Solid-Phase Synthesis and Combinatorial Technologies

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details). This basic set was screened on several targets in a qualitative mode, that is without trying to quantify the trNOE phase switch of the ligand cross peaks but considering it a signal of ligand-target interactions; a reliable “yes-or-no” answer was unequivocably obtained for each SHAPES library individual. Several weak binding frameworks (microto low millimolar constants), which would have been missed by conventional biological screening protocols, were used to focus later larger screening efforts on biased collections or libraries containing these or similar frameworks; the frequency positives from SHAPES-derived screening sets was significantly increased if compared with random screening (90), proving the validity of the approach. The method does not require labeled receptors and is ideally suited for large macromolecules (>60 kDa); the amount of macromolecular target needed for a SHAPES screen is relatively limited (tens of mgs of large proteins); a complete NMR assignation for the macromolecular target is not required; the library size allows to use small mixtures of ligands (one to four ligands per sample), thus increasing reliability and simplicity; the SHAPES NMR screen takes only from several hours to a few days; a visual inspection of the spectra is enough to discriminate among binders and non-interacting compounds. Even though the information acquired is not quantitative (but can be quantified by progressing the positives through PFG-NMR experiments, as was done in ref. 90) and the binding specificity must be determined, this method will surely become a major asset for the early, information-poor phases of ligand fishing related to difficult targets for which no ligands have been found using traditional HTS campaigns. The method could in future be tailored also for non-pharmaceutical applications.

NOE pumping (94) was recently reported by Chen and Shapiro. After the application of a diffusion filter suppressing all the ligand signals, the NOE experiment started to “pump” the magnetization from the target to the binders in the ligand mixture. By increasing the mixing time tm of the target and the ligand mixture the magnetization transfer gradually decreased the target signals (horse serum albumin in ref. 94), and gradually increased the ligand signals (salicylic acid) without interferences from nonbinders (glucose and ascorbic acid). Similar characteristics in respect with trNOE (no target labelling, no MW cut-offs for targets, no need of a complete NMR assignment for the target) make also this technique particularly appealing for NMRassisted screening of mixtures.

Saturation transfer difference NMR (STD-NMR) was recently reported by Mayer and Meyer (95) as a fast and reliable screening method and was used to spot the binding of saccharides to wheat germ agglutinin (WGA); the same assay was repeated successfully anchoring WGA on SP and recording spectra from heterogeneous systems using magic angle spinning STD-NMR (96).

Several reviews dealing with analytical techniques and off-bead structure determination methods are available to the reader. The application of MS techniques to target-assisted, off-bead structure determination of library actives has been recently reviewed (97, 98), as happened also for NMR screening methods for positive identification (99–102). Two more general, screening-focused reviews dealing with targetassisted methods using immobilized (supported) or confined targets (segregated in a

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compartment) targets for identification of positives from mixtures have recently appeared (103, 104).

The majority of the above-mentioned examples dealt with small, assembled mixtures of commercial compounds, containing known ligands and inactive compounds, which were used to validate the ability of the target-assisted screening to detect known interactions. Other examples have tested small peptide or peptidomimetic libraries, and in general, all the reported studies have proved the applicability of off-bead target-assisted screening of model pool libraries. It is easy to predict, though, that the coming years will see an expansion of the efforts in this area. Eventually target-assisted screening and identification of positives for specific applications, especially in the pharmaceutical area, will become an important screening option.

7.2.3 On-Bead Direct Structure Determination of Positives

The identification of positives from an SP pool library usually requires cleavage from the beads, a screening campaign, and finally the crucial step of linking the observed activity with the structure of one, or more, of the library components. A powerful approach, generally named bead based, consists of submitting the libraries to assays that allow detection of the activities of single beads, and thus eventually to determine the structure of the individual that was loaded on the specific, active bead. Some formats for bead-based structure determination will be examined in detail in Sections 7.4 and 7.5, while here we will present the so-called on-bead approach.

Screening of a library is usually intended as a solution process, where both the library individuals and the target/receptor are dissolved in the same assay medium. An obvious drawback is that when the library aliquot (usually a few library equivalents, to ensure coverage all the chemical diversity) is cleaved and tested, only a few active compounds (if any) are found, but the whole library aliquot is then lost and a second screening campaign on a different target will require a new resin-bound library aliquot. If, however, the resin-bound library is on-bead screened, two major advantages are gained. First, the complex between the target and the library individual is formed at the solid–solution interface and, by using denaturating conditions, the target can be washed away and the same library aliquot can be tested on other targets. Second, positive beads are easily spotted, for example, by building a colorimetric or fluorescent detection method able to spot the target–ligand complex; after removal from the other library members of the spotted beads and complex denaturation, the structures of the ligand library individuals are directly determined by sensitive analytical methods.

On-bead screening and structure determination of positives from pool libraries was first reported by Lam et al. (2) using an enzyme-linked colorimetric assay. This detection method was subsequently used by the same group (105–110) and others (111–115) to probe the specificity of random peptide or peptidomimetic libraries. Other reports have used fluorescence-labeled (110, 116, 117) or radionucleotidelabeled (118, 119) targets. Aside from the above-mentioned binding assays, functional assays have also been performed on-bead (120–123), and hybrid off-bead/on-bead assays have been reported (124). The use of chromogenic test substrates to screen on-bead for Zr4+-binding peptides as ligands to promote phosphate hydrolysis was

7.2 DIRECT STRUCTURE DETERMINATION OF POSITIVES 285

also reported (125). All these examples were able to detect one or more peptide sequences, which were reconfirmed after their identification as true binders/inhibitors interacting with the desired target. Recently an example of large oligocarbamate libraries (one cyclic trimer library, 19,863 individuals, and two tetramer libraries, 531,441 individuals) screened to search for human thrombin ligands (126) and another of glycopeptide libraries (around 300,000 individuals) screened for lectin binding (127) somewhat enlarged the applicability of on-bead screening to library structures other than peptides.

Several considerations need to be made regarding the usefulness of on-bead screening and structure determination. The interested reader will find these treated in detail in some recent reviews (14, 105, 128). To date, on-bead screening has been used to detect and identify biological interactions using isolated receptors, enzymes, and antibodies as targets and aqueous environments. The use of hydrophilic supports is necessary to create the suitable aqueous environment for the target–ligand interaction while allowing proper swelling of the beads. Relevant studies (115, 119, 129–131) have been performed to determine the accessibility of the resin sites to macromolecular targets; enzymes such as trypsin (23.5 kDa), papain (23 kDa) and chymotrypsin (22 kDa) were used to cleave resin-bound peptide sequences (119, 129, 130) and to bind to resin-bound inhibitors (131).

The results of these studies showed that hydrophilic PS swelling resins, such as Tentagel or Argogel, are usually accessible to the enzymes only on their surface, while access to inner sites is possible (115, 131) but only through slow, complex exchange mechanisms with the surface sites and when the ligand has a potent binding activity. Other polyacrylamide-based PEGA hydrophilic resins (132) are completely accessible to the above-mentioned macromolecules, even at the inner sites and, at the time of writing, represent the resin of choice for on-bead screening and structure determination. Any other hydrophilic resin, though, can be used for colorimetric assays where binding to the surface stains the positive beads.

The technical details of on-bead screening are also extremely important, because false positives (beads that turned up positive but failed to reconfirm activity as resynthesized pure individuals) due to a specific binding of library individuals may be a major problem when testing millions of beads. Many possible causes have been reported for such a specific binding: extremely hydrophilic molecules or highly charged peptides, artificial dimeric or trimeric resin-bound individuals, which were not active as monomers, and even interactions with parts of the bead structure could produce a significant amount of false positives. Some general precautionary procedures [use of high-ionic-strength buffers or addition of proteins such as bovine serum albumin (BSA) to prevent aspecific lipophilic binding] have been suggested, and several protocols have also been developed where a double-staining procedure is performed to minimize aspecific interactions. These involve repeating the assay twice on positive beads after complex denaturation or after incubating them in the presence of a known ligand to show the competition for the receptor (107, 110). A dual-color detection scheme has also been designed and successfully applied (106).

The main issue is support anchoring the library individuals onto the SP. For peptides, adjustments can be made to support the molecules on a side-chain function that does not interfere with the binding and/or a spacer can be inserted between the

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resin and the peptide sequence. Small organic molecules, though, generally contain just a few functional groups, which are often essential for the activity on the target. Their use to support the library components will prevent binding and consequently the detection of positives. The same may also be true if the linking function is adjacent to a relevant portion of the active library compound, thus preventing the approach of the target and the correct docking between the two partners. It would be desirable to perform on-bead screening and structure determination on cleavable SP SOM pool libraries and then to cleave the library and test it in solution. A comparison of the results would show if on-bead screening is successful (similar screening results) or not (no on-bead activity, actives in solution). An even better solution to save precious library equivalents would be, when possible, to support a known active compound, belonging to the chemical class that will constitute the library, in exactly the same manner as planned in the SP scheme. If the active standard results on-bead active the library can be tested on-bead; otherwise an off-bead screening must be performed, followed by a more classical structure determination approach. Unfortunately, when active standards are available, the need of a large primary library is reduced, and a small–medium discrete focused library is usually preferred.

A comparison between different library screening formats was carried out using three identical, 576-member peptide polyamine conjugate libraries (133); one of them was screened in solution, one anchored onto Tentagel beads and the last as a PEGAsupported pool library. The libraries were screened for inhibition of trypanothione reductase used as a soluble target weighing around 55 kDa; in accordance with previous experiences (131), the PEGA-supported resin provided several medium–low nanomolar inhibitors, as did the soluble library, thus showing the good properties of this support for on-bead screening. In this specific example, the constraint imposed by the support and by the linkage to the bead did not prevent the biological interaction between the library individuals and the enzyme. The library supported onto Tentagel, on the contrary, did not show any result from screening, even if theoretically the identification of surface sites on the beads should have allowed to spot the positive beads. After this successful experiment, the same screening protocol applied to small organic molecule libraries supported onto PEGA-like resins is crucial to judge the future potential of on-bead direct screening. In the next section we will describe in detail an excellent example where peptide-derived libraries were screened on-bead and valuable active structures were rapidly determined.

7.2.4 An Example: On-Bead Screening and Structure Determination of Positives from SH3 Domain-Directed Libraries

Schreiber and co-workers recently reported (134, 135) the synthesis of two SP pool libraries L3 and L4 based on the structures of a phage display-derived dodecapeptide ligand of the SH3 domain of the tyrosine kinase Src (7.14, Fig. 7.13) (136), of its truncated version (7.15), and of a nonpeptide ligand derived from an encoded primary library (7.16, Fig. 7.13) (137). The authors kept the common motif proline-leucine- proline (PLP) in the biased library structures and explored the left (library L3, 2500 members, Fig. 7.13) and the right part of the ligand sequences (library L4, 125,000

L3

2.500 individuals, encoded pool library M1: 50 monomers (amino acids)

M2: 50 monomers (isocyanates, isothiocyanates, acyl chlorides, acids, anhydrides, amino acids)

Lin = linker

L4

125.000 individuals, encoded pool library M1-M3: 50 monomers (amino acids)

7.17

Figure 7.13 On-bead structure determination: structures of known SH3 peptidic and nonpeptidic ligands (7.14–7.16) of designed SP peptidomimetic libraries (L3, L4) and of the planned, optimized ligand 7.17.

members, Fig. 7.13). Subsequent combination of the best findings from the two libraries should have produced a new, hybrid generic structure 7.17 (Fig. 7.13).

Both libraries were prepared using automated standard peptide coupling conditions for monomer additions using either commercially available or easily prepared monomers. The biasing library elements were either initially coupled to the resin (iNpePLPPLP, L3) and then elongated or used as a final capping reagent (VSLARRPLP, L4) for the trimeric randomized sequence. Randomizations were performed using

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mix-and-split techniques (2, 3), and each library component was encoded (111) to accelerate the structure determination of library individuals loaded onto positive beads.

Library L3 was screened using a soluble N-biotinylated N-terminal residue of the Src SH3 domain (134), which had been previously incubated with the commercially available alkaline phosphatase conjugate with streptavidin (SAAP). The strong interaction between biotin and streptavidin formed a tight receptor complex, which was subsequently incubated with two batches of around 12,500 beads (five library equivalents), which had been previously incubated with the screening buffer to reduce nonspecific binding. After 12 h of incubation, the receptor solution was filtered off, the beads were thoroughly washed with the screening buffer and then treated with the alkaline phosphatase substrate BCIP (5-bromo, 6-chloro, 3-indolyl phosphate), and the staining agent nitro blue tetrazolium (NBT). The complexed alkaline phosphatase on positive beads dephosphorylated BCIP, and this eventually reduced NBT to the insoluble, deep blue diformazan, which mostly precipitated in proximity to the phosphatase, that is, on the active beads. These colored beads were pipetted out of the assay medium, resuspended in a new assay medium and then washed with a destaining, denaturating 6 N guanidinium hydrochloride solution to recondition them for new assay cycles. Treatment with SAAP alone allowed removal of beads interacting only with the alkaline phosphatase, while the others were submitted to a second, identical SH3 screening to reconfirm their activity.

O

R=

Figure 7.14 On-bead structure determination: resynthesized positives7.18–7.25 from the SP peptidomimetic library L3.

7.2 DIRECT STRUCTURE DETERMINATION OF POSITIVES 289

On-bead screening of L3 produced 32 positive beads, which were rapidly decoded (111). Thirty out of 32 beads contained a specific monomer in position M1, while position M2 showed a higher tolerance for different monomers. The structures of resynthetized positives 7.18–7.25 with their binding constants are reported in Fig. 7.14. The larger library L4 was tested and decoded using the very same protocol, and, while some sequences were discarded because they were unrelated to any other structure, a few related sequences (7.26–7.28, Fig. 7.15) were determined. The

ligands from L4:

Figure 7.15 On-bead structure determination: resynthesized positives7.26–7.28 from the SP peptidomimetic library L4 and hybrid structure of an optimized L3/L4 derived ligand 7.29.

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combination of structural information derived from L3 (7.18) and L4 (7.28) produced the “hybrid” ligand 7.29 (Fig. 7.15), which possessed a comparable binding activity to the original dodecapeptide 7.14 having replaced 9 of the 12 α-amino acids with nonnatural building blocks.

7.3 DECONVOLUTION METHODS FOR SOLID-PHASE POOL LIBRARIES

7.3.1 Iterative Deconvolution

While the structure determination methods seen in the previous section are based on the analytical identification of the positive library individuals, other methods exist to perform the so-called deconvolution of the library complexity and allow the eventual identification of one or more positives from the library without determining their structure by analytical methods. The most common approach is based on iterative synthesis cycles of less complex pools deriving from the original active pool(s) until single compounds are prepared and tested (iterative deconvolution) (3).

A detailed scheme for the iterative deconvolution of a 625-member hypothetical library, where four monomer sets, each composed of five representatives, are added to form the library members in four consecutive steps (Fig. 7.16), is reported in Fig. 7.17. Monomers P–T, K–O, and F–J are added respectively in the first, second, and third randomization steps using the mix-and-split technique; then monomers A–E are added in the fourth step and are kept divided. The library is thus prepared as five pools A–E, each containing 125 individuals, and its screening (step a, Fig. 7.17) produces four inactive pools and one active pool D, which is selected (step b). The four pools A–C and E are now discarded, and a first synthesis iteration (step c) produces an 125-member library where monomer D is always present in the fourth position. The five prepared pools F–J, each containing 25 individuals, have a determined monomer in the third position. Their screening (step a) produces two active pools, G (less active) and J (more active), and three inactive pools, from which the most active pool J is selected (step b). The second iterative synthesis (step d) produces a 25-member, DJ-biased library with five pools K–O. Among them pools L (more active) and N (less active) are active (step a) and the former is selected (step b). Finally the third synthesis iteration (step e) prepares 5 discretes DJLP–DJLT, and three actives DJLP, DJLS (most active), and DJLT are found (step b, Fig. 7.17). An attractive modification, named recursive deconvolution (138), archives an aliquot of each intermediate pool during the mix-and- split synthesis. This allows rapid iterative deconvolution cycles using archived samples rather than repreparing deconvolution libraries from the beginning. This modification has been successfully applied to a small carbohydrate library (139).

Iterative deconvolution selects the pools solely according to the screening results, and no analytical characterizations are performed at any deconvolution stage. This is particularly useful when sophisticated analytical instrumentation is not available. The measured activity is always the sum of each individual contribution. Assuming that the SP assessment studies, the monomer rehearsal, and the model library synthesis