased the substrate affinity

a:incubation with integrin-coated Petri dishes, 30ºC, 1 hr; b: washing of the unbound clones;

c:elution of the bound phages with pH 2.2 buffer solution;

d:amplification of the selected population in E. coli; e: steps a-d, three additional cycles;

f:DNA sequencing, identification of selected peptides.

Figure 10.8 Screening of the phage libraries L1 and L2 for integrin binding: the selection/amplification iterative process.

10.3

Figure 10.11 Single-point mutations to improve the specificity of the prostate specific antigen (PSA) substrate 10.2.

of the three peptides with the receptors in the presence of the reducing agent dithiothreitol (DTT), known to inhibit disulfide bond formation in peptides. The binding affinity of compounds 10.5–10.7 was significantly decreased proportionally to the increase of DTT. Other examples of constrained, cyclic peptide libraries displayed on phage surfaces have been reported recently (75–79).

Small peptide phage libraries have been used to screen for binding to organic molecules to identify consensus motifs that are then compared to known protein sequences to eventually determine biologically relevant interactions. Rodi et al. (80) screened a commercially available 12-mer library L6 fused to phage pIII coat protein for its binding with a biotinylated derivative of taxol supported onto streptavidin-coated petri dishes (Fig. 10.15). The isolated sequences after one, two, and three rounds of selection and amplification were compared for common structural motifs. Two 5-mer motifs were identified, and a search in protein databases found six binding candidates for taxol (Fig. 10.15). More sophisticated

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L1 = 4 AAs-flexible spacer, GGAG

L2 = 6 AAs-linker, GGAGSS

= 8 AAs-random sequence

=known epitope for 3-E7 mAb

Figure 10.12 Screening of the phage library L3 to improve the specificity of PSA substrates: the selection/amplification process.

similarity analyses of the selected peptides and of the whole sequence of the six proteins confirmed only the anti-apoptotic protein Bcl-2 as a binding partner for taxol. Further investigations elucidated the location of the peptide binding region in Bcl-2, the structural changes deriving from the binding with taxol and the nanomolar binding affinity of library individuals. The absence of homology between the selected peptides and tubulin, the primary binding target for taxol, was attributed to constraints imposed by the phage structure on the dodecapeptides, which could not assume any tubulinlike conformation for the binding.

SUBSTRATE MOTIF

P5: R,L>others; P4: S>A>others; P3: S>A,R,T>others; P2: Y>others;

P1: Y>L>others; P1': S,T,A>Q>others; P2': S>A,R>others; P3': A,S>others.

P5: hydrophobic>others; P4: S>T,A>others; P3: S>others; P2: Y>V,L>others;

P1: Y>others; P1': S>others; P2': G>A>others; P3': A>others.

10.4

a: kinetic measurement and selection of best substrates; b: determination of the 8-mer consensus motif and synthesis of the resulting consensus 14-mer peptide.

Figure 10.13 Screening of the phage library L3 to improve the specificity of PSA substrates: characterization of selected clones and structure of the most active substrate10.4.

10.1.4 Phage Display Libraries: Large Peptide Sequences as Receptors, Antibodies, or Enzymes

The number of hexapeptides obtained by complete randomization of the 20 natural α-amino acids is 6.4 × 107, whereas the population of a large phage display library is made up of 108–109 clones and cannot exceed this number to allow the handling of the library during screening, selection, and amplification. For this reason the complete randomization of long peptide chains is not realizable, and alternative methods for the display and optimization of large peptides, antibodies, or protein structures on phages have been developed.

In analogy with constrained peptide libraries, several reports have described the use of small proteins, protein domains, or antibodies as scaffolds for the display of random polypeptide sequences to obtain novel binding proteins or antibodies. Koide et al. (81) used the tenth FN3 sequence, a 94-amino-acid fibronectin domain (82, 83) known to be involved in molecular recognition, as a scaffold to build a phagemid 3+3 library L7 (Fig. 10.16) where less than a copy of modified FN3 was present on each phage capsid. The ≈108-member library was screened using plates coated with ubiquitin, a small protein for which native FN3 does not have any affinity. The library was made by randomizing five amino acids in positions 26–30 (BC) and five amino acids in

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a:incubation with PDZ-coated plates, rt, 2 hrs; b: washing of the unbound clones;

c:elution of the bound phages with pH 2.0 buffer solution;

d:amplification of the selected population in E. coli;

e:steps a-d, two additional cycles; f: DNA sequencing, identification of peptide structures.

Figure 10.14 Screening of the cyclic phage libraries L4 and L5 for PDZ domains (PDZ) syntrophin binding: the selection/amplification process and the structures of the best binders

10.5–10.7.

positions 77–81 and deleting the 82–84 Pro–Ala–Ser sequence of FN3 (FG, Fig. 10.16) sequences, which corresponded to two nonconserved loops of FN3 and the absence of which was not likely to reduce the protein stability. After five selection/amplification cycles 11 clones were selected (steps a–f, Fig. 10.15) among which the sequence 10.8 (Ubi4) was dominant. This novel motif (only the residue in position 30 of wild-type FN3 was conserved; Fig. 10.16) was confirmed as interacting with ubiquitin with an IC50 of 5 M. An alanine scan, in which each randomized amino acid was replaced in turn with L-alanine, showed a general decrease of affinity between ubiquitin and the mutated protein domains. Other examples of scaffolded phage libraries from antibodies (84–90) or proteins (91–96) with small randomization sequences have been reported recently. This subject has also been reviewed extensively (97, 98).

Phage display libraries of more heavily randomized antibodies aimed at improving the binding affinity for an antigen using a library DNA encoding method schematized in Fig. 10.16 have been reported (99). Recombinant phage libraries of antibodies have been assembled from two distinct vector libraries, one containing a repertoire of VH (heavy-chain gene fragments) and another containing a repertoire of VL (light-chain