The advantages of combinatorial chemistry for the rapid generation of chemical compounds for pharmaceutical screening are well established. The single greatest strength of combinatorial chemistry is that it makes possible the generation of libraries of huge numbers of compounds in a relatively short period of time.
Several methods have been employed for the preparation of such libraries, e.g., solution-phase synthesis; solid-phase synthesis on polymer beads or other divided supports; synthesis on soluble, precipitatable polymer supports; and synthesis on planar supports. All of these methods are capable of generating large libraries (i.e., libraries containing a large number of compounds), but none of them are amenable to rapid screening of the libraries for binding activity, biological activity, or other desirable properties.
Current methods for screening libraries include those in which individual library members, or small groups of members, are assayed in microtiter plates, e.g., by screening for a desired activity or for binding to a specific binding partner, such as a receptor or antibody or other ligand, but the number of compounds that can be assayed at once is on the order of 102 to 104: one compound per well in a 96-well microtiter plate screens at most 96 compounds, while twenty compounds per well in a 384-well plate screens about 7680 compounds. The latter approach, while permitting higher throughput, requires secondary screening to identify the active species in any given well. For example, screening a library containing all of the 3.2 million possible pentapeptides which could be made from the twenty natural amino acids (i.e., a 3.2-million-member library) by these methods would require 500 to 3400 plates. Screening a library of the 64 million possible hexapeptides would require 10,000 to 68,000 plates. Robotic systems are available for microtiter plate assays, but screening a large library by such a method remains a massive undertaking. Furthermore, in a pharmaceutical discovery environment this represents only one of the many assays an organization might wish to conduct.
An alternative to testing individual compounds is the testing of complex mixtures, with various “deconvolution” strategies being employed to deduce the active species. These strategies have in common the re-synthesis and re-testing of successively less-complex mixtures. In addition to the great effort involved, the testing of complex mixtures is limited by the low concentration of any individual species in the mixture, and is susceptible to false positive results from the additive effects of large numbers of weakly active species. In practice, deconvolution has had limited success in probing large libraries of compounds. See L. Wilson-Lingaro, J. Med. Chem., 39, 2720-2726 (1996); and D. A. M. Konings, J. Med. Chem., 39, 2710-2719 (1996), and references therein, for a discussion of deconvolution strategies.
Ideally, the probing of a combinatorial library would be conducted in a single operation, with the active members of the library being in some way “pointed out” of the vast population of compounds by the assay. Two of the current methods meet this requirement. Both methods employ solid-phase synthesis, and both require that the library members remain attached to the solid support. In one method, a library of compounds bound to polystyrene beads is prepared by the split-mix method. The library is assayed in a single batch, by being exposed to a molecule of interest, such as a receptor, enzyme, or other specific binding partner. Any beads to which the molecule binds are visualized (e.g., by a colorimetric assay), and beads so identified are selected and the structure of the library member attached to the bead is determined. This can be done by sequencing if the compound is a peptide or nucleic acid, as described, e.g., in U.S. Pat. No. 5,382,513 (incorporated herein by reference). The structure of the compound on the bead may in some cases be deduced from spectroscopic evidence (see, e.g., U.S. Pat. No. 5,382,513), or by decoding a chemical tag that reveals the chemical history of the bead, as described in patent application WO 95/24186 (incorporated herein by reference). The method is in principle capable of screening very large libraries, limited only by the number of beads one is willing to examine. In practice, libraries of 104 to 106 members can be dealt with in this fashion.
The second approach involves physically locating a compound or compounds in a spatially addressable array of compounds on a planar support. In this approach, a compound's identity is revealed by its location in the array. One method of this type employs an array of compounds generated by light-directed synthesis, as first disclosed by Fodor et al. in Science, 251, 767-773 (1991), in which a fraction of sites on a planar support carrying photo-detachable protecting groups is exposed to light through a photolithographic mask, and the fraction of sites thus deprotected are functionalized with a specific monomer or building block, itself carrying a photo-detachable protecting group. The process is repeated with the mask in a different position or orientation, or with a different mask, and a second monomer or building block is attached to the support and/or to the first monomer residues. After numerous such cycles, with careful attention to the pattern of masking, an array of compounds is built up on the support. The final array is completely deprotected, and exposed to the ligand of interest. Binding of the ligand is visualized by immunofluorescence, using antibodies against the ligand which are tagged with a fluorescent dye. Under a fluorescence microscope, any location in the array to which the ligand has bound is visible as a fluorescent area, and the x-y coordinates of the area reveals the identity of the library member to which the ligand was bound. This technology, as applied to polypeptide and oligonuceotide synthesis, is known as Very Large Scale Immobilized Polymer Synthesis, or VLSIPS. It is described in U.S. Pat. Nos. 5,143,854, 5,413,939, 5,424,186, and 5,527,681, all of which are incorporated herein by reference.
The photolithographic method of synthesis, however, is cumbersome, and requires a substantial investment in very specialized equipment. A further investment in a fluorescence microscope or a specialized scanner is required for the assay, and highly skilled technicians are required, at least to conduct the synthesis aspect of the process. The scale of library synthesis is limited by the size of the masks and by the translational reach of the scanning device, which together limit the accessible surface for synthesis to a few square centimeters. In practice this technique is presently limited to arrays of 104 to 105 compounds. For these reasons the method is not routinely employed; see G. Jung and A. G. Beck Sickinger in Angewandte Chemie, 31, 367 (1992).
As an alternative to photolithography, the use of directed laser light to conduct light-directed synthesis has been described in U.S. Pat. Nos. 4,719,615 and 5,318,679 (both of which are incorporated herein by reference). In the latter patent, a rectangular array support is either held stationary or translated, while a laser beam is scanned across the array by means of a rotating mirror, in the manner of an ordinary laser printer. This provides an alternative to the photolithographic masking approach, but fluorescence microscopy is still relied upon as an assay method.
Another alternative method for the synthesis of spatially addressable arrays utilizes ink-jet printing technology to spray micro-droplets of reagent solutions onto a substrate. This method is disclosed in U.S. Pat. Nos. 5,474,796 and 5,449,754, both of which are incorporated herein by reference. This method is in theory capable of preparing arrays of 107 compounds, but a method of indexing such large arrays is not disclosed. Again, fluorescence microscopy is the preferred means of conducting an assay.
There remains a need for reliable methods of generating very large, very high-density arrays of chemical compounds, on the order of about 108 or more compounds, along with a method of rapidly screening such arrays for chemical properties of interest, such as binding to antibodies, cellular receptors or other ligands, catalytic activity, or inhibition of enzymes.