Protein Structure-Based Combinatorial Chemistry: Discovery of Non-Peptide Binding Elements to Src SH3 Domain Andrew P. Combs, ² Tarun M. Kapoor, ² Sibo Feng, James K. Chen, Lygia F. Daude ´-Snow, and Stuart L. Schreiber* Howard Hughes Medical Institute Department of Chemistry, HarVard UniVersity 12 Oxford Street, Cambridge, Massachusetts 02138 ReceiVed NoVember 7, 1995 Small molecule ligands can be used to cause a conditional loss or gain of function of their protein receptors and, therefore, can be viewed as equivalents of conditional alleles. 1-3 In order to extend their use, methods to identify such ligands de novo are required. We have previously reported the use of biased combinatorial libraries to discover peptide ligands to proteins, 4 and the coupled use of combinatorial chemistry and structural biology to understand the nature of protein-ligand interactions. 5-7 More recently, we have been exploring whether the knowledge of protein structure can facilitate the design of monomers and linking elements leading to vast numbers of potential ligands targeted to a particular protein. We now report a first illustration of this strategy resulting in the discovery of novel and specific ligands containing non-peptide structural elements. Structural investigations of SH3-peptide complexes have revealed that SH3 domains bind peptide ligands in either of two orientations (classes I and II; these differ in the directionality of the backbone amides 5,6 ) involving the three pockets depicted in Figure 1. 4-7 We designed a library of ligands predisposed to adopt the class I orientation by attaching a common low- affinity (K d > 1 mM) biasing sequence PLPPLP (P ) Pro, L ) Leu) to a solid support. This sequence was expected to fill the two pockets (labeled 1 and 2) that bind Leu-Pro dipeptides. Furthermore, structural analyses show that the N-terminal proline should be positioned to orient elements attached to its pyrrolidine nitrogen into the third pocket (labeled 3), which is lined by the nSrc and RT loops common to all SH3 domains and is the primary determinant of ligand specificity. 8 We synthesized an encoded 9 combinatorial library derived from 32 monomers incorporated during three consecutive cycles of split-and-pool synthesis 10,11 following the synthesis of the common PLPPLP sequence (synthesized in the C to N direction) and terminating with one of 32 capping reagents (Figure 2). We purposefully incorporated an encoded blank (“skip-codon”) 12 during mono- mer and cap incorporation in order to increase library diversity significantly by creating sublibraries with deletions at any one or more of the three monomer and one cap sites. A sensitive binding assay for the SH3 domain from the protein tyrosine kinase Src was developed using N-terminally biotinyl- ated Src SH3 (cf. ref 4) and streptavidin-alkaline phosphatase as a colorimetric reporter. 13 A three-stage screen allowed for the removal of false positives and ensured the selection of the darkest beads from the library. 14 Screening a biased library containing ∼1.1 million discrete compounds identified 15 beads containing ligands specific for the Src SH3 domain. Decoding revealed two consensus sequences (Table 1). Position 3 (M3) was decoded as monomer 1 (see Figure 2A) in 12 of 15 beads. Position 2 (M2) was occupied by either monomer 29 or monomer 18 in 13 of 15 beads. The monomers at the M1 site * Address correspondence to this author. ² These authors contributed equally to this research. (1) Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.; Schreiber, S. L. Nature 1994, 369, 756-768. (2) Spencer, D. M.; Wandless, T. J.; Schreiber, S. L.; Crabtree, G. R. Science 1993, 262, 1019-1024. (3) Pruschy, M. N.; Spencer, D. M.; Kapoor, T. M.; Miyake, H.; Crabtree, G. R.; Schreiber, S. L. Chem. Biol. 1994, 1, 163-172. (4) Chen, J. K.; Lane, W. S.; Brauer, A. W.; Tanaka, A.; Schreiber, S. L. J. Am. Chem. Soc. 1993, 115, 12591-12592. (5) Yu, H.; Chen, J. K.; Feng, S.; Dalgarno, D. C.; Brauer, A. W.; Schreiber, S. L. Cell 1994, 76, 933-945. (6) Feng, S.; Chen, J. K.; Yu, H.; Simon, J. A.; Schreiber, S. L. Science 1994, 266, 1241-1247. (7) Chen, J. K.; Schreiber, S. L. Angew. Chem., Int. Ed. Engl. 1995, 34, 953-969. (8) Feng, S.; Kasahara, C.; Rickles, R. J.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12408-12415. (9) Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.; Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10922-10926. (10) Furka, A Ä .; Sebestye ´n, F.; Asgedom, M.; Dibo ´, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (11) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (12) This procedure generates a complete representation of the subli- braries: C-M-M-M-PLPPLP, C-M-M-PLPPLP, C-M-PLPPLP, C-PLPPLP. (13) The library was initially incubated with the preformed complex of N-terminally biotinylated (via semicarbazide linkage) Src SH3 domain and streptavidin-alkaline phosphatase (SAAP) and then washed extensively. Treatment of the library with substrate for alkaline phosphatase gave varying degrees of color deposition on the beads. The darkest beads (∼350) were physically removed with a syringe, destained, and then reassayed with SAAP alone in order to eliminate false positives. None of the beads were identified as SAAP binders. A final high-stringency assay of all ∼350 beads was performed with lower concentrations of Src SH3-SAAP in order to identify the highest affinity ligands. Figure 1. Schematic of the strategy used for the rational design of a library of ligands that direct non-peptide binding elements into the specificity pocket of SH3 proteins. W, D, and Y are one-letter amino acid codes for tryptophan, aspartic acid, and tyrosine, respectively. Figure 2. (A) Sample structures of 33 monomers (M1, M2, and M3) used in library synthesis (standard FMOC chemistry). (B) Sample structures of 33 caps (C) used in library synthesis as sequence- terminating reagents. A complete listing of monomers and capping agents can be obtained in the supporting information. 287 J. Am. Chem. Soc. 1996, 118, 287-288 0002-7863/96/1518-0287$12.00/0 © 1996 American Chemical Society