JP2000508624A - Method for determining the affinity of a protein for a chemical during screening of combinatorial libraries - Google Patents

Abstract

  (57) [Summary] A method for determining the affinity of a protein target for a chemical during screening of a combinatorial library. The library is synthesized on a solid support with a coupling site gradient so that the target has a compound concentration within a certain range. The distribution of the protein bound to the immobilized compound is determined by quantitative imaging analysis. Compounds are ranked in order of binding affinity by comparison of their ability to bind across the gradient.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for Measuring the Affinity of Proteins for Chemicals During Screening of Combinatorial Libraries Background of the Invention Potential Observations During Screening of Large Combinatorial Libraries Containing Target Proteins Given the large number of positive proteins, it is preferable to apply stringent criteria to select key activators to follow. Such criteria include one or more of the following: selectivity for a target versus mutant or closely related protein, binding affinity for a protein target, or active site of an enzyme versus allosteric or non-specific site. For binding to. Current assessment methods for these criteria for compounds require the production of a soluble form of the compound and a separate test. For example, to determine the binding affinity of an agonist or antagonist ligand for a receptor, multiple aliquots of the compound need to be incubated with the receptor over a range of compound concentrations. This operation is tedious, time consuming and requires relatively large amounts of compounds. There is a need in the art for a rapid method of directly measuring the binding affinity of a compound during the screening step. A novel method for determining the affinity of a compound for a protein target in a screen of a combinatorial library on a solid support is described below. SUMMARY OF THE INVENTION The present invention relates to improved methods for biologically evaluating combinatorial libraries using methods that allow the direct determination of the binding affinity of a compound for a biological target. A preferred embodiment of the present invention is a method of determining the affinity of a target protein for a chemical during screening of a combinatorial library, wherein the target protein is provided with a constant gradient of compound concentration on a solid support, A method comprising measuring the amount of bound target at a concentration is provided. The invention also relates to compounds identified using this method. Preferably, the amount of bound target is measured by imaging the distribution of protein bound to the support. The present invention further relates to a support for solid-phase synthesis of a compound in which chemical coupling sites are non-uniformly distributed, and a compound library synthesized on the support. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1a shows the structure of a polymer disc in which the coupling density is distributed radially by coaxial extrusion. When used in FIG. 1a, Sc indicates an equal local concentration of the coupling site. FIG. 1b is a front view of the coaxial extruded body of FIG. 1a. FIG. 2 shows the structure of a rod having a linear coupling concentration. The principle of the method detailed description the invention is that the compound concentrations series of positions localized to the protein target to supply, to determine the amount of target bound at each concentration. In the case of a solid phase library, this method is achieved by varying the coupling density of the compound to the support, forming a compound gradient on a gradient surface. Binding of the protein to the compound with a constant gradient is determined by using reagents that generate optical readings corresponding to the bound protein, and then imaging the distribution of bound protein using compound density. Important elements of the invention include solid supports configured with constant gradient chemical coupling sites for compound attachment, protein targets for pharmaceuticals, and accessory reagents for generating optical signals. And a quantitative imaging device for measuring optical signals. The solid support is an important element in this method and is configured to have a predictable, constant gradient of chemical coupling sites. The gradient is constructed on a disc, rod, ellipsoid, bead or other shaped substrate shape. The preferred form is a disc or rod (FIGS. 1, 2). The number of chemical coupling sites on a particular polymer can be determined by either "doping" a chemoselective linker, photochemically activating / inactivating a sensitizing coupling group, or iodiding a reactive site. Removal of the reactive site from the Wang linker using an alkylating agent such as methyl, or removal of the reactive site from the Merrifie 1d linker using an alkoxide such as potassium methoxide, or a reactive amine-containing linker Is controlled by controlled blocking of undesired sites by chemical means such as removal with an acylating agent such as benzoyl chloride. Preferred substrates are Rapp Tentagel, polyethylene glycol polymers or Perseptive polystyrene-polyethylene glycol polymers, as they can be wetted with aqueous reagents. Discs with discontinuous gradient coupling densities are constructed by subjecting a polymer having a specific density of coupling sites to coaxial extrusion of a cylinder (FIG. 1), followed by cutting. The polymer in each layer is doped with a known concentration of linker by adding a specific concentration of linker to the polymer prior to extrusion. For example, if a resin based on polyethylene glycol (PEG) with a concentration of 3.3 mM of the compound is required, bromoethyl wrap tentagel may be used, which may be combined with a 10: 1 ratio of parahydroxytoluene and parahydroxybenzyl. Reaction with an alcohol and a base such as sodium hydride in a solvent such as DMF yields an available one-like linker site at a 10: 1 dilution. If a polyethylene glycol-based resin in which the compound is present in a concentration of 0.33 mM is required, use bromoethyl wrap tentagel, which is 100: 1 ratio of parahydroxytoluene and parahydroxybenzyl alcohol with sodium hydride. Reaction in a solvent such as DMF, using a base of the formula, to obtain a 100: 1 dilution of available one-like linker sites. If a polyethylene glycol based resin in which the compound is present at a concentration of 33 μM is desired, use bromoethyl wrap tentagel, which is combined with a 1000: 1 ratio of parahydroxytoluene and parahydroxybenzyl alcohol with a base such as sodium hydride. React in a solvent such as DMF to obtain 1000: 1 dilution of available one-like linker sites. Alternatively, the number of available linker sites in the construct of Perseptive PEG polystyrene-like particles can be reduced to polystyrene particles in PEG units in a similar manner by using a 10: 1 ratio of unfunctionalized PEG: functionalized PEG. During the deposition step. If the linker site is further diluted 10-fold, a 100: 1 ratio of unfunctionalized PEG: functionalized PEG is used during the step of attaching the PEG units to the polystyrene particles. If the linker site is further diluted 100-fold, a 1000: 1 ratio of unfunctionalized PEG: functionalized PEG is used during the step of attaching the PEG units to the polystyrene particles. Alternatively, a rod of polymer containing a photocleavable linker can be extruded and the light level changed during extrusion to inactivate the linker at the desired location (FIG. 2). This method allows for the formation of a linear or exponential gradient. Another method is to photoactivate or photoinactivate the light-sensitive linker on the photoreactive substrate in a desired gradient pattern, followed by die-cutting to produce a pattern-engraved particle. The use of lithographic technology. The gradient can be linear, non-linear or discontinuous and the density range is chosen according to the needs of the screen. For example, if a compound with an affinity for the protein target in the range of 10-100 nM is desired, the gradient is configured to achieve an equal local concentration of the compound in the range of 1-1000 nM. The area occupied by the gradient depends on the resolution of the imaging device. Typically, this resolution range is about 100 μm for macro imaging with most CCD cameras, where the gradient is at least as large as a rectangular gradient to allow a 100 nM step resolution at equal compound concentrations. Must occupy an area of 0.1 x 1 mm. The use of high-density CCD arrays and / or magnification optics allows for higher resolution or smaller gradient areas at the expense of processing power. In the latter case, it is limited by the field area that can be imaged. Equal local concentrations of the compound are estimated from the concentration of the coupling site in the polymer. For example, unmodified Rapp Tentagel contains ３００300 picomoles of coupling sites per 260 micromolar (swelling) beads. This corresponds to a compound concentration in the polymer of ３33 mM, assuming a monovalent linker is used and all coupling sites are occupied. The type of linker is selected according to the desired chemistry of the subsequent combinatorial synthesis. Examples of known linkers are one or Merrifield linkers. Once such a collection of particles has been constructed, the library is synthesized using conventional ligation and cleavage protocols. A collection of particles, each having a local density gradient of the individual compound, is thus obtained. The particles are screened against the protein target by adding the particles to a solution of the protein target and measuring the optical signal associated with the protein. The protein target is dissolved or bound to the membrane. This may be directly labeled with a substance capable of forming an optical signal. Preferably, the optical signal is fluorescent or luminescent. The fluorophore is attached to the protein by chemical means. A well-known example is the use of fluorescein isothiocyanate, which attaches fluorescein to amino groups in proteins. Alternatively, the protein may be indirectly labeled by providing a fluorescently labeled antibody that recognizes the protein itself or a suitable tag incorporated into the sequence of the protein. Techniques for these and related protein labels are well known to those skilled in the art. For membrane bound targets, lipophilic fluorescent dyes are available from commercial sources, such as 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine, Molecular, Oregon. Available from Probes of Eugene. Such dyes dissolve in membrane lipids and are used to label vesicles with receptors or other membrane-bound protein targets. Prior to addition of the protein target, the particles may be treated with a high concentration of a blocking reagent to occupy non-specific protein binding sites. Examples of such blocking reagents are 1% bovine serum albumin or casein. After addition of the protein target, the particle suspension is incubated to equilibrate binding. Incubation conditions will vary for each protein target, but generally 2 hours at 37 ° C. will be sufficient for most targets. The optimal protein concentration depends on the sensitivity of the optical detection device and the binding affinity and number of active compounds in the library and is determined empirically. After incubation, the particles are washed with a suitable buffer, for example, 10 mM HEPES (pH 7.6), 0.15 mM sodium chloride, 0.1% NP-40. The particles are then spread on filter paper and imaged. The optical signal is detected using a CCD camera, for example, a Tundra device obtained from Imaging Research Inc., St. Catherine, Ontario, Canada. Positive particles are selected by measuring the decrease in optical signal along the gradient axis. Compounds that bind proteins with high affinity produce signals even at low compound densities, and thus the signals extend to areas of low density gradients of such compounds. Compounds are ranked in order of binding affinity by comparing the optical signal of each particle at a given time within the gradient. After selection of the particles with the high affinity ligand as described above, the compounds are identified. This identification is performed by cleaving the compound from each particle and subjecting the compound to analysis by mass spectrometry. Alternatively, the compound may be tagged during synthesis with a particular chemical marker, and the nature of the compound may be encoded by the tag. Such tagging methods are well-known to those skilled in the art. One example of mass spectrometric compound identification is a mass spectrometric solution to the address problem of combinatorial libraries [Brummel-CL; Lee-IN; Zhou-Y; Benkovic-SJ; Winograd-N, Science. 1994. 264 (5157): 399-402]. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Although preferred embodiments of the present invention have been described above, it is to be understood that the invention is not limited to what is disclosed herein, but that all rights within the scope of the following claims are retained. Can be

Claims (1)

[Claims]   1. During screening of combinatorial libraries, A method for measuring an affinity for a chemical substance,   a) providing the target protein with a constant gradient of compound concentration on the solid support;   b) Determine the amount of target bound at each concentration A method consisting of:   2. Imaging the amount of bound target and the distribution of protein bound to the support The method according to claim 1, wherein the measurement is performed by performing the following.   3. Support for solid-phase compound synthesis with non-uniform distribution of chemical coupling sites body.   4. A compound library synthesized in the support according to claim 3.   5. A compound identified using the method of claim 1.