High-throughput screening of small molecules compound collections based on a functional assay can now be performed in many academic and industrial settings. However, these screens are quite expensive. Moreover, most compound collections have not been assembled with ease of hit optimization in mind, necessitating tedious medicinal chemistry campaigns to achieve high affinity. So as a general source of large numbers of high affinity protein ligands, it is not clear that function-based high-throughput screening of small molecule libraries is a good place to start. An alternative to functional screening to mine one bead one compound (OBOC) libraries for protein ligands using a simple binding assay, [3
]. In most such cases, the library will have been made by split and pool synthesis and thus the identity of the compound on any particular bead will not be known a priori. Thus, the nature of the immobilized molecule must allow their direct structural characterization, usually by mass spectrometry, or they must be encoded [7
]. Encoding, especially using DNA-based methods [9
], opens the way for the creation of very large libraries of drug-like molecules in an OBOC format.
OBOC libraries are generally screened by incubating a labeled protein with the library and identifying candidate protein ligands by following the label. This is done most conveniently when the library is synthesized on a hydrophilic resin such as TentaGel, which does not bind proteins non-specifically [11
]. Alternatively, compounds can be cleaved from polystyrene resins and displayed in other formats, such as a small molecule microarray [13
]. OBOC libraries that are screened when attached to a solid surface have the advantage that one knows where they can be modified without concern for ablating the ability of the molecule to bind the target protein: the point of attachment of the molecule to the surface.
The hits that arise from such screening efforts are almost always micromolar or, at best, high nanomolar ligands [15
]. At least a 1000-fold improvement in the affinity of these hits is desirable in order to mimic the binding of a good antibody. Many different strategies have been employed to achieve this jump in affinity, but it is fair to say that no general strategy has yet been demonstrated to be general and capable of high-throughput. This is a critical issue in the field. The following discussion is not intended to be comprehensive, but rather highlight generally interesting methodologies that have the potential to contribute to the eventual design of a convenient and high-throughput system for protein ligand development.
One approach is to join together two modest affinity, non-competitive ligands with an appropriate linker chain to create a bivalent compound, which can exhibit much better affinity than the individual ligands. The simplest strategy is to identify ligand pairs that bind the protein of interest non-competitively, then link them together via a suitable linker arm. A version of this approach, called “SAR by NMR” [16
], in which an NMR solution structure of the ligand protein complex was employed to guide linker design, was one of the first successful approaches of this type, but since then, many other strategies for the creation of these bivalent ligands have been reported. [17
A special case of this type of approach suitable for the creation of high affinity protein capture agents is to simply co-immobilize two non-competitive ligands for the same protein on a densely functionalized surface () [18
]. The idea is that some fraction of the two different immobilized molecules will have the appropriate spatial relationship to engage the target protein in a bivalent fashion, resulting in high affinity. In this case, the surface itself serves as a kind of “library of linkers”, allowing some fraction of the immobilized ligands to act as high affinity bivalent ligands.
Figure 1 Interactions between a bivalent ligand and a target protein(s) supported by a surface. A. Co-immobilization of two non-competitive ligands for a target protein (light blue oval) can provide a high affinity capture agent by virtue of the fact that some (more ...)
A philosophically similar idea, but in reverse, has been reported by Hruby and co-workers as a strategy to create high affinity and selectivity cell surface binding reagents that recognize two different receptors () [20
]. The idea behind this strategy is that the cell membrane will act as a two-dimensional surface to template binding of covalently linked ligands to two different receptors even if the receptors are not physically associated. [22
Another approach to bivalent ligands that eliminates the need for linker optimization is to employ a target protein as a template for an uncatalyzed Huisgen cycloaddition between members of a library of alkynes and a library of azides. The cycloaddition of alkynes and azides is highly favorable thermodynamically, but extremely slow kinetically. Thus, when an azide and an alkyne are mixed together, little or no reaction will occur in a reasonable period of time in the absence of a catalyst unless two molecules are brought into close proximity by binding to nearby surfaces of the protein. However, if independent binding of an azide and an alkyne to nearby surfaces of a protein occurs, coupling can take place, creating a high affinity, bivalent ligand in situ. This strategy was used to great effect to find a pM ligand for carbonic anhydrase (CA) [23
The problem with this clever approach is that the identity of the resultant bivalent compound had to be determined directly from the screening mixture. Only stoichiometric amounts of ligand (with respect to the protein), at best, could be created in one experiment owing to the slow off rate of the bivalent ligand. This meant that very large amounts of protein were required for the screen, making this technique impractical for most protein targets. However, a re-engineering of this system by Heath, Sharpless and co-workers has been reported that alleviates this problem and provides a powerful and much more practical system for multivalent ligand discovery () [24••
]. In this case, an OBOC peptide library was first screened using a standard approach to identify a ligand for CA. This peptide, which exhibited a weak affinity (KD
≈ 500 μM) for CA, was then synthesized with an N-terminal azide appendage. A new OBOC library was then created that terminated with an alkyne-containing residue. The library, the soluble original hit and the labeled target protein, but now at a lower concentration were then mixed and new hits were identified. As before, higher affinity hits were obtained through a protein-templated Huisgen cycloaddition, leading to retention of the labeled protein. In this case however, only a small fraction of the peptide molecules on the resin entered into the reaction. The remainder could easily be sequenced via Edman degradation, allowing the identity of the so-called bi-ligand to be deduced. One of these bi-ligands was found to be improved about 160-fold (KD
≈ 3 μM) over the original hit. Finally, the process was repeated again, this time using the azide-modified biligand as the soluble partner and a tri-ligand was identified with a KD
of about 40 nM. This begins to approach the antibody range, yet the mass of the molecule is more than a 100-fold lower than that of an antibody. The important point is that only modest amounts of fluorescently labeled protein are required for this procedure, removing the major drawback of the original assay. Although this study employed peptides, presumably any class of molecules whose sequence could be determined from a single bead or by encoding could be employed in this kind of protocol.
Figure 2 An iterative, protein-templated Huisgen cycloaddition strategy for protein ligand discovery. Step 1: An OBOC peptide library is screened for a ligand to fluorescently labeled Carbonic Anhydrase (CA). Step 2: The OBOC alkyne-terminated peptide library (more ...)
A related approach, but which did not employ a protein-templated reaction, was reported independently and at about the same time by our laboratory [25
]. In this case, a previously identified, low affinity (KD
≈ 300 μM) peptoid ligand for the KIX domain [26
] of the transcriptional coactivator CBP was modified with an azide functionality, then incubated with a microarray-displayed peptoid library terminating in an alkyne functionality. A copper catalyst was added to “click” the soluble lead molecule onto each of the immobilized library compounds. The array was then incubated with labeled target protein. This procedure resulted in the identification of a 300-fold improved bivalent ligand.